Intestinal delivery of encapsulated bacteriocin peptides in cross-linked alginate microcapsules.

Oral delivery of larger bioactive peptides (>20 amino acids) to the small intestine remains a challenge due to their sensitivity to proteolytic degradation and chemical denaturation during gastrointestinal transit. In this study, we investigated the capacity of crosslinked alginate microcapsules (CLAMs) formed by spray drying to protect Plantaricin EF (PlnEF) (C-EF) in gastric conditions and to dissolve and release PlnEF in the small intestine. PlnEF is an unmodified, two-peptide (PlnE: 33 amino acids; PlnF: 34 amino acids) bacteriocin produced by Lactiplantibacillus plantarum with antimicrobial and gut barrier protective properties. After 2 h incubation in simulated gastric fluid (SGF) (pH 1.5), 43.39 % ± 8.27 % intact PlnEF was liberated from the CLAMs encapsulates, as determined by an antimicrobial activity assay. Transfer of the undissolved fraction to simulated intestinal fluid (SIF) (pH 7) for another 2 h incubation resulted in an additional release of 16.13 % ± 4.33 %. No active PlnEF was found during SGF or sequential SIF incubations when pepsin (2,000 U/ml) was added to the SGF. To test PlnEF release in C-EF contained in a food matrix, C-EF was mixed in peanut butter (PB) (0.15 g C-EF in 1.5 g PB). A total of 12.52 % ± 9.09 % active PlnEF was detected after incubation of PB + C-EF in SGF without pepsin, whereas no activity was found when pepsin was included. Transfer of the remaining PB + C-EF fractions to SIF yielded the recovery of 46.67 % ± 13.09 % and 39.42 % ± 11.53 % active PlnEF in the SIF following exposure to SGF and to SGF with pepsin, respectively. Upon accounting for the undissolved fraction after SIF incubation, PlnEF was fully protected in the CLAMs-PB mixture and there was not a significant reduction in active PlnEF when pepsin was present. These results show that CLAMs alone do not guard PlnEF bacteriocin peptides from gastric conditions, however, mixing them in PB protected against proteolysis and improved intestinal release.

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.

Lytic polysaccharide monooxygenase activity increases productive binding capacity of cellobiohydrolases on cellulose.

Cellobiohydrolases are crucial for cellulose breakdown, but their efficiency on crystalline cellulose is hampered by limited access to single chain ends to initiate hydrolysis. As a result, they depend on enzymes like lytic polysaccharide monooxygenases (LPMOs), which directly target the crystalline cellulose surface. This study investigated how LPMO pretreatment affected the productive binding capacity of a Trichoderma longibrachiatum cellobiohydrolase, TlCBHI, on crystalline cellulose by applying an amperometric cellobiose dehydrogenase biosensor. After the 24-hour of LPMO pretreatment, the productive binding capacity of TlCBHI significantly increased in all reactions. However, with a shorter 5-hour LPMO pretreatment, minimal to no effect on productive binding capacity was observed. Of note, all LPMO reactions were inactivated around this time point. This delayed LPMO effect suggests that the improved binding capacity for cellulases does not directly result from cellulose chain cleavage by LPMOs but rather from the cellulose decrystallization following the oxidative cleavage.

The role of a moisture-barrier latex in controlling retention, stability and release of D-limonene from complex coacervated matrix microparticles formed during spray drying.

Limonene from citrus peel oil is valued as fragrance and flavor additives in food and beverages; however, D-limonene is highly volatile and oxygen-sensitive, thus present storage and stability challenges in food products. A novel, industrially-scalable microencapsulation by in situ complex coacervation during spray drying process (CoCo process) was applied to encapsulate limonene in alginate-gelatin matrix microparticles. Specifically, we investigated the potential to improve upon prior work demonstrating volatile retention and enteric release of limonene from the complex coacervated (CoCo) microcapsules by incorporating ethylcellulose to improve moisture and oxygen barrier properties of the encapsulation matrix. We hypothesized that ethylcellulose, commonly used as a water-barrier coating with pharmaceuticals, would enhance the ability of CoCo microcapsules to retain and shelf-stabilize limonene. The CoCo process alone could achieve limonene retention of 77.7% ± 1.3% during spray drying, with only ∼10% limonene loss and low oxidation rate after 3-weeks of storage in ambient conditions. Contrary to expectations, incorporating ethylcellulose with the CoCo formulation increased volatile losses of limonene during spray drying and during prolonged storage. Moreover, CoCo powders with ethylcellulose accelerated limonene release in water and simulated gastric fluid, and decelerated release in simulated intestinal fluid-a result that was contrary to targeting enteric release. Instead of simply forming a protective water barrier film in the microparticles during spray drying as envisioned, ethylcellulose appeared to bring limonene to the particle surfaces, thereby enhancing volatile losses, facilitating oxidation and accelerating release in acidic aqueous media. Using ethylcellulose as a model, this study demonstrated the potential to formulate CoCo microparticles using latex excipients to control burst release of the payload followed by long-lasting sustained release in air and in aqueous environments.

