Highly efficient phase boundary biocatalysis with enzymogel nanoparticles.

The enzymogel nanoparticle made of a magnetic core and polymer brush shell demonstrates a novel type of remote controlled phase-boundary biocatalysis that involves remotely directed binding to and engulfing insoluble substrates, high mobility, and stability of the catalytic centers. The mobile enzymes reside in the polymer brush scaffold and shuttle between the enzymogel interior and surface of the engulfed substrate in the bioconversion process. Biocatalytic activity of the mobile enzymes is preserved in the enzymogel while the brush-like architecture favors the efficient interfacial interaction when the enzymogel spreads over the substrate and extends substantially the reaction area as compared with rigid particles.

Modeling production of antifungal compounds and their role in biocontrol product inhibitory activity.

Partial least squares (PLS) regression modeling was used to relate the antifungal activity of Bacillus subtilis solid-state fermentation extracts to the individual high-performance liquid chromatography (HPLC) peaks from those extracts. A model was developed that predicted bioassay inhibition based on the extract HPLC profile (R(2) = 0.99). Concentrations of the members of the antifungal lipopeptide families iturin A and fengycin were found to correlate positively with extract inhibition, but a peak with unidentified chemical composition (designated as peak 48) showed the strongest correlation with extract inhibition. HPLC data were used to construct models for the production of iturin A, fengycin, and peak 48 as a function of the substrate moisture content, incubator temperature, and aeration rate in the solid-state bioreactors. Maximum production of all compounds occurred at the highest moisture content (1.7 g/g dry basis) and lowest incubator temperature (19 degrees C) tested. Optimal aeration rates for the production of the two known lipopeptides and peak 48 were 0.1 and 1.5 L/min, respectively.

Optimization of spore and antifungal lipopeptide production during the solid-state fermentation of Bacillus subtilis.

Bacillus subtilis strain TrigoCor 1448 was grown on wheat middlings in 0.5-l solid-state fermentation (SSF) bioreactors for the production of an antifungal biological control agent. Total antifungal activity was quantified using a 96-well microplate bioassay against the plant pathogen Fusarium oxysporum f. sp. melonis. The experimental design for process optimization consisted of a 2(6-1) fractional factorial design followed by a central composite face-centered design. Initial SSF parameters included in the optimization were aeration, fermentation length, pH buffering, peptone addition, nitrate addition, and incubator temperature. Central composite face-centered design parameters included incubator temperature, aeration rate, and initial moisture content (MC). Optimized fermentation conditions were determined with response surface models fitted for both spore concentration and activity of biological control product extracts. Models showed that activity measurements and spore production were most sensitive to substrate MC with highest levels of each response variable occurring at maximum moisture levels. Whereas maximum antifungal activity was seen in a limited area of the design space, spore production was fairly robust with near maximum levels occurring over a wider range of fermentation conditions. Optimization resulted in a 55% increase in inhibition and a 40% increase in spore production over nonoptimized conditions.

Effects of enzyme loading, densification, and storage on AFEX-pretreated biomass for ethanol production.

Corn stover, switchgrass, and prairie cordgrass were treated with an ammonia fiber expansion (AFEX) process and a novel densification method (ComPAKco). Separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) were used to evaluate impacts of densification. ComPAKco densification is characterized by low-temperature and low-energy requirements, resulting in compacted biomass briquettes (CBB) 1-2 cm square, with a bulk density of 380-460 kg/m(3). Feedstocks were evaluated before and following AFEX pretreatment, after densification, and after storage. Two enzyme doses were tested. The low rate used 5 filter paper units (FPU) of Spezyme CP (cellulase) and 21.3 cellobiase units (CBU) of Novozyme 188 (aka NS50010 [ß-glucosidase]) per gram of glucan. The high dosage rate was three times higher and resulted in 40-56 % and 33-82 % higher ethanol yields with SHF and SSF, respectively. Trials revealed no adverse effect on ethanol yield following densification or 6-month storage of densified, AFEX-pretreated feedstocks.

Modeling the Effect of pH and Temperature for Cellulases Immobilized on Enzymogel Nanoparticles.

