Production of high protein yeast using enzymatically liquefied almond hulls.

Animal feed ingredients, especially those abundant in high quality protein, are the most expensive component of livestock production. Sustainable alternative feedstocks may be sourced from abundant, low value agricultural byproducts. California almond production generates nearly 3 Mtons of biomass per year with about 50% in the form of hulls. Almond hulls are a low-value byproduct currently used primarily for animal feed for dairy cattle. However, the protein and essential amino acid content are low, at ~30% d.b.. The purpose of this study was to improve the protein content and quality using yeast. To achieve this, the almond hulls were liquefied to liberate soluble and structural sugars. A multi-phase screening approach was used to identify yeasts that can consume a large proportion of the sugars in almond hulls while accumulating high concentrations of amino acids essential for livestock feed. Compositional analysis showed that almond hulls are rich in polygalacturonic acid (pectin) and soluble sucrose. A pectinase-assisted process was optimized to liquefy and release soluble sugars from almond hulls. The resulting almond hull slurry containing solubilized sugars was subsequently used to grow high-protein yeasts that could consume nutrients in almond hulls while accumulating high concentrations of high-quality protein rich in essential amino acids needed for livestock feed, yielding a process that would produce 72 mg protein/g almond hull. Further work is needed to achieve conversion of galacturonic acid to yeast cell biomass.

Interfacial molecular interactions of cellobiohydrolase Cel7A and its variants on cellulose.

BACKGROUND: Molecular-scale mechanisms of the enzymatic breakdown of cellulosic biomass into fermentable sugars are still poorly understood, with a need for independent measurements of enzyme kinetic parameters. We measured binding times of cellobiohydrolase Trichoderma reesei Cel7A (Cel7A) on celluloses using wild-type Cel7A (WTintact), the catalytically deficient mutant Cel7A E212Q (E212Qintact) and their proteolytically isolated catalytic domains (CD) (WTcore and E212Qcore, respectively). The binding time distributions were obtained from time-resolved, super-resolution images of fluorescently labeled enzymes on cellulose obtained with total internal reflection fluorescence microscopy. RESULTS: Binding of WTintact and E212Qintact on the recalcitrant algal cellulose (AC) showed two bound populations: ~ 85% bound with shorter residence times of < 15 s while ~ 15% were effectively immobilized. The similarity between binding times of the WT and E212Q suggests that the single point mutation in the enzyme active site does not affect the thermodynamics of binding of this enzyme. The isolated catalytic domains, WTcore and E212Qcore, exhibited three binding populations on AC: ~ 75% bound with short residence times of ~ 15 s (similar to the intact enzymes), ~ 20% bound for < 100 s and ~ 5% that were effectively immobilized. CONCLUSIONS: Cel7A binding to cellulose is driven by the interactions between the catalytic domain and cellulose. The cellulose-binding module (CBM) and linker increase the affinity of Cel7A to cellulose likely by facilitating recognition and complexation at the substrate interface. The increased affinity of Cel7A to cellulose by the CBM and linker comes at the cost of increasing the population of immobilized enzyme on cellulose. The residence time (or inversely the dissociation rates) of Cel7A on cellulose is not catalysis limited.

Conversion of cassava leaf to bioavailable, high-protein yeast cell biomass.

BACKGROUND: Cassava leaves are an abundant global agricultural residue because the roots are a major source of dietary carbohydrates. Although cassava leaves are high in protein, the protein is not bioavailable. This work aimed to convert cassava leaves to a bioavailable protein-rich animal feed ingredient using high-protein yeasts. RESULTS: The structural proteins (ca 200 g kg-1 d.b.) from sundried cassava leaves were solubilized by mild alkali pretreatment, and the resulting cassava leaf hydrolysate (CLH) was used to screen for growth of 46 high-protein yeasts from 30 species. Promising candidates from the initial screen cultivated at a 10 mL scale demonstrated increases in relative abundance of essential amino acids over that of CLH. In particular, lysine, growth-limiting for some livestock, was increased up to 226% over the CLH content. One yeast, Pichia kudriavzevii UCDFST 11-602, was grown in 3 L of CLH in a bioreactor to examine the scale-up potential of the yeast protein production. While glucose was completely consumed, yeast growth exited log phase before depleting either carbon or nitrogen, suggesting other growth-limiting factors at the larger scale. CONCLUSIONS: High-value animal feed with enriched essential amino acid profiles can be produced by yeasts grown on agricultural residues. Yeasts convert structural protein solubilized from cassava leaves to essential amino acid-enriched, digestible protein. The low carbohydrate content of the leaves (ca 200 g kg-1 d.b.), however, necessitated glucose supplementation for yeast growth. © 2018 Society of Chemical Industry.