Optimizing viability and yield and improving stability of Gram-negative, non-spore forming plant-beneficial bacteria encapsulated by spray-drying.

This study investigates methods to commercialize safer alternatives to chemical pesticides that pose risks to human safety and the environment. Spray-drying encapsulation of the plant-protective, antifungal bacterium Collimonas arenae Cal35 in in situ cross-linked alginate microcapsules (CLAMs) was optimized to minimize losses during spray-drying and maximize yield of spray-dried powder. Only inlet temperature significantly affected survival during spray-drying, while inlet temperature, spray rate, and alginate concentration significantly affected yield of spray-dried powder. Lowering inlet temperature to 95 °C provided the greatest survival during spray-drying, while increasing inlet temperature and lowering spray rate and alginate concentration produced the highest yield. Without the CLAMs formulation, Cal35 did not survive spray-drying. When Cal35 was encapsulated in CLAMs in the presence of modified starch, shelf survival was extended to 3 weeks in a low oxygen, low humidity storage environment. Cal35 retained its antifungal activity throughout spray-drying and shelf storage, supporting its potential use as a formulated biofungicide product.

How alginate properties influence in situ internal gelation in crosslinked alginate microcapsules (CLAMs) formed by spray drying.

Alginates gel rapidly under ambient conditions and have widely documented potential to form protective matrices for sensitive bioactive cargo. Most commonly, alginate gelation occurs via calcium mediated electrostatic crosslinks between the linear polyuronic acid polymers. A recent breakthrough to form crosslinked alginate microcapsules (CLAMs) by in situ gelation during spray drying ("CLAMs process") has demonstrated applications in protection and controlled delivery of bioactives in food, cosmetics, and agriculture. The extent of crosslinking of alginates in CLAMs impacts the effectiveness of its barrier properties. For example, higher crosslinking extents can improve oxidative stability and limit diffusion of the encapsulated cargo. Crosslinking in CLAMs can be controlled by varying the calcium to alginate ratio; however, the choice of alginates used in the process also influences the ultimate extent of crosslinking. To understand how to select alginates to target crosslinking in CLAMs, we examined the roles of alginate molecular properties. A surprise finding was the formation of alginic acid gelling in the CLAMs that is a consequence of simultaneous and rapid pH reduction and moisture removal that occurs during spray drying. Thus, spray dried CLAMs gelation is due to calcium crosslinking and alginic acid formation, and unlike external gelation methods, is insensitive to the molecular composition of the alginates. The 'extent of gelation' of spray dried CLAMs is influenced by the molecular weights of the alginates at saturating calcium concentrations. Alginate viscosity correlates with molecular weight; thus, viscosity is a convenient criterion for selecting commercial alginates to target gelation extent in CLAMs.

Chelator Regulation of In Situ Calcium Availability to Enable Spray-Dry Microencapsulation in Cross-Linked Alginates.

A recently patented one-step in situ cross-linked alginate microencapsulation (CLAM) by spray-drying (i.e., the UC Davis CLAMs technology) can overcome the high cost of scale-up that limits commercial applications. While increasing calcium loading in the CLAMs process can increase the extent of cross-linking and improve retention and protection of the encapsulated cargo, the potential for residual undissolved calcium salt crystals in the final product can be a concern for some applications. Here, we demonstrate an alternate one-step spray-dry CLAMs process using pH-responsive chelation of calcium. The "Chelate CLAMs" process is an improvement over the patented process that controls ion availability based on pH-responsive solubility of the calcium salt. Hyaluronic acid was encapsulated in CLAMs to minimize swelling and release in aqueous formulations. CLAMs with 61% (d.b.) hyaluronic acid (HA-CLAMs) demonstrated restricted plumping, limited water absorption capacity, and reduced leaching, retaining up to 49% hyaluronic acid after 2 h in water. Alternatively, "Chelate HA-CLAMs" formed by the improved process exhibited nearly full retention of hyaluronic acid over 2 h in water and remained visibly insoluble after 1 year of storage in water at 4 °C. Successful hyaluronic acid retention in CLAMs is likely due in part to its ability to cross-link with calcium.

