UC Davis

There are over 1.1 million tons of almond produced in California resulting in over two million tons of almond hulls generated each year. Currently, those hulls are mostly used for dairy feed and bedding at low value. However, the almond industry is growing faster than the dairy industry, producing large amounts of excess hulls. Finding alternative uses for almond hulls while increasing their value is essential for the almond industry to achieve its zero-waste goal and improve sustainability. Due to their high sugar and fiber contents, almond hulls have the potential to be utilized for producing edible filamentous fungal biomass for food applications. Fungal biomass can serve as an important source of protein and other nutrients for feeding the increasing population. The overall goal of this research was to develop an efficient system for producing edible fungal biomass from almond hulls by integrating sugar extraction, enzymatic hydrolysis, and submerged cultivation.In the first study, almond hulls of three varieties collected from almond processors were characterized for chemical and physical properties, such as total solids, fiber, protein and fat contents. The three varieties were Independence, Nonpareil, and Monterey. Sugars were extracted from hulls and used for the production of edible fungi. Two different grinding methods, continuous and successive grinding, were compared to get desired particle size for sugar extraction using water as an extraction medium. A kinetic model was developed, based on Fick’s law, for sugar extraction from Independence almond hulls using three different liquid-solid ratios. The model was validated with data collected from extraction experiments conducted at different temperatures. Results showed that successive grinding was chosen to prepare almond hulls with particle sizes in the range of 2.36 to 3.38 mm. The established model was applied to predict sugar extraction from Nonpareil and Monterey almond hulls. The model provided a theoretical framework to understand the extraction process, which is helpful in designing sugar extraction processes for different purposes. Experiments were also conducted to optimize sugar extraction from Independence almond hulls. Response Surface Methodology (RSM) based on Box-Behnken Design was utilized to determine the optimal combination of time, temperature, and liquid-solid ratio for achieving the maximum total reducing sugar yield. The optimum extraction conditions were determined to be as follows: extraction time of 86.6 minutes, temperature of 77.4°C, and a liquid-solid ratio of 14. Under these conditions, the experimental sugar yield was 38.1%, which was well matched with the predicted yield 39.3%. The almond hull extract was used for fungal biomass production of Aspergillus awamori (A. awamori). In 250 mL glass bottles or flasks, A. awamori’s growth in pellet form, was investigated under different conditions including inoculum level, aeration, nitrogen source and carbon to nitrogen ratio (C/N ratio). Subsequently, the fungal cultivation was scaled up to a 2 L bioreactor, where configurations that could favor pellet formation were studied. The structure and morphology of fungal pellets at various growth stages were observed and compared using an environmental scanning electron microscope. Results indicated that yeast extract and NH4Cl worked better than peptone and NaNO3, respectively, as supplemental nitrogen sources. To reduce the cost of raw materials, a combination of yeast extract and NH4Cl was chosen to adjust the initial C/N ratio. The highest concentration and yield of A. awamori biomass were observed when the fungus was cultivated at a C/N ratio of 15, compared to ratios of 30 and 45. At a C/N ratio of 15, the crude protein content in pellets was at its highest at 18.10% (dry basis, d.b.). While the fat content was at its lowest at 2.28% (d.b.). The effects of pH control and agitation on A. awamori growth were investigated in the bioreactor. Ultimately, the optimal conditions for A. awamori growth in almond hull extract were determined to be a C/N ratio of 15, an inoculum level of 103 spores/mL, without pH control, and an agitation rate of 150 rpm over a period of five days in a bioreactor with aeration at 1 air volume per working volume per minute to control dissolved oxygen above 25% of the saturation. Under these conditions, the biomass yield was around 0.85 g VSS/g sugar, with an average pellet size of 3.75 mm. In addition to using the water-soluble extracts from almond hulls for fungal biomass cultivation, the potential of the residual almond hull solids (RASH) after sugar extraction for fungal cultivation was studied by using enzymatical hydrolysis. The effect of three different enzymes (Cellic CTec2, Viscozyme L and Pectinex Ultra SPL) and their combinations on hydrolysis performance were investigated and compared. The optimum loadings of enzymes were determined. The