Enzymatic hydrolysis of almond hulls for cultivation of edible filamentous fungi

Abstract

Enzymatic hydrolysis and liquefaction of the residual almond hull solids (RAHS) after sugar extraction were studied. The hydrolysis performance of three different enzymes (Cellic CTec2, Viscozyme L and Pectinex Ultra SPL) were investigated. The ability of Aspergillus awamori and Aspergillus oryzae in utilizing the hydrolysate was evaluated. In 50 mL Falcon tubes, the combination of 200 $\:{\upmu\:}$L/g RAHS of Cellic Ctec2 and 60 $\:{\upmu\:}$L/g RAHS of Viscozyme L enzymes resulted in the optimal performance, with total sugar yield of 47.21%, total fiber conversion of 86.01%, and liquefaction efficiency of 51.61%. Applying these optimal conditions in 250 mL glass bottles resulted in higher liquefaction efficiency of 72.53%, while total fiber conversion was same as that in Falcon tubes. Through a combination of sugar extraction and subsequent enzymatic hydrolysis, a total of 0.55 g of sugars could be released per gram of almond hulls (dry basis, d.b.). The cultivation of Aspergillus awamori and Aspergillus oryzae in hydrolysate for five days produced uniform fungal pellets, with biomass yields of 0.89 g total suspended solids (TSS)/g sugar and 0.43 g TSS/g sugar, respectively. Both fungal strains were able to utilize sugars in the hydrolysate, including galacturonic acid. This research demonstrates that enzymatic hydrolysis of RAHS enables the recovery of additional sugars from almond hulls, thereby increasing the yield of fungal biomass and improving the overall utilization of almond hulls.

Keywords

Almond hull / Enzymatic hydrolysis / Sugar recovery / Liquefaction / Filamentous fungi / Submerged cultivation

References

[1] Almond Board of California (2024) Almond Almanac – 2024. https://www.almonds.com/sites/default/files/2024-12/Digital_Almanac_2024.pdf

[2] Almond Board of California (2019, September 12). Almond Hulls in Chicken Feed? Capstone Project Explores Feasibility. https://www.almonds.com/almond-industry/industry-news/almonds-hulls-chicken-feed-capstone-project-explores-feasibility

[3] APHA, Rice, E., Baird, R., Eaton A (2017) Standard Methods for the Examination of Water and Wastewater ed-23rd. Washington DC: American Public Health Association (APHA), American Water Works Association (AWWA) and Water Environment Federation (WEF)

[4] AxelrodR, Palma MinerL, VanderGheynstJS, SimmonsCW, Fernandez-BayoJD. Soil application of almond residue biomass following black soldier fly larvae cultivation. Front Sustainable Food Syst, 2021, 5664635.

[5] BarzeeTJ, MashadE, CaoHM, ChioL, PanA, ZhangR. Cell-cultivated food production and processing: A review. Food Bioeng, 2022, 1(1): 4-25.

[6] CaoLProduction of fungal biomass (Aspergillus awamori) from almond hulls for food applications, 2023, Davis. University of California.

[7] CaoL, BarzeeTJ, MashadE, PanHM, ZhangR. Potential of utilizing almond hull extract for filamentous fungi production by submerged cultivation. Food Bioeng, 2024, 3(1): 3-13.

[8] Cao L, Mashad E, Pan HM, Z., Zhang R (2025) Production of filamentous fungal biomass (Aspergillus awamori) in pellet form from almond hull extract. Food Bioprocess Technology. https://doi.org/10.1007/s11947-025-03955-x

[9] CardonaMJ, TozziEJ, KarunaN, JeohT, PowellRL, McCarthyMJ. A process for energy-efficient high-solids fed-batch enzymatic liquefaction of cellulosic biomass. Bioresour Technol, 2015, 198: 488-496.

[10] ComboAMM, AguedoM, GoffinD, WatheletB, PaquotM. Enzymatic production of pectic oligosaccharides from polygalacturonic acid with commercial pectinase preparations. Food Bioprod Process, 2012, 90(3): 588-596.

