Simultaneous enzymatic saccharification and comminution for the valorization of lignocellulosic biomass toward natural products.

BACKGROUND: Large-scale processing of lignocellulosics for glucose production generally relies on high temperature and acidic or alkaline conditions. However, extreme conditions produce chemical contaminants that complicate downstream processing. A method that mainly rely on mechanical and enzymatic reaction completely averts such problem and generates unmodified lignin. Products from this process could find novel applications in the chemicals, feed and food industry. But a large-scale system suitable for this purpose is yet to be developed. In this study we applied simultaneous enzymatic saccharification and communition (SESC) for the pre-treatment of a representative lignocellulosic biomass, cedar softwood, under both laboratory and large-scale conditions. RESULTS: Laboratory-scale comminution achieved a maximum saccharification efficiency of 80% at the optimum pH of 6. It was possible to recycle the supernatant to concentrate the glucose without affecting the efficiency. During the direct alcohol fermentation of SESC slurry, a high yield of ethanol was attained. The mild reaction conditions prevented the generation of undesired chemical inhibitors. Large-scale SESC treatment using a commercial beads mill system achieved a saccharification efficiency of 60% at an energy consumption of 50 MJ/kg biomass. CONCLUSION: SESC is very promising for the mild and clean processing of lignocellulose to generate glucose and unmodified lignin in a large scale. Economic feasibility is highly dependent on its potential to generate high value natural products for energy, specialty chemicals, feed and food application.

Integrated process development for grass biomass utilization through enzymatic saccharification and upgrading hydroxycinnamic acids via microbial funneling.

In grass biomass, hydroxycinnamic acids (HCAs) play crucial roles in the crosslinking of lignin and polysaccharides and can be easily extracted by mild alkaline pretreatment, albeit heterogeneously. Here, HCAs were extracted from bamboo and rice straw as model grass biomass with different HCAs composition, and microbial funneling was then conducted to produce 2-pyrone-4,6-dicarboxylic acid (PDC) and (4S)-3-carboxymuconolactone (4S-3CML), promising building blocks for bio-based polymers, respectively. Pseudomonas putida PpY1100 engineered for efficient microbial funneling completely converted HCAs to PDC and 4S-3CML with high titers of 3.9-9.3 g/L and molar yields of 92-99%, respectively. The enzymatic saccharification efficiencies of lignocellulose after HCAs extraction were 29.5% in bamboo and 73.8% in rice straw, which are 8.9 and 6.8 times higher than in alkaline-untreated media, respectively. These results provide a green-like process for total valorization of grass biomass through enzymatic saccharification integrated with upgrading heterogeneous HCAs to a valuable single chemical via microbial funneling.

Production of flavorful alcohols from woods and possible applications for wood brews and liquors.

This work explores the utilization of wood for high-value production of novel alcoholic brews and liquors with natural flavors. The process capitalizes on our original wet-type bead milling (WBM) technology that enables direct enzymatic saccharification and alcohol fermentation of wood without chemical and heat treatment, resulting in the absence of toxic compounds. When alcohol-based products from various wood species, including Cryptomeria japonica (cedar), Cerasus × yedoensis (cherry), and Betula platyphylla (birch), were analyzed by SPME-GC-MS, different natural flavor components were found in each. Correlation analysis using Heracles NEO and ASTREE V5 showed that the alcohols from wood have different flavor and taste characteristics when compared with those of existing commercial liquors. From pilot-scale experiments, the yield of alcoholic brew per biomass amount was determined. Pilot-scale runs established the importance of optimum wood particle size during WBM for efficient alcohol production. Although the alcohol produced from wood must first be established as safe for human consumption, this is the first description of drinking alcohols produced from wood. This work may open up important avenues for the exploitation of wood resources toward food production to further advance the current state of forestry.

Purification, molecular cloning, and enzymatic properties of a family 12 endoglucanase (EG-II) from fomitopsis palustris: role of EG-II in larch holocellulose hydrolysis.

A family 12 endoglucanase with a molecular mass of 23,926 Da (EG-II) from the brown-rot basidiomycete Fomitopsis palustris was purified and characterized. One of the roles of EG-II in wood degradation is thought to be to loosen the polysaccharide network in cell walls by disentangling hemicelluloses that are associated with cellulose.