Production costs of cellulosic biofuels can be lowered if cellulases are recovered and reused using particulate carriers that can be extracted after biomass hydrolysis. Such enzyme recovery was recently demonstrated using enzymogel nanoparticles with grafted polymer brushes loaded with cellulases. In this work, cellulase (NS50013) and ß-glucosidase (Novozyme 188) were immobilized on enzymogels made of poly(acrylic acid) polymer brushes grafted to the surface of silica nanoparticles. Response surface methodology was used to model effects of pH and temperature on hydrolysis and recovery of free and attached enzymes. Hydrolysis yields using both enzymogels and free cellulase and ß-glucosidase were highest at the maximum temperature tested, 50 °C. The optimal pH for cellulase enzymogels and free enzyme was 5.0 and 4.4, respectively, while both free ß-glucosidase and enzymogels had an optimal pH near 4.4. Highest hydrolysis sugar concentrations with cellulase and ß-glucosidase enzymogels were 69 and 53 % of those with free enzymes, respectively. Enzyme recovery using enzymogels decreased with increasing pH, but cellulase recovery remained greater than 88 % throughout the operating range of pH values less than 5.0 and was greater than 95 % at pH values below 4.3. Recovery of ß-glucosidase enzymogels was not affected by temperature and had little impact on cellulase recovery.

Impact of enzyme loading on the efficacy and recovery of cellulolytic enzymes immobilized on enzymogel nanoparticles.

Cellulase and ß-glucosidase were adsorbed on a polyacrylic acid polymer brush grafted on silica nanoparticles to produce enzymogels as a form of enzyme immobilization. Enzyme loading on the enzymogels was increased to a saturation level of approximately 110 µg (protein) mg(-1) (particle) for each enzyme. Enzymogels with varied enzyme loadings were then used to determine the impact on hydrolysis rate and enzyme recovery. Soluble sugar concentrations during the hydrolysis of filter paper and Solka-Floc with the enzymogels were 45 and 53%, respectively, of concentrations when using free cellulase. ß-Glucosidase enzymogels showed lower performance; hydrolyzate glucose concentrations were just 38% of those using free enzymes. Increasing enzyme loading on the enzymogels did not reduce net efficacy for cellulase and improved efficacy for ß-glucosidase. The use of free cellulases and cellulase enzymogels resulted in hydrolyzates with different proportions of cellobiose and glucose, suggesting differential attachment or efficacy of endoglucanases, exoglucanases, and ß-glucosidases present in cellulase mixtures. When loading ß-glucosidase individually, higher enzyme loadings on the enzymogels produced higher hydrolyzate glucose concentrations. Approximately 96% of cellulase and 66 % of ß-glucosidase were recovered on the enzymogels, while enzyme loading level did not impact recovery for either enzyme.

Effect of initial particle size and densification on AFEX-pretreated biomass for ethanol production.

Switchgrass (SG), corn stover (CS), and prairie cordgrass (PCG) pretreated with ammonia fiber expansion (AFEX) were densified using a novel low-temperature, low-pressure densification method. Simultaneous saccharification and fermentation (SSF) and separate hydrolysis and fermentation (SHF) were performed with loose and densified AFEX-treated biomass to determine the effect of post-AFEX densification. Biomass particle size reduction before pretreatment increased 144-h SSF ethanol yields from densified material by 8-9 % although no significant differences were seen in the first 72 h. Grinding material after densification had no impact on final ethanol yields but increased production rates in the first 24-48 h. Low-pressure, post-AFEX densification had no adverse effects on SSF ethanol yields from SG or CS but reduced yields from densified PCG by 16 %. Glucose concentrations after hydrolysis (SHF) showed similar trends. Ethanol yields after SHF, however, showed that densification had no significant impact on CS or PCG but reduced final ethanol yields from SG.

Effect of hemicellulase addition during enzymatic hydrolysis of switchgrass pretreated by soaking in aqueous ammonia.

The focus of this study was to determine the effect of supplementing cellulase with hemicellulase during enzymatic hydrolysis of switchgrass pretreated by soaking in aqueous ammonia (SAA) under a range of conditions. SAA was performed using 15% aqueous ammonia for 8 or 24h at temperatures of 40 or 60°C. The combined effect of cellulase and hemicellulase loadings on glucose yield during enzymatic hydrolysis was modeled for each pretreatment condition. Glucose yields greater than 85% of theoretical were achieved for pretreatment at 40°C for 24h and for 60°C for 8h. Hemicellulase supplementation was not sufficient to achieve these glucose yields at lower severity SAA pretreatment. High severity SAA pretreatment also led to low yields despite improved delignification.

Combined effect of pelleting and pretreatment on enzymatic hydrolysis of switchgrass.