[11] DąbkowskaK, MechM, KopećK, PilarekM. Enzymatic activity of some industrially-applied cellulolytic enzyme preparations. Ecol Chem Eng S, 2017, 24(1): 9-18.

[12] de Alencar GuimarãesNC, GlienkeNN, GaleanoRMS, RullerR, ZanoeloFF, MasuiDC, GiannesiGC. Polygalacturonase from Aspergillus japonicus (PGAj): enzyme production using low-cost carbon source, biochemical properties and application in clarification of fruit juices. Biocatal Agric Biotechnol, 2022, 39102233.

[13] DePetersEJ, SwansonKL, BillHM, AsmusJ, HeguyJM. Nutritional composition of almond hulls. Appl Anim Sci, 2020, 36(6): 761-770.

[14] DeyTB, AdakS, BhattacharyaP, BanerjeeR. Purification of polygalacturonase from Aspergillus Awamori Nakazawa MTCC 6652 and its application in Apple juice clarification. LWT-Food Sci Technol, 2014, 59(1): 591-595.

[15] DuJ, ZhangF, LiY, ZhangH, LiangJ, ZhengH, HuangH. Enzymatic liquefaction and saccharification of pretreated corn Stover at high-solids concentrations in a horizontal rotating bioreactor. Bioprocess Biosyst Eng, 2014, 37: 173-181.

[16] HamadH, AlmaM, ISMAELH, GÖÇERİA. The effect of some sugars on the growth of Aspergillus Niger. KSÜ Doğa Bilimleri Dergisi, 2014, 17(4): 7-11.

[17] HoltmanKM, OffemanRD, Franqui-VillanuevaD, BayatiAK, OrtsWJ. Countercurrent extraction of soluble sugars from almond hulls and assessment of the bioenergy potential. J Agric Food Chem, 2015, 63(9): 2490-2498.

[18] IchishimaE. Development of enzyme technology for Aspergillus oryzae, A. sojae, and A. luchuensis, the National microorganisms of Japan. Biosci Biotechnol Biochem, 2016, 80(9): 1681-1692.

[19] KianiA, TorabiP, MousaviZE. Green recovery of phenolic compounds from almond hull waste using ultrasound-assisted extraction: phenolics characterization and antimicrobial investigation. J Food Sci Technol, 2024, 61(10): 1930-1942.

[20] Kim Y, Lee V (2021) Enzyme-assisted protein extraction from alfalfa leaves. 2021 ASABE Annual International Virtual Meeting, 1

[21] KoukoumakiDI, TsoukoE, PapanikolaouS, IoannouZ, DiamantopoulouP, SarrisD. Recent advances in the production of single cell protein from renewable resources and applications. Carbon Resour Convers, 2024, 72100195.

[22] Li D, Hua X, Luo J, Xu Y (2023) Quantitative determination of galac turonic acid in pectin and pectin products by combined pectinase hydrolysis and HPLC determination. Food Additives & Contaminants: Part A, 40(3):319–327. https://doi.org/10.1080/19440049.2023.2165171

[23] LiuL, YouY, DengH, GuoY, MengY. Promoting hydrolysis of Apple pomace by pectinase and cellulase to produce microbial oils using engineered Yarrowia lipolytica. Biomass Bioenergy, 2019, 126: 62-69.

[24] McKenzieFC, WilliamsJ. Sustainable food production: constraints, challenges and choices by 2050. Food Secur, 2015, 7: 221-233.

[25] ModenbachAA, NokesSE. Enzymatic hydrolysis of biomass at high-solids loadings–a review. Biomass Bioenergy, 2013, 56: 526-544.

[26] Monroe HE (2021) Conversion of almond hulls into protein-enriched animal feed supplements. University of California. https://escholarship.org/uc/item/0707x3kv Davis

[27] NajariZ, KhodaiyanF, YarmandMS, HosseiniSS. Almond hulls waste valorization towards sustainable agricultural development: production of pectin, phenolics, pullulan, and single cell protein. Waste Manag, 2022, 141: 208-219.

[28] NguyenCL, NguyenHVH. The quality of mulberry juice as affected by enzyme treatments. Beverages, 2018, 4241.

