Alberto Gallifuoco

Effect of inhibitors released during steam-explosion treatment of poplar wood on subsequent enzymatic hydrolysis and SSF.

Steam‐exploded (SE) poplar wood biomass was hydrolyzed by means of a blend of Celluclast and Novozym cellulase complexes in the presence of the inhibiting compounds produced during the preceding steam‐explosion pretreatment process. The SE temperature and time conditions were 214 °C and 6 min, resulting in a log R0 of 4.13. In enzymatic hydrolysis tests at 45 °C, the biomass loading in the bioreactor was 100 gDW/L (dry weight) and the enzyme‐to‐biomass ratio 0.06 g/gDW. The enzyme activities for endo‐glucanase, exo‐glucanase, and β‐glucosidase were 5.76, 0.55, and 5.98 U/mg, respectively. The inhibiting effects of components released during SE (formic, acetic, and levulinic acids, furfural, 5‐hydroxymethyl furfural (5‐HMF), syringaldehyde, 4‐hydroxy benzaldehyde, and vanillin) were studied at different concentrations in hydrolysis runs performed with rinsed SE biomass as model substrate. Acetic acid (2 g/L), furfural, 5‐HMF, syringaldehyde, 4‐hydroxybenzaldehyde, and vanillin (0.5 g/L) did not significantly effect the enzyme activity, whereas formic acid (11.5 g/L) inactivated the enzymes and levulinic acid (29.0 g/L) partially affected the cellulase. Synergism and cumulative concentration effects of these compounds were not detected. SSF experiments show that untreated SE biomass during the enzymatic attack gives rise to a nonfermentable hydrolysate, which becomes fermentable when rinsed SE biomass is used. The presence of acetic acid, vanillin, and 5‐HMF (0.5 g/L) in SSF of 100 gDW /L biomass gave rise to ethanol yields of 84.0%, 73.5%, and 91.0% respectively, with respective lag phases of 42, 39, and 58 h.

Comparison of SHF and SSF processes for the bioconversion of steam-exploded wheat straw

Two processes for ethanol production from wheat straw have been evaluated — separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF). The study compares the ethanol yield for biomass subjected to varying steam explosion pretreatment conditions: temperature and time of pretreatment was 200°C or 217°C and at 3 or 10 min. A rinsing procedure with water and NaOH solutions was employed for removing lignin residues and the products of hemicellulose degradation from the biomass, resulting in a final structure that facilitated enzymatic hydrolysis. Biomass loading in the bioreactor ranged from 25 to 100 g l−1 (dry weight). The enzyme-to-biomass mass ratio was 0.06. Ethanol yields close to 81% of theoretical were achieved in the two-step process (SHF) at hydrolysis and fermentation temperatures of 45°C and 37°C, respectively. The broth required addition of nutrients. Sterilisation of the biomass hydrolysate in SHF and of reaction medium in SSF can be avoided as can the use of different buffers in the two stages. The optimum temperature for the single-step process (SSF) was found to be 37°C and ethanol yields close to 68% of theoretical were achieved. The SSF process required a much shorter overall process time (≈30 h) than the SHF process (96 h) and resulted in a large increase in ethanol productivity (0.837 g l−1 h−1 for SSF compared to 0.313 g l−1 h−1 for SHF). Journal of Industrial Microbiology & Biotechnology (2000) 25, 184–192.

On the use of chitosan-immobilized β-glucosidase in wine-making: kinetics and enzyme inhibition

Abstract The kinetics of chitosan-immobilized β-glucosidase and enzyme inhibition by several components of wine and must (glucose, fructose and terpenols) were studied. Optimum immobilization conditions were: temperature 25°C, pH between 5·5 and 6·0, polymeric support dimension in the range 38–75 μm, cross-linking time 30 min, glutaraldehyde concentration 0·5–1·0% w/v, 1 g of chitosan per 1000 units of β-glucosidase. The immobilized enzyme retained 29% of the wet biocatalyst activity when freeze-dried and showed good stability (half-life roughly 2 years) when stored at 4°C. Kinetics were tested at 25°C following the hydrolysis of p-nitrophenyl β- d glucopyranoside and obey the Michaelis-Menten rate equation. Km = 1·3 mM and the activation energy, 62·84 kJ mol−1, are close to those of the free enzyme. The operational half-life was roughly 500 h.Glucose only depressed the enzyme activity according to a reversible non-competitive inhibition mechanism with Ki = 11·2 mM.

High-yield continuous production of nicotinic acid via nitrile hydratase-amidase cascade reactions using cascade CSMRs.

