Sugar Production from Hybrid Poplar Sawdust: Optimization of Enzymatic Hydrolysis and Wet Explosion Pretreatment.

Wet explosion pretreatment of hybrid poplar sawdust (PSD) for the production of fermentable sugar was carried out in the pilot-scale. The effects of pretreatment conditions, such as temperature (170-190 °C), oxygen dosage (0.5-7.5% of dry matter (DM), w/w), residence time (10-30 min), on cellulose and hemicellulose digestibility after enzymatic hydrolysis were ascertained with a central composite design of the experiment. Further, enzymatic hydrolysis was optimized in terms of temperature, pH, and a mixture of CTec2 and HTec2 enzymes (Novozymes). Predictive modeling showed that cellulose and hemicellulose digestibility of 75.1% and 83.1%, respectively, could be achieved with a pretreatment at 177 °C with 7.5% O2 and a retention time of 30 min. An increased cellulose digestibility of 87.1% ± 0.1 could be achieved by pretreating at 190 °C; however, the hemicellulose yield would be significantly reduced. It was evident that more severe conditions were required for maximal cellulose digestibility than that of hemicellulose digestibility and that an optimal sugar yield demanded a set of conditions, which overall resulted in the maximum sugar yield.

Oxidative degradation of biorefinery lignin obtained after pretreatment of forest residues of Douglas Fir.

Harvested forest residues are usually considered a fire hazards and used as "hog-fuel" which results in air pollution. In this study, the biorefinery lignin stream obtained after wet explosion pretreatment and enzymatic hydrolysis of forestry residues of Douglas Fir (FS-10) was characterized and further wet oxidized under alkaline conditions. The studies indicated that at 10% solids, 11.7wt% alkali and 15min residence time, maximum yields were obtained for glucose (12.9wt%), vanillin (0.4wt%) at 230°C; formic acid (11.6wt%) at 250°C; acetic acid (10.7wt%), hydroxybenzaldehyde (0.2wt%), syringaldehyde (0.13wt%) at 280°C; and lactic acid (12.4wt%) at 300°C. FTIR analysis of the solid residue after wet oxidation showed that the aromatic skeletal vibrations relating to lignin compounds increased with temperature indicating that higher severity could result in increased lignin oxidation products. The results obtained, as part of the study, is significant for understanding and optimizing processes for producing high-value bioproducts from forestry residues.

Pretreatment of forest residues of Douglas fir by wet explosion for enhanced enzymatic saccharification.

The logging and lumbering industry in the Pacific Northwest region generates huge amount of forest residues, offering an inexpensive raw material for biorefineries. Wet explosion (WEx) pretreatment was applied to the recalcitrant biomass to optimize process conditions including temperature (170-190 °C), time (10-30 min), and oxygen loading (0.5-7.5% of DM) through an experimental design. Optimal pH for enzymatic hydrolysis of the optimized samples and a complete mass balance have been evaluated. Results indicated that cellulose digestibility improved in all conditions tested with maximum digestibility achieved at 190 °C, time 30 min, and oxygen loading of 7.5%. Glucose yield at optimal pH of 5.5 was 63.3% with an excellent recovery of cellulose and lignin of 99.9% and 96.3%, respectively. Hemicellulose sugars recovery for xylose and mannose was found to be 69.2% and 76.0%, respectively, indicating that WEx is capable of producing relative high sugar yield even from the recalcitrant forest residues.

Making lignin accessible for anaerobic digestion by wet-explosion pretreatment.

Lignin is a major part of the recalcitrant fraction of lignocellulose and in nature its degradation occurs through oxidative enzymes along with microbes mediated oxidative chemical actions. Oxygen assisted wet-explosion pretreatment promotes lignin solubility and leads to an increase biodegradation of lignin during anaerobic digestion processes. The pretreatment of feedlot manure was performed in a 10L reactor at 170°C for 25min using 4bars oxygen and the material was fed to a continuous stirred tank reactor operated at 55°C for anaerobic digestion. Methane yield of untreated and pretreated material was 70±27 and 320±36L/kg-VS/Day, respectively, or 4.5 times higher yield as a result of the pretreatment. Aliphatic acids formed during the pretreatment were utilized by microbes. 44.4% lignin in pretreated material was actually converted in the anaerobic digestion process compared to 12.6% for untreated material indicating the oxygen assisted explosion promoted lignin degradation.

On-site enzymes produced from Trichoderma reesei RUT-C30 and Aspergillus saccharolyticus for hydrolysis of wet exploded corn stover and loblolly pine.

Cellulase production by two filamentous fungi Trichoderma reesei RUT-C30 and novel fungal strain, Aspergillus saccharolyticus on pretreated corn stover was investigated. Cellulase production was followed by the hydrolysis of two feedstocks, wet-exploded corn stover (WECS) and wet-exploded loblolly pine (WELP) by on-site produced enzyme cocktails containing cellulase from T. reesei RUT-C30 and ß-glucosidase from A. saccharolyticus. The sugar yields using the on-site enzyme cocktails were compared with commercial enzymes preparations, Celluclast 1.5L and Novozym 188 at two substrate concentrations, 5% and 10% (w/w) and enzyme loading at 5 and 15 FPU/g glucan for WECS and WELP. The highest sugar yields were obtained at 5% (w/w) substrate concentration and 15 FPU/g glucan for both feedstocks. Glucose yields of 81% and 88% were obtained from on-site and commercial enzymes, respectively using WECS as feed stock. The sugar yields were 55% and 58% for WELP samples hydrolyzed with on-site and commercial enzymes, respectively.