[29] OffemanRD, DaoGT, HoltmanKM, OrtsWJ. Leaching behavior of water-soluble carbohydrates from almond hulls. Ind Crops Prod, 2015, 65: 488-495.

[30] OlivieriG, WijffelsRH, MarzocchellaA, RussoME. Bioreactor and bioprocess design issues in enzymatic hydrolysis of lignocellulosic biomass. Catalysts, 2021, 116680.

[31] PadayacheeA, DayL, HowellK, GidleyMJ. Complexity and health functionality of plant cell wall fibers from fruits and vegetables. Crit Rev Food Sci Nutr, 2017, 57(1): 59-81.

[32] PalmqvistB, WimanM, LidénG. Effect of mixing on enzymatic hydrolysis of steam-pretreated spruce: a quantitative analysis of conversion and power consumption. Biotechnol Biofuels, 2011, 4: 1-8.

[33] ParkH-S, JunS-C, HanK-H, HongS-B, YuJ-H. Diversity, application, and synthetic biology of industrially important Aspergillus fungi. Adv Appl Microbiol, 2017, 100: 161-202.

[34] Paz-CedenoFR, CarcellerJM, IborraS, DonatoRK, RodriguezAFR, MoralesMA, Solorzano-ChavezEG, RoldánIUM, de PaulaAV, MasarinF. Stability of the cellic CTec2 enzymatic Preparation immobilized onto magnetic graphene oxide: assessment of hydrolysis of pretreated sugarcane Bagasse. Ind Crops Prod, 2022, 183114972.

[35] PrgometI, GonçalvesB, Domínguez-PerlesR, Pascual-SevaN, BarrosAI. A box-behnken design for optimal extraction of phenolics from almond by-products. Food Anal Methods, 2019, 12: 2009-2024.

[36] SheaE, Fernandez-BayoJ, SimmonsC. Effect of almond residue soil amendments and irrigation regiment on organic acid development and transport in soil. ACS Agricultural Sci Technol, 2024, 4(9): 899-906.

[37] Sitepu IR, Hitomi A, Wu W, Wu A, Jeoh T, Boundy-Mills K (2023) Production of high protein yeast using enzymatically liquefied almond hulls. PLoS ONE 18(11):e0293085

[38] Souza FilhoPF, NairRB, AnderssonD, LennartssonPR, TaherzadehMJ. Vegan-mycoprotein concentrate from pea-processing industry byproduct using edible filamentous fungi. Fungal Biology Biotechnol, 2018, 5(1): 1-10.

[39] Spadoni AndreaniE, KarbouneS. Comparison of enzymatic and microwave-assisted alkaline extraction approaches for the generation of oligosaccharides from American cranberry (Vaccinium macrocarpon) pomace. J Food Sci, 2020, 85(8): 2443-2451.

[40] TaniguchiK, MatsudaK, TeramuraH, WakeK. Effect of acetic acid on growth of Aspergillus Niger. J Fac Sci Hokkaido Univ Ser 5 Bot, 1977, 10(4): 189-198

[41] Umsza-GuezMA, DíazAB, de OryI, BlandinoA, GomesE, CaroI. Xylanase production by Aspergillus Awamori under solid state fermentation conditions on tomato pomace. Brazilian J Microbiol, 2011, 42: 1585-1597.

[42] U.S. Food & Drug (2018) Microorganisms & Microbial- Derived Ingredients Used in Food (Partial List). https://www.fda.gov/food/generally-recognized-safe-gras/microorganisms-microbial-derived-ingredients-used-food-partial-list

[43] Vivek K, Venkitasamy C (2023) Role and applications of fungi in food and fermentation technology. In Fungal resources for sustainable economy: current status and future perspectives (pp. 71–87). Springer. https://doi.org/10.1007/978-981-19-9103-5_3

[44] ZicariSMEnzymatic liquefaction of sugar beets as a feedstock for biofuel production, 2016, Davis. University of California.

Funding

California Department of Food and Agriculture(20-0001-036-SF)

Resnick Agricultural Innovation Research Fund(Resnick Agricultural Innovation Research Fund)

RIGHTS & PERMISSIONS

The Author(s)