Switchgrass was pelleted to evaluate the effect of densification on acidic and alkaline pretreatment efficacy. Bulk density and durability of pellets were 724 kg/m(3) and 95%, respectively. Ground switchgrass (D(90) = 21.7 mm) was further ground to a fine power (D(90) = 0.5mm) in the pellet mill prior to densification. This grinding increased enzymatic hydrolyzate glucose yields of non-pretreated materials by 210%. Pelleting had no adverse impact on dilute acid pretreatment efficacy. Grinding and pelleting increased hydrolyzate glucose yields of switchgrass pretreated by soaking in aqueous ammonia (SAA) by 37%. Xylose yields from SAA-pretreated switchgrass pellets were 42% higher than those from the original biomass. Increases in sugar yields from SAA-pretreated pelleted biomass are attributed to grinding and heating of biomass during the pelleting process. Potential transportation, storage, and handling benefits of biomass pelleting may be achieved without negatively affecting the downstream processing steps of pretreatment or enzymatic hydrolysis.

Effects of protein separation conditions on the functional and thermal properties of canola protein isolates.

UNLABELLED: Canola meal protein isolates were prepared from defatted canola meal flour using alkaline solubilization and acid precipitation. A central composite design was used to model 2nd-order response surfaces for the protein yield and the functional properties of protein isolates. The solubilization pH and precipitation pH were used as design factors. The models showed that the protein yield and functional properties of isolates, such as water absorption and fat absorption, were sensitive to both solubilization pH and precipitation pH, whereas the emulsification was sensitive to only solubilization pH. Gel electrophoresis analysis of protein fractions gave evidence to the compositional changes between proteins isolated under different conditions. Differences in glass transition temperatures suggest that proteins tend to be more denatured when solubilized at highly alkaline conditions. These conformational and compositional changes due to different protein separation conditions have contributed to the changes in functional properties of protein isolates. PRACTICAL APPLICATION: Protein isolation conditions may be determined primarily through optimization of total protein yield. Improvements in protein functional properties may be achieved with a relatively small sacrifice in yield by altering isolation conditions.

Deficiency of cellulase activity measurements for enzyme evaluation.

Switchgrass was used as a model feedstock to determine the influence of pretreatment conditions and biomass quality on enzymatic hydrolysis using different enzyme products. Dilute sulfuric acid and soaking in aqueous ammonia pretreatments were used to produce biomass with varied levels of hemicellulose and lignin sheathing. Pretreated switchgrass solids were tested with simple enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) with three commercial enzyme products: Accellerase 1000 (Genencor), Spezyme CP (Genencor)/Novozyme 188 (Novozymes), and Celluclast/Novozyme 188 (Novozymes). Enzymes were loaded on a common activity basis (FPU/g cellulose and CBU/g cellulose). Despite identical enzyme loadings, glucose yields were significantly different for both acid and alkaline pretreatments but differences diminished as hydrolysis progressed for acid-pretreated biomass. Cellobiose concentrations in Accellerase treatments indicated an initial beta-glucosidase limitation that became less significant over time. SSF experiments showed that differences in glucose and ethanol yields could not be attributed to enzyme product inhibition. Yield discrepancies of glucose or ethanol in acid pretreatment, alkaline pretreatment, and acid pretreatment/SSF were as much as 15%, 19%, and 5%. These results indicate that standardized protocols for measuring enzyme activity may not be adequate for assessing activity using pretreated biomass substrates.

Nonylphenol in anaerobically digested sewage sludge from New York State.

Nonylphenols (NPs) have been identified as xenoestrogens and have been found at high concentrations in Canadian and European biosolids. While nonylphenol polyethoxylates (NPEOs) are being phased out and regulated in several European countries, there is currently no regulation of these compounds in the United States, and little information is available concerning the presence of NPs in U.S. biosolids. Anaerobically digested sewage sludge from five wastewater treatment plants in central New York State was analyzed for the presence of NPs. Samples were taken from treatment plants in both small municipalities and larger metropolitan areas with a range of industrial inputs. Samples were extracted via Soxhlet apparatus and analyzed by GC/MS. The various isomers of NP were summed yielding total NP concentrations as high as 1840 mg/kg with a mean of 1500 mg/kg on a dry weight basis. These values are two to five times as high as previously reported concentrations for U.S. and Canadian biosolids from plants using similar treatment schemes.