ability of A. awamori to grow on the resulting hydrolysate was evaluated. The most effective hydrolysis in terms of liquefaction and sugar yield was achieved through the combination of Cellic CTec2 and Viscozyme L. Using 200 uL/g RASH of Cellic CTec2 and 60 uL/g RASH of Viscozyme L resulted in a total sugar yield, total fiber conversion, and liquefaction efficiency of 41.36%, 86.01%, and 51.61%, respectively. Applying these optimal conditions at a larger scale resulted in improved liquefaction efficiency, reaching 72.53%. While the sugar yield was similar to small scale hydrolysis. After cultivating A. awamori in hydrolysate for five days, uniform yellow fungal pellets were observed, resulting in a biomass yield of 0.8 g VSS/g sugar. This study suggests that RASH could be a promising source for fungal-based food production, thus optimizing almond hull utilization and increasing the yield of fungal biomass from almond hulls. A techno-economic analysis was conducted for an industrial-scale system for the production of A. awamori fungal biomass. Almond hulls were utilized as the feedstocks, with an input of 50 MT/batch (wet basis, w.b.). SuperPro Designer software was used for process simulation and economic assessment. Two different scenarios were evaluated: the first system was using sugar extract alone as a source of nutrients needed for fungi cultivation and the second was similar to the first system plus the nutrients produced from the enzymatically hydrolyzed residual almond hull solids after sugar extraction. A sensitivity analysis was carried out for studying different parameters, affecting the breakeven price of fungal biomass, such as almond hull price and facility processing capacity. Using the same amount of hulls, the simulated systems produced approximately 2,971 MT/year fungal biomass using the sugars extracted from the hulls using hot water; and around 4,653 MT/year fungal biomass using the sugars extracted from the hulls using hot water plus the sugars produced from the enzymatic hydrolysis of the hulls with the same amount of almond hulls. The breakeven price of the fungal biomass from both systems ranged from $6 to $7 per kg of fungal biomass (d.b.). In both scenarios, the breakeven price of fungal biomass is more sensitive to the capacity at small scale (lower than 50 MT/batch) and more sensitive to almond hull price at larger scale. The results of this study indicate that almond hulls are very good feedstock for the production of myco-foods that can be competitive in the markets of proteins, probiotics, and other food categories, benefiting both the almond and food industries. Finally, the potential of using A. awamori pellets for developing different products (myco-foods) was explored. The color and characteristic of the pellets were studied using both artificial food dyes and natural colored media derived from agricultural and industrial byproducts. Pellets’ colors obtained from natural colored media were more stable after one-month storage. A method to create multilayered color pellets was developed successfully by deactivating pre-cultured pellets and then growing new spores on them. The texture of pellets was improved by coating with potato dextrose agar (PDA) and re-growing to form condensed mycelium inside fungal pellets. The potential for developing A. awamori biomass into dried products was also evaluated. A mice study was conducted to determine the health benefits of the developed products. It was found that the biomass could potentially be processed into protein/fiber powder-based products through freeze drying or transformed into crispy, snack-like myco-chips through hot air drying. Lighter color was obtained after freeze drying, while hot air drying led to darker color of the fungal biomass. No mycotoxins were detected in the A. awamori-based foods, and the foods showed significant health benefits in the mice study. The spent media collected after pellet production contained enzymes, suggesting the potential for its utilization in the development of beverages. The findings from this study provided important information of the potential of developing A. awamori biomass into novel functional food. In summary, based on the findings from this study, California’s two million tons of almond hulls can be potentially transformed into 0.6 million tons of fungal biomass. This conversion could increase the revenue generated from almond hulls, from a previous 0.4 billion dollars (when sold as dairy feed) to 4.2 billion dollars in fungal products. This research demonstrated converting almond hulls into fungi-based food could significantly add economic value to the almond hulls. Further investigations can be undertaken to scale up the production of fungal biomass from almond hulls to large scale. Additionally, exploring the viability of utilizing almond hulls for cultivating various fungal strains could be a promising way forward for future research.