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.

Industrially-Scalable Microencapsulation of Plant Beneficial Bacteria in Dry Cross-Linked Alginate Matrix.

Microencapsulation of plant-beneficial bacteria, such as pink pigmented facultative methylotrophs (PPFM), may greatly extend the shelf life of these Gram-negative microorganisms and facilitate their application to crops for sustainable agriculture. A species of PPFM designated Methylobacterium radiotolerans was microencapsulated in cross-linked alginate microcapsules (CLAMs) prepared by an innovative and industrially scalable process that achieves polymer cross-linking during spray-drying. PPFM survived the spray-drying microencapsulation process with no significant loss in viable population, and the initial population of PPFM in CLAMs exceeded 1010 CFU/g powder. The PPFM population in CLAMs gradually declined by 4 to 5 log CFU/g over one year of storage. The extent of alginate cross-linking, modulated by adjusting the calcium phosphate content in the spray-dryer feed, did not influence cell viability after spray-drying, viability over storage, or dry particle size. However, particle size measurements and light microscopy of aqueous CLAMs suggest that enhanced crosslinking may limit the release of encapsulated bacteria. This work demonstrates an industrially scalable method for producing alginate-based inoculants that may be suitable for on-seed or foliar spray applications.

Undefined cellulase formulations hinder scientific reproducibility.

In the shadow of a burgeoning biomass-to-fuels industry, biological conversion of lignocellulose to fermentable sugars in a cost-effective manner is key to the success of second-generation and advanced biofuel production. For the effective comparison of one cellulase preparation to another, cellulase assays are typically carried out with one or more engineered cellulase formulations or natural exoproteomes of known performance serving as positive controls. When these formulations have unknown composition, as is the case with several widely used commercial products, it becomes impossible to compare or reproduce work done today to work done in the future, where, for example, such preparations may not be available. Therefore, being a critical tenet of science publishing, experimental reproducibility is endangered by the continued use of these undisclosed products. We propose the introduction of standard procedures and materials to produce specific and reproducible cellulase formulations. These formulations are to serve as yardsticks to measure improvements and performance of new cellulase formulations.

Mechanistic kinetic models of enzymatic cellulose hydrolysis-A review.

Bioconversion of lignocellulose forms the basis for renewable, advanced biofuels, and bioproducts. Mechanisms of hydrolysis of cellulose by cellulases have been actively studied for nearly 70 years with significant gains in understanding of the cellulolytic enzymes. Yet, a full mechanistic understanding of the hydrolysis reaction has been elusive. We present a review to highlight new insights gained since the most recent comprehensive review of cellulose hydrolysis kinetic models by Bansal et al. (2009) Biotechnol Adv 27:833-848. Recent models have taken a two-pronged approach to tackle the challenge of modeling the complex heterogeneous reaction-an enzyme-centric modeling approach centered on the molecularity of the cellulase-cellulose interactions to examine rate limiting elementary steps and a substrate-centric modeling approach aimed at capturing the limiting property of the insoluble cellulose substrate. Collectively, modeling results suggest that at the molecular-scale, how rapidly cellulases can bind productively (complexation) and release from cellulose (decomplexation) is limiting, while the overall hydrolysis rate is largely insensitive to the catalytic rate constant. The surface area of the insoluble substrate and the degrees of polymerization of the cellulose molecules in the reaction both limit initial hydrolysis rates only. Neither enzyme-centric models nor substrate-centric models can consistently capture hydrolysis time course at extended reaction times. Thus, questions of the true reaction limiting factors at extended reaction times and the role of complexation and decomplexation in rate limitation remain unresolved. Biotechnol. Bioeng. 2017;114: 1369-1385. © 2017 Wiley Periodicals, Inc.

The productive cellulase binding capacity of cellulosic substrates.

