Almond shells turned into prebiotic sugars and a valuable acid without added enzymes

Almond shells, a bulky by-product of nut processing that is mostly burned or turned into charcoal, can be converted into two higher-value products in a single integrated process, according to a study published in Bioresources and Bioprocessing. The research team combined hot-water pretreatment with fermentation by an engineered fungus to produce xylooligosaccharides, short-chain sugars with reported prebiotic activity, and cellobionic acid, a sugar acid with potential uses in food, pharmaceutical, and chemical manufacturing. The work was carried out on shells collected from California orchards during the 2023 harvest season.

The central idea was to split the biomass into two useful streams rather than targeting only one component. Almond shells contain roughly 26 to 38 percent cellulose, 23 to 28 percent hemicellulose, and 21 to 29 percent lignin, a composition that makes them attractive as a feedstock but also difficult to break down. The researchers hypothesized that hydrothermal treatment with nothing but water could selectively dissolve hemicellulose as xylooligosaccharides while leaving cellulose intact in the solid residue, which the fungus could then convert directly into cellobionic acid without any externally added cellulase enzymes.

Pretreatment conditions were optimized using response surface methodology, a statistical technique that maps how temperature and residence time interact to shape the outcome. In small tube reactors loaded with 10 percent solids, temperatures between 165.9 and 194.1 degrees Celsius and times between 5.9 and 34.1 minutes were tested. A second-order polynomial model fitted the data well, with a coefficient of determination of 0.98 and a non-significant lack-of-fit test, meaning the model adequately described XOS production across the tested range. All linear, interaction, and quadratic terms significantly affected the yield.

The model predicted an optimum near 176.7 degrees Celsius for 17.9 minutes, but experimental validation at those conditions produced only 19.8 grams per liter of xylooligosaccharides along with elevated xylose, a degradation product that signals over-hydrolysis. The researchers therefore selected 180 degrees Celsius for 8 minutes as the practical optimum, a compromise that delivered 18.1 grams per liter of xylooligosaccharides, a yield of 68.9 percent based on the initial xylan content, while keeping xylose formation down to 0.4 grams per liter. Glucose formation was negligible, indicating that the treatment preserved cellulose rather than attacking it.

Scaling the process from laboratory tubes to larger stirred vessels introduced a complication. The 14-milliliter tube reactor needed only one minute to heat from 100 to 180 degrees Celsius, whereas a 200-milliliter Parr reactor took 10 minutes and a 1-liter reactor took 28 minutes to reach the same temperature. The slower heating produced slightly lower xylooligosaccharide concentrations, 17.4 and 17.2 grams per liter respectively, along with more xylose and degradation products. The researchers attributed this to thermal lag, which effectively exposes the biomass to more severe conditions even when the holding temperature and time are identical, and suggested that continuous-flow systems or direct steam injection could help maintain selectivity at scale.

Analysis of the sugar chain lengths in the hydrolysate showed that the product was dominated by short-chain oligomers. Degrees of polymerization ranged from 2 to 10, with xylotriose and xylotetraose, the three- and four-unit sugars, as the predominant species. After purification using charcoal adsorption and ethanol desorption, the chromatograms showed clean, well-separated peaks with minimal background, confirming a narrow and functionally relevant distribution. Short-chain xylooligosaccharides are the forms most associated with prebiotic effects, stimulating beneficial gut microorganisms, and the literature also reports antioxidant, anti-inflammatory, antimicrobial, and antitumor activities, though those biological effects come from prior studies rather than from direct testing in this work.

To gauge how much the pretreatment had loosened the biomass structure, the researchers compared enzymatic hydrolysis of raw and pretreated shells against microcrystalline cellulose as a reference. Raw almond shells were highly recalcitrant, yielding only 6.9 percent glucose conversion at a moderate enzyme loading and 9.8 percent at a high loading of 60 filter paper units of cellulase per gram of cellulose. Pretreated shells reached 32.4 percent and 46.6 percent under the same two conditions, a nearly fivefold improvement that reflects partial hemicellulose removal and disruption of the lignocellulosic matrix. Even so, conversion stayed below 50 percent at the high enzyme loading, underscoring how stubborn the remaining substrate remained.

The second stage of the process handed the pretreated solids to Thermothelomyces heterothallica TH-11, a thermophilic fungus engineered in the laboratory by deleting the genes for beta-glucosidase, phosphorylase, and transporter proteins that would otherwise consume cellobionic acid. Because the fungus produces its own cellulase suite, it can grow on the biomass, secrete enzymes, hydrolyze cellulose to cellobiose, and oxidize that cellobiose to cellobionic acid all in one vessel. Fermentation ran at 40 degrees Celsius for seven days with no added enzymes, using a solid loading equivalent to 15 grams per liter of cellulose.

The results showed the fungus outperforming the commercial enzyme cocktail on the same substrate. Cellobiose peaked on day two and then fell to zero as the fungus converted it further, while cellobionic acid accumulated to 31.8 millimolar. Cellulose conversion reached 56.4 percent, and the yield of cellobionic acid from the cellulose actually consumed was 89.0 percent, meaning that most of the cellulose the fungus managed to digest was channeled efficiently into the target product. Peak cellulase activity of 13.1 filter paper units per gram of cellulose occurred on day two, indicating that most hydrolysis happened within the first three days of fermentation.

The authors note that their cellulose conversion and product yields were lower than those reported for an engineered Neurospora crassa strain growing on alkaline-pretreated wheat straw, which achieved yields approaching 90 percent. They attribute the gap to the higher lignin content of almond shells and to the way hydrothermal treatment redistributes and condenses lignin on the biomass surface, creating physical barriers that block enzyme access and non-productively adsorb cellulase proteins. Alkaline pretreatment removes lignin more effectively, leaving a more open cell-wall structure. The team suggests that chemical-free physical methods such as wet disk refining or intensive milling could improve accessibility in future work, and they flag that cellulose conversion to cellobionic acid remains the main bottleneck, with further optimization, mass and energy balances, and techno-economic analysis planned to identify where the process loses value.

Subject of Research: Integrated biorefinery conversion of almond shells into xylooligosaccharides and cellobionic acid

Article Title: Integrated hydrothermal and microbial valorization of almond shells into xylooligosaccharides and cellobionic acid

Article References: Sharma, S., Shapouri, H., Pournaki, S. K., Barile, D., Kasuga, T., & Fan, Z. (2026). Integrated hydrothermal and microbial valorization of almond shells into xylooligosaccharides and cellobionic acid. Bioresources and Bioprocessing, 13(1), Article 142. https://doi.org/10.1186/s40643-026-01125-1

Image Credits: AI Generated

DOI: 10.1186/s40643-026-01125-1

Keywords: almond shells, xylooligosaccharides, cellobionic acid, hydrothermal pretreatment, biorefinery, Thermothelomyces heterothallica, lignocellulose, prebiotics, response surface methodology, circular bioeconomy, fungal fermentation, agri-food waste