Efficient secretion of (R)-3-hydroxybutyric acid from Halomonas sp. KM-1 cultured with saccharified Japanese cedar under microaerobic conditions.

In the presence of d-glucose, consumption of pentoses such as d-xylose is somewhat repressed by most bacteria. However, in Halomonas sp. KM-1, simultaneous utilization of a pure hexose and pentose for growth and PHB production has been observed. Moreover, this strain has been shown to preferentially utilize d-xylose from a mixture of hexose and pentose. In addition, the KM-1 strain produced (R)-3-hydroxybutyric acid ((R)-3-HB) by using saccharified Japanese cedar (Cryptomeria japonica) wood. The concentration of intracellular PHB after aerobic cultivation for 24h was 8.4 g/L, and after shifting to microaerobic conditions and further cultivation for 18 h, the concentration of (R)-3-HB in the medium reached 8.0 g/L. These results show that the KM-1 strain can efficiently utilize saccharified Japanese cedar and secreted (R)-3-HB under microaerobic conditions.

Regulated patterns of bacterial movements based on their secreted cellulose nanofibers interacting interfacially with ordered chitin templates.

Gluconacetobacter xylinus, a gram-negative bacterium that synthesizes and extrudes a cellulose nanofiber in SH media moves in random manners, resulting in 3D-network structure of the secreted nanofibers termed a pellicle. In this study, the bacterial movement was successfully regulated to be in a waving manner when cultured on ordered templates made of chitin. Interestingly, by addition of more cellulose into the chitin ordered templates, the waving pattern was getting close to a linear or straight manner. Real time video analysis and other visualization techniques clarified that the regulation of the moving manners was due to the interfacial interaction between the secreted nanofibers and the template surfaces. Furthermore, the changing of the pattern due to the cellulose content in the ordered templates appeared to depend on the magnitude of the interaction between the template and nanofibers. This regulated autonomous deposition of the fibers will build patterned 3D-structure with unique properties on the surface of the templates, leading to a novel type of nanotechnology using biological systems with biomolecular nano-templates to design 3D-structures.

Effects of growth stage on enzymatic saccharification and simultaneous saccharification and fermentation of bamboo shoots for bioethanol production.

Bamboo is a fast-growing renewable biomass that is widely distributed in Asia. Although bamboo is recognised as a useful resource, its utilization is limited and further development is required. Immature bamboo shoots harvested before branch spread were found to be a good biomass resource to achieve a high saccharification yield. The saccharification yield of the shoots increased (up to 98% for immature Phyllostachys bambusoides) when xylanase was used in addition to cellulase. Simultaneous saccharification and fermentation (SSF) processing converted immature shoots of P. bambusoides and Phyllostachys pubescens to ethanol with an ethanol yield of 169 and 139 g kg(-1), respectively (98% and 81%, respectively, of the theoretical yields based on hexose conversion) when 12 FPU g(-1) enzyme and the yeast Saccharomyces cerevisiae were used.

Biodirected epitaxial nanodeposition of polymers on oriented macromolecular templates.

Biodirected epitaxial nanodeposition of polymers was achieved on a template with an oriented molecular surface. Acetobacter xylinum synthesized a ribbon of cellulose I microfibrils onto a fixed, nematic ordered substrate of glucan chains with unique surface characteristics. The substrate directed the orientation of the motion due to the inverse force of the secretion during biosynthesis, and the microfibrils were aligned along the orientation of the molecular template. Using real-time video analysis, the patterns and rates of deposition were elucidated. Field emission scanning electron microscopy revealed that a strong molecular interaction allowed for the deposition of nascent biosynthesized 3.5-nm cellulose microfibrils with inter-microfibrillar spacings of 7-8 nm on the surface of the template. The cellulose was deposited parallel to the molecular orientation of the template. Directed cellulose synthesis and ordered movement of cells were observed only by using a nematic ordered substrate made from cellulose, and not from ordered crystalline cellulose substrates or ordered cellulose-related synthetic polymers such as polyvinyl alcohol. This unique relationship between directed biosynthesis and the ordered fabrication from the nano to the micro scales could lead to new methodologies for the design of functional materials with desired nanostructures.