Cellulosic biomass is the most promising feedstock for renewable biofuel production; however, the mechanisms of the heterogeneous cellulose saccharification reaction are still unsolved. As cellulases need to bind isolated molecules of cellulose at the surface of insoluble cellulose fibrils or larger aggregated cellulose structures in order to hydrolyze glycosidic bonds, the "accessibility of cellulose to cellulases" is considered to be a reaction limiting property of cellulose. We have defined the accessibility of cellulose to cellulases as the productive binding capacity of cellulose, that is, the concentration of productive binding sites on cellulose that are accessible for binding and hydrolysis by cellulases. Productive cellulase binding to cellulose results in hydrolysis and can be quantified by measuring hydrolysis rates. In this study, we measured the productive Trichoderma reesei Cel7A (TrCel7A) binding capacity of five cellulosic substrates from different sources and processing histories. Swollen filter paper and bacterial cellulose had higher productive binding capacities of ∼6 µmol/g while filter paper, microcrystalline cellulose, and algal cellulose had lower productive binding capacities of ∼3 µmol/g. Swelling and regenerating filter paper using phosphoric acid increased the initial accessibility of the reducing ends to TrCel7A from 4 to 6 µmol/g. Moreover, this increase in initial productive binding capacity accounted in large part for the difference in the overall digestibility between filter paper and swollen filter paper. We further demonstrated that an understanding of how the productive binding capacity declines over the course of the hydrolysis reaction has the potential to predict overall saccharification time courses. Biotechnol. Bioeng. 2017;114: 533-542. © 2016 Wiley Periodicals, Inc.

The Effect of Ionic Liquid Pretreatment on the Bioconversion of Tomato Processing Waste to Fermentable Sugars and Biogas.

Tomato pomace is an abundant lignocellulosic waste stream from industrial tomato processing and therefore a potential feedstock for production of renewable biofuels. However, little research has been conducted to determine if pretreatment can enhance release of fermentable sugars from tomato pomace. Ionic liquids (ILs) are an emerging pretreatment technology for lignocellulosic biomass to increase enzymatic digestibility and biofuel yield while utilizing recyclable chemicals with low toxicity. In this study, pretreatment of tomato pomace with the ionic liquid 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) was investigated. Changes in pomace enzymatic digestibility were affected by pretreatment time and temperature. Certain pretreatment conditions significantly improved reducing sugar yield and hydrolysis time compared to untreated pomace. Compositional analyses suggested that pretreatment primarily removed water-soluble compounds and enriched for lignocellulose in pomace, with only subtle changes to the composition of the lignocellulose. While tomato pomace was effectively pretreated with [C2mim][OAc] to improve enzymatic digestibility, as of yet, unknown factors in the pomace caused ionic liquid pretreatment to negatively affect anaerobic digestion of pretreated material. This result, which is unique compared to similar studies on IL pretreatment of grasses and woody biomass, highlights the need for additional research to determine how the unique chemical composition of tomato pomace and other lignocellulosic fruit residues may interact with ionic liquids to generate inhibitors for downstream fermentation to biofuels.

Room-temperature storage of microalgae in water-in-oil emulsions: influence of solid particle type and concentration in the oil phase.

Water-in-oil emulsions containing silica nanoparticles (Aerosil R974) have the potential to stabilize microalgae for long-term storage. Studies were completed to determine if smectite clays could be used as an alternative to Aerosil R974. Emulsions were prepared with Aerosil R974, and hectorite and bentonite clays in the continuous phase and Chlorella sorokiniana was added to the aqueous phase to monitor the effects of solid particles on emulsion stability. Biological stability (cell viability) was determined using cell density measurements, and physical stability was measured from water droplet size distributions obtained by light scattering measurements and by examining phase separation over time. Measurements were also made to determine the effects of particles in the oil phase on emulsion viscosity. Particle concentrations greater than 0.25 wt% in the oil phase were required for maintaining physical stability. In emulsions containing 1 wt% solid particles and microalgae, biological stability of cells could be sustained for 340 days, regardless of particle type. At 1 wt% particles in the oil phase, apparent viscosity was 165% greater for samples containing hectorite and bentonite clays compared to samples containing Aerosil R974. The higher viscosity would need to be considered in large-scale production of emulsions for commercial application.

Purification and characterization of recombinant Cel7A from maize seed.

The corn grain biofactory was used to produce Cel7A, an exo-cellulase (cellobiohydrolase I) from Hypocrea jecorina. The enzymatic activity on small molecule substrates was equivalent to its fungal counterpart. The corn grain-derived enzyme is glycosylated and 6 kDa smaller than the native fungal protein, likely due to more sugars added in the glycosylation of the fungal enzyme. Our data suggest that corn seed-derived cellobiohydrolase (CBH) I performs as well as or better than its fungal counterpart in releasing sugars from complex substrates such as pre-treated corn stover or wood. This recombinant protein product can enter and expand current reagent enzyme markets as well as create new markets in textile or pulp processing. The purified protein is now available commercially.

The impact of alkali pretreatment and post-pretreatment conditioning on the surface properties of rice straw affecting cellulose accessibility to cellulases.

Rice straw was pretreated with sodium hydroxide and subsequently conditioned to reduce the pH to 5-6 by either: (1) extensive water washing or (2) acidification with hydrochloric acid then water washing. Alkali pretreatment improved the enzymatic digestibility of rice straw by increasing the cellulose accessibility to cellulases. However, acidification after pretreatment reversed the gains in cellulose accessibility to cellulases and enzymatic digestibility due to precipitation of solubilized compounds. Surface composition analyses by ToF-SIMS confirmed a reduction in surface lignin by pretreatment and water washing, and suggested that acidification precipitated a chemically modified form of lignin on the surfaces of rice straw. The spin-spin relaxation times (T2) of the samples indicated increased porosity in alkali pretreated rice straw. The acidified pretreated rice straw had reduced amounts of water in the longer T2 proton pools associated with water in the pores of the biomass likely due to back-filling by the precipitated components.

The role of endoglucanase and endoxylanase in liquefaction of hydrothermally pretreated wheat straw.

The role of endocellulases and endoxylanase during liquefaction and saccharification of hydrothermally pretreated wheat straw was studied. The use of a flow-loop setup with in-line magnetic resonance imaging enabled frequent measurements of viscosity at 55°C during saccharification at 6% total solids content. Viscosity data were complemented with off-line measurements of fiber lengths and release of soluble sugars. A clear correlation between fiber attrition and a decrease in viscosity was found. Fiber lengths and viscosity dropped quickly within the first hour and then stagnated, while sugar yields increased substantially thereafter, illustrating that liquefaction and saccharification are separate mechanisms. Both endoglucanase and endoxylanase were shown to have a significant effect on viscosity during liquefaction while the addition of endoxylanase also increased sugar yield.

Assessing cellulose microfibrillar structure changes due to cellulase action.

There is a need to understand how cellulose structural properties impact productive cellulase-cellulose interactions toward solving the mechanisms of the heterogeneous reaction. We coupled biochemical studies of cellulose hydrolysis by a purified Trichoderma reesei Cel7A (TrCel7A) cellobiohydrolase with atomic force microscopy (AFM) to study the impact of the cellulolytic activity on the fibrillar structure of cellulose. Bacterial cellulose (BC) fibrils were hydrolyzed by TrCel7A then immobilized by hydrophobic interactions on glass for AFM imaging. Commonly used methods to culture and isolate cellulose fibrils resulted in significant oxidation of the reducing-ends but minimal oxidation along the fibrils. We observed extensive fibrillation of BC fibrils to ∼3 nm microfibrils during the course of hydrolysis by TrCel7A, leaving thinned un-fibrillated recalcitrant fibrils at >80% hydrolysis extents. Additionally, this remaining fraction appeared to be segmented along the fibril length.

Binding and movement of individual Cel7A cellobiohydrolases on crystalline cellulose surfaces revealed by single-molecule fluorescence imaging.

The efficient catalytic conversion of biomass to bioenergy would meet a large portion of energy requirements in the near future. A crucial step in this process is the enzyme-catalyzed hydrolysis of cellulose to glucose that is then converted into fuel such as ethanol by fermentation. Here we use single-molecule fluorescence imaging to directly monitor the movement of individual Cel7A cellobiohydrolases from Trichoderma reesei (TrCel7A) on the surface of insoluble cellulose fibrils to elucidate molecular level details of cellulase activity. The motion of multiple, individual TrCel7A cellobiohydrolases was simultaneously recorded with ∼15-nm spatial resolution. Time-resolved localization microscopy provides insights on the activity of TrCel7A on cellulose and informs on nonproductive binding and diffusion. We measured single-molecule residency time distributions of TrCel7A bound to cellulose both in the presence of and absence of cellobiose the major product and a potent inhibitor of Cel7A activity. Combining these results with a kinetic model of TrCel7A binding provides microscopic insight into interactions between TrCel7A and the cellulose substrate.