ß-glucosidases from a new Aspergillus species can substitute commercial ß-glucosidases for saccharification of lignocellulosic biomass.

ß-Glucosidase activity plays an essential role for efficient and complete hydrolysis of lignocellulosic biomass. Direct use of fungal fermentation broths can be cost saving relative to using commercial enzymes for production of biofuels and bioproducts. Through a fungal screening program for ß-glucosidase activity, strain AP (CBS 127449, Aspergillus saccharolyticus ) showed 10 times greater ß-glucosidase activity than the average of all other fungi screened, with Aspergillus niger showing second greatest activity. The potential of a fermentation broth of strain AP was compared with the commercial ß-glucosidase-containing enzyme preparations Novozym 188 and Cellic CTec. The fermentation broth was found to be a valid substitute for Novozym 188 in cellobiose hydrolysis. The Michaelis-Menten kinetics affinity constant as well as performance in cellobiose hydrolysis with regard to product inhibition were found to be the same for Novozym 188 and the broth of strain AP. Compared with Novozym 188, the fermentation broth had higher specific activity (11.3 U/mg total protein compared with 7.5 U/mg total protein) and also increased thermostability, identified by the thermal activity number of 66.8 vs. 63.4 °C for Novozym 188. The significant thermostability of strain AP ß-glucosidases was further confirmed when compared with Cellic CTec. The ß-glucosidases of strain AP were able to degrade cellodextrins with an exo-acting approach and could hydrolyse pretreated bagasse to monomeric sugars when combined with Celluclast 1.5L. The fungus therefore showed great potential as an onsite producer for ß-glucosidase activity.

Onsite enzyme production during bioethanol production from biomass: screening for suitable fungal strains.

Cellulosic ethanol production from biomass raw materials involves process steps such as pre-treatment, enzymatic hydrolysis, fermentation, and distillation. Use of streams within cellulosic ethanol production was explored for onsite enzyme production as part of a biorefinery concept. Sixty-four fungal isolates were in plate assays screened for lignocellulolytic activities to discover the most suitable fungal strain with efficient hydrolytic enzymes for lignocellulose conversion. Twenty-five were selected for further enzyme activity studies using a stream derived from the bioethanol process. The filter cake left after hydrolysis and fermentation was chosen as substrate for enzyme production. Five of the 25 isolates were further selected for synergy studies with commercial enzymes, Celluclast 1.5L and Novozym 188. Finally, IBT25747 (Aspergillus niger) and strain AP (CBS 127449, Aspergillus saccharolyticus) were found as promising candidates for onsite enzyme production where the filter cake was inoculated with the respective fungus and in combination with Celluclast 1.5 L used for hydrolysis of pre-treated biomass.

Aspergillus saccharolyticus sp. nov., a black Aspergillus species isolated in Denmark.

A novel species, Aspergillus saccharolyticus sp. nov., belonging to the Aspergillus section Nigri group is described. This species was isolated in Denmark from treated hardwood. Its taxonomic status was determined using a polyphasic taxonomic approach including phenotypic (morphology and extrolite profiles) and molecular (ß-tubulin, internal transcribed spacer and calmodulin gene sequences, and universally primed PCR fingerprinting) analysis. Phenotypic and molecular data enabled this novel species to be clearly distinguished from other black aspergilli. A. saccharolyticus is a uniseriate Aspergillus species that is morphologically similar to Aspergillus japonicus and Aspergillus aculeatus, but has a totally different extrolite profile compared to any known Aspergillus species. The type strain of A. saccharolyticus sp. nov. is CBS 127449(T) (=IBT 28509(T)).

Hydrolysis of Miscanthus for bioethanol production using dilute acid presoaking combined with wet explosion pre-treatment and enzymatic treatment.

Miscanthus is a high yielding bioenergy crop. In this study we used acid presoaking, wet explosion, and enzymatic hydrolysis to evaluate the combination of the different pre-treatment methods for bioethanol production with Miscanthus. Acid presoaking is primarily carried out in order to remove xylose prior to wet explosion. The acid presoaking extracted 63.2% xylose and 5.2% glucose. Direct enzymatic hydrolysis of the presoaked biomass was found to give only low sugar yields of 24-26% glucose. Wet explosion is a pre-treatment method that combines wet-oxidation and steam explosion. The effect of wet explosion on non-presoaked and presoaked Miscanthus was investigated using both atmospheric air and hydrogen peroxide as the oxidizing agent. All wet explosion pre-treatments showed to have a disrupting effect on the lignocellulosic biomass, making the sugars accessible for enzymatic hydrolysis. The combination of presoaking, wet explosion, and enzymatic hydrolysis was found to give the highest sugar yields. The use of atmospheric air gave the highest xylose yield (94.9% xylose, 61.3% glucose), while hydrogen peroxide gave the highest glucose yield (82.4% xylose, 63.7% glucose).