High yields of nicotinic acid from 3-cyanopyridine bioconversion were obtained by exploiting the in situ nitrile hydratase-amidase enzymatic cascade system of Microbacterium imperiale CBS 498-74. Experiments were carried out in continuously stirred tank UF-membrane bioreactors (CSMRs) arranged in series. This reactor configuration enables both enzymes, involved in the cascade reaction, to work with optimized kinetics, without any purification, exploiting their differing temperature dependences. To this end, the first CSMR, optimized for the properties of the NHase, was operated (i) at low temperature (5°C), limiting inactivation of the more fragile enzyme, nitrile hydratase, (ii) with a high residence time (24 h) to overcome reaction rate limitation. The second CSMR, optimized for the properties of the AMase, was operated (i) at a higher temperature (50°C), (ii) with a lower residence time (6h), and (iii) with a lower substrate (3-cyanopyridine) concentration to control excess substrate inhibition. The appropriate choice of operational conditions enabled total conversion of 3-cyanpyridine (up to 200 mM) into nicotinic acid to be achieved at steady-state and for long periods. Higher substrate concentrations required two CSMRs optimized for the properties of the NHase arranged in series to drive the first reaction to completion.

Immobilized β-glucosidase for the winemaking industry: study of biocatalyst operational stability in laboratory-scale continuous reactors

Abstract The stability of β-glucosidase immobilized on chitosan pellets was studied under operational conditions in continuous stirred tank membrane reactors. The rate of enzyme deactivation was monitored at 25°C using p -nitrophenyl β- d -glucopyranoside as model substrate. The medium was also supplemented with chemicals present in the wines, namely fructose, ethanol, nerol, linanol and geraniol. Fructose did not decrease biocatalyst stability, while alcohol affected enzyme half-life from 2586 h at 3% (w/v) ethanol to 1378 h at 12% (w/v). The addition of terpenols to solution containing 10% (w/v) alcohol reduced the half-life by a further 10%. Enzyme stability was not dependent on substrate concentration and was considered satisfactory for an industrial process (half-life 1.2 years). These results were independent of the use of wet stored pellets or of samples freeze-dried (24 h at –60°C). No added chemical influenced enzyme specific activity up to the tested limits: fructose 20 mM, terpenols 5 ppm each, ethanol 12% (w/v).

Selective water adsorption from aqueous ethanol-containing vapours by phillipsite-rich volcanic tuffs

Abstract The use of Neapolitan yellow tuff (NYT), a phillipsite/chabazite-containing volcaniclastic rock, rich in phillipsite, is proposed for an inexpensive dehydration process of ethanol vapours. NYT selective water adsorption kinetics was investigated both in a continuous microbalance apparatus and in laboratory-scale fixed-bed columns. The influence of operational parameters such as tuff grain dimensions, column temperature and pressure, volumetric flow-rate and partial pressure of adsorbates was studied. A preliminary study on the fluid dynamics of the column is also described. Finally, evidence is reported that a partial, but economical bed regeneration can be attained at the column working temperature by simply flushing the system with dry nitrogen.

Biosaccharification of cellulosic biomass in immiscible solvent–water mixtures

The enzymatic hydrolysis of wheat straw was carried out in bi-phasic media prepared with acetate esters and Na-acetate buffer. The volume percentage of the organic chemicals was 75%. The biomass was pretreated in a steam explosion plant at 217°C and for 3 min. A cellulase complex from commercial source was utilised and the experiments were run at 45°C and at constant enzyme to biomass weight ratio (0.06). Biomass loadings ranged from 6.25 to 100 g per litre of reactor. The amount of glucose formed per litre of reactor and hour and the glucose yield (grams of product per gram of biomass) were close to the values attained in pure buffer. The glucose concentration in the aqueous phase was in bi-phasic media much higher than in pure buffer and reached the value of 146 g lH2O−1 during 72 h of saccharification. The results were poorly dependent on the physical–chemical properties of the solvents. Nevertheless, butyl acetate could be slightly preferred to propyl and i-amyl acetate. The use of bi-phasic media did not require stirring rate higher than in pure buffer. The presence of acetate ester traces did not alter markedly the production of ethanol in the fermentation stage, but determined the extension of the lag phase.

Nitrile, amide and temperature effects on amidase-kinetics during acrylonitrile bioconversion by nitrile-hydratase/amidase in situ cascade system.

In this study the amidase kinetics of an in situ NHase/AMase cascade system was explored as a function of operational parameters such as temperature, substrate concentration and product formation. The results indicated that controlling amidase inactivation, during acrylonitrile bioconversion, makes it possible to recover the intermediate product of the two-step reaction in almost a pure form, without using purified enzyme. It has been demonstrated, in long-term experiments performed in continuous stirred UF-membrane bioreactors, that amidase is kinetically controlled by its proper substrate, depending on the structure, and by acrylonitrile. Using acrylamide, AMase-stability is temperature dependent (5°C, kd=0.008 h(-1); 30°C kd=0.023 h(-1)). Using benzamide, amidase is thermally stable up to 50°C and no substrate inhibition/inactivation occurs. With acrylonitrile, AMