Geotrichum candidum IBB69: a high-yield microbial protein producer with superior nutritional profile and industrial potential

Abstract

Identifying alternative protein sources is crucial in view of the shortage of protein resources. A new strain Geotrichum candidum IBB69 was isolated for microbial protein production in this study. The protein yield, biomass, and specific protein production (the ratio of total intracellular and extracellular protein to biomass) of G. candidum IBB69 were 7.6 g/L, 18.42 g/L and 60.8% after optimization through shaking flask fermentation, response surface methodology, and 5L scale-up fermentation. Compared to flask fermentation, the protein yield and content increased by 83.13% and 91.08%, respectively. The protein product of IBB69 was composed of 18 amino acids with a ratio of 37.51% (EAA to TAA). Transcriptome analysis revealed that, compared with ammonium sulfate, the addition of urea upregulated the expression levels of key genes in the carbon–nitrogen metabolism process of G. candidum IBB69, promoting amino acid synthesis and cell growth. This study tapped a new microbial protein producing strain, and optimized results set the stage for the industrial development and application of G. candidum for sustainable alternative proteins.

Keywords

Geotrichum candidum / Single-cell protein / Fermentation optimization / Nutritional composition / Transcriptome analysis

References

[1] AbdullahiN, DandagoMA, YunusaAK. Review on production of single-cell protein from food wastes. TURJAF, 2021, 9: 968-974

[2] SelinNE. Global biogeochemical cycling of mercury: a review. Annu Rev Environ Resour, 2009, 34: 43-63.

[3] DijkMV, MorleyT, RauML, SaghaiY. A meta-analysis of projected global food demand and population at risk of hunger for the period 2010–2050. Nat Food, 2021, 2: 494-501.

[4] WadhwaM, BakshiM. Utilization of fruit and vegetable wastes as livestock feed and as substrates for generation of other value-added products. Rap Publ, 2013, 467

[5] KadimIT, MahgoubO, BaqirS, FayeB, PurchasR. Cultured meat from muscle stem cells: a review of challenges and prospects. J Integr Agric, 2015, 14: 222-233.

[6] HülsenT, HsiehK, LuY, TaitS, BatstoneDJ. Simultaneous treatment and single cell protein production from agri-industrial wastewaters using purple phototrophic bacteria or microalgae–a comparison. Bioresour Technol, 2018, 254: 214-223.

[7] GiecA, SkupinJ. Single cell protein as food and feed. Food Nahrung, 1988, 32: 219-229.

[8] KieliszekM, KotAM, Bzducha-WróbelA, BłażejakS, GientkaI, KurczA. Biotechnological use of Candida yeasts in the food industry: a review. Fungal Biol Rev, 2017, 31: 185-198.

[9] NasseriA, Rasoul-AminiS, MorowvatMH, GhasemiY. Single cell protein: production and process. Am J Food Technol, 2011, 6: 103-116.

[10] MissousG, ThammavongsB, LaunayG, GuéguenM, DieuleveuxV, PanoffJ-M. Relationship between growth behaviour, micro and macroscopic morphologies and freezing sensitivity of the ripening starter Geotrichum candidum is strain specific and mostly related to the morphotypes: the arthrospores/hyphae parameter. J Dairy Res, 2010, 77: 425-431.

[11] LinkoM, HaikaraA, RitalaA, PenttiläM. Recent advances in the malting and brewing industry. J Biotechnol, 1998, 65: 85-98.

[12] BambaT, HoriY, UmebayashiK, SohC, HakozakiT, ToyamaK, OsumiM, KondoA, HasunumaT. Comprehensive metabolic profiling of Geotrichum candidum and comparison with Saccharomyces cerevisiae. J Biosci Bioeng, 2024, 137: 9-15.

[13] BorisovaAS, EneyskayaEV, BobrovKS, JanaS, LogachevA, PolevDE, LapidusAL, IbatullinFM, SaleemU, SandgrenM. Sequencing, biochemical characterization, crystal structure and molecular dynamics of cellobiohydrolase Cel7A from Geotrichum candidum 3C. FEBS J, 2015, 282: 4515-4537.

[14] BoutrouR, GuéguenM. Interests in Geotrichum candidum for cheese technology. Int J Food Microbiol, 2005, 102: 1-20.

[15] CaldwellI, TrinciA. The growth unit of the mould Geotrichum candidum. Arch Microbiol, 1973, 88: 1-10.

[16] PiegzaM, WitkowskaD, StempniewiczR. Enzymatic and molecular characteristics of Geotrichum candidum strains as a starter culture for malting. J Inst Brew, 2014, 120: 341-346.

[17] BertoliniMC, LarameeL, ThomasDY, CyglerM, SchragJD, VernetT. Polymorphism in the lipase genes of Geotrichum candidum strains. FEBS J, 1994, 219: 119-125.

[18] TianY, LiJ, MengJ, LiJ. High-yield production of single-cell protein from starch processing wastewater using co-cultivation of yeasts. Bioresour Technol, 2023, 370. 128527

[19] World Health O. Laboratory biosafety manual. 4th ed ed. World Health Organization, Geneva. 2020.

[20] BourdichonF, CasaregolaS, FarrokhC, FrisvadJC, GerdsML, HammesWP, HarnettJ, HuysG, LaulundS, OuwehandA. Food fermentations: microorganisms with technological beneficial use. Int J Food Microbiol, 2012, 154: 87-97.

[21] QiaoH, CaiG, SunJ. Selection of Geotrichum and its preliminary application. J Food Sci Biotechnol, 2011, 30: 295-301(In Chinese with English absrtract)

[22] HuX, YuC, LiX, ZouJ, DuY, PatersonDM. Biomineralization of heavy metals based on urea transport and hydrolysis within a new bacterial isolate B intermedia. TSBOI J Hazard Mater, 2024, 469. 134049

[23] ZhouY, YangX, LiQ, PengZ, LiJ, ZhangJ. Optimization of fermentation conditions for surfactin production by B. subtilis YPS-32. BMC Microbiol., 2023, 23117.

[24] TongS, ChenW, HongR, ChaiM, SunY, WangQ, LiD. Efficient mycoprotein production with low CO₂ emissions through metabolic engineering and fermentation optimization of Fusarium venenatum. J Agric Food Chem., 2023, 72: 604-612.

[25] YuQ, GuoM, ZhangB, WuH, ZhangY, ZhangL. Analysis of nutritional composition in 23 kinds of edible fungi. J Food Qual., 2020, 20208821315.

[26] ZhouW, TongS, AminFR, ChenW, CaiJ, LiD. Heterologous expression and biochemical characterization of a thermostable endoglucanase (Mt EG5-1) from Myceliophthora thermophila. Fermentation, 2023, 9462.

[27] Ozturk UrekR, IlginS. Production and partial characterization of the exopolysaccharide from Pleurotus sajor caju. Ann Microbiol, 2019, 69: 1201-1210.

[28] LiQ, WangJ, ZhaX, PanL, ZhangH, LuoJ. Structural characterization and immunomodulatory activity of a new polysaccharide from jellyfish. Carbohydr Polym, 2017, 159: 188-194.

[29] LiR, FanX, JiangY, WangR, GuoR, ZhangY, FuS. From anaerobic digestion to single cell protein synthesis: a promising route beyond biogas utilization. Water Res., 2023.

[30] ZhangQ, ZhangT. Determination of polysaccharide content by phenol-sulfuric acid method. SDIFST, 2004, 13: 17-18(In Chinese with English absrtract)

[31] HongR, TongS, ChaiM, ChenW, LiuX, ChenY, LiDemao. Enhancing mycoprotein yield: metabolic modulation of chitin synthase in Fusarium venenatum. J Agric Food Chem., 2024, 72: 27274-27283

[32] LiY, ChenL, ChenW, ZhuJ, ChenY, LiD. Transcriptomic analysis of the metabolic regulatory mechanism of Schizochytrium limacinum B4D1 using sodium acetate to produce DHA. Biochem Eng J, 2023, 197. 108963

[33] HuJ, ZhangY, XuY, SunQ, LiuJ, FangW, XiaoY, KüesU, FangZ. Gongronella sp. w5 elevates Coprinopsis cinerea laccase production by carbon source syntrophism and secondary metabolite induction. Appl Microbiol Biotechnol., 2019, 103: 411-425.

[34] ZhangJ, ZhaoN, GuoL, LiP, GuS, YuanJ, FanM. In-depth exploration on the effects of organic nitrogen addition during different fermentation periods on the quality of cider. LWT, 2023, 187. 115376

[35] NaikRP, PreetamVC, KumariNN, RajuM, PrakashB, ReddyM. Effect of different zinc sources and concentrations on the biomass yield of Saccharomyces cerevisiae yeast. Biol Trace Elem Res., 2022.

[36] ObataH, HayashiA, TodaT, UmebayashiM. Effects of zinc deficiency on the growth, proteins, and other constituents of yeast, Saccharomyces cerevisiae, cells. Soil Sci Plant Nutr, 1996, 42: 147-154.

[37] OkK, FilipovicMR, MichelSL. Targeting zinc finger proteins with exogenous metals and molecules: lessons learned from tristetraprolin, a CCCH type zinc finger. Eur J Inorg Chem, 2021, 2021: 3795-3805.

[38] NyyssöläA, SuhonenA, RitalaA, Oksman-CaldenteyKM. The role of single cell protein in cellular agriculture. COBIOT., 2022.

[39] NordlundE, Silventoinen-VeijalainenP, Hyytiäinen-PabstT, NyyssöläA, ValtonenA, RitalaA, LienemannM, Rosa-SibakovN. In vitro protein digestion and carbohydrate colon fermentation of microbial biomass samples from bacterial, filamentous fungus and yeast sources. Food Res Int, 2024, 182: 0963-9969.

[40] MengY, WeiZ, XueC. Protein fibrils from different food sources: a review of fibrillation conditions, properties, applications and research trends. Trends Food Sci Technol, 2022, 121: 0924-2244.

[41] WiebeM. Myco-protein from Fusarium venenatum: a well-established product for human consumption. Appl Microbiol Biotechnol, 2002, 58: 421-427.

[42] JacobsonMF, DePorterJ. Self-reported adverse reactions associated with mycoprotein (Quorn-brand) containing foods. Ann Allergy Asthma Immunol, 2018, 120(6): 626-630.

[43] AhmedA, SohailM. Characterization of pectinase from Geotrichum candidum AA15 and its potential application in orange juice clarification. J King Saud Univ Sci, 2020, 32: 955-961.

[44] MuhammadA, BokhariSAI, VernouxJ-P, AliMI, FaryalR, DesmasuresN, ImranM. Purification, characterization and thermodynamic assessment of an alkaline protease by Geotrichum candidum of dairy origin. Iran J Biotechnol, 2019.

[45] PichethGF, PirichCL, SierakowskiMR, WoehlMA, SakakibaraCN, de SouzaCLF, MartinAA, da SilvaR, de FreitasRA. Bacterial cellulose in biomedical applications: a review. Int J Biol., 2017, 104: 97-106.

[46] WuC, JiangN, WangR, JiangS, YuanZ, XueL, WuJ, ShiH, WuR. Linoleic acid enrichment of cheese by okara flour and Geotrichum candidum overexpressing Δ12 fatty acid desaturase. J Sci Food Agric, 2023, 103: 2960-2969.

[47] YamakawaCK, QinF, MussattoSI. Advances and opportunities in biomass conversion technologies and biorefineries for the development of a bio-based economy. Biomass Bioenergy, 2018, 119: 54-60.

[48] LiuK, HuangS, ZhangL, XiongY, WangX, BaoY, LiD, LiJ. Efficient production of single cell protein from biogas slurry using screened alkali-salt-tolerant Debaryomyces hansenii. Bioresour Technol, 2024, 393. 130119

[49] HeY, QinH, WenJ, WangL, CaoW, FanS, LuW, LiJ, LiC. Characterization of amino acid composition, nutritional value, and taste of fruits from different Actinidia arguta resources. J Food Qual, 2024, 20241005194.

[50] DingH, LiG, DengF, HuangS, ZhouP, LiuX, LiZ, LiD. Ammonia nitrogen recovery from biogas slurry by SCP production using Candida utilis. JEM, 2023, 325: 0301-4797.

[51] ZhouP, ZhangL, DingH, GaoX, ChenY, LiD. Optimization of culture conditions of screened Galactomyces candidum for the production of single cell protein from biogas slurry. Electron J Biotechnol, 2022, 55: 47-54.

[52] NyyssöläA, SuhonenA, RitalaA, Oksman-CaldenteyKM. The role of single cell protein in cellular agriculture. COBIOT, 2022.

[53] NordlundE, Silventoinen-VeijalainenP, Hyytiäinen-PabstT, NyyssöläA, ValtonenA, RitalaA, LienemannM, Rosa-SibakovN. In vitro protein digestion and carbohydrate colon fermentation of microbial biomass samples from bacterial, filamentous fungus and yeast sources. Food Res Int, 2024, 182: 0963-9969.

[54] MengY, WeiZ, XueC. Protein fibrils from different food sources: a review of fibrillation conditions, properties, applications and research trends. Trends Food Sci Technol, 2022, 121: 0924-2244.

[55] WikandariR, TanugrahaDR, YastantoAJ, ManikhardaGR, TeixeiraJA. Development of meat substitutes from filamentous fungi cultivated on residual water of tempeh factories. Molecules., 2023, 28997.

[56] NavarathnaDH, DasA, MorschhauserJ, NickersonKW, RobertsDD. Dur3 is the major urea transporter in Candida albicans and is co-regulated with the urea amidolyase Dur 1, 2. Microbiology., 2011, 157: 270-279.

[57] YangX, YangY, HuangJ, ManD, GuoM. Comparisons of urea or ammonium on growth and fermentative metabolism of Saccharomyces cerevisiae in ethanol fermentation. World J Microbiol Biotechnol, 2021, 3798.

[58] SmithDP, ThrashJC, NicoraCD, LiptonMS, Burnum-JohnsonKE, CariniP, SmithRD, GiovannoniSJ. Proteomic and transcriptomic analyses of “Candidatus Pelagibacter ubique” describe the first PII-independent response to nitrogen limitation in a free-living Alphaproteobacterium. mBio., 2013.

[59] LuZ, HeS, KashifM, ZhangZ, MoS, SuG, DuL, JiangC. Effect of ammonium stress on phosphorus solubilization of a novel marine mangrove microorganism Bacillus aryabhattai NM1-A2 as revealed by integrated omics analysis. BMC Genom, 2023, 241550.

[60] ZhuH, YeZ, XuZ, WeiL. Transcriptomic analysis reveals the effect of urea on metabolism of Nannochloropsis oceanica. Life, 2024, 14797.

[61] NasrA, BekheetIA, IbrahimR. The effect of different nitrogen and carbon sources on amino acid synthesis in Ulva. Dictyota Pteroclad Hydrobiol, 1968, 31: 7-7.

[62] BeylerliO, SufianovaG, ShumadalovaA, ZhangD, GareevI. MicroRNAs-mediated regulation of glucose transporter (GLUT) expression in glioblastoma. Non-coding RNA Res, 2022, 7: 205-211.

Funding

National Key Research and Development Program of China(2023YFD1201600)

RIGHTS & PERMISSIONS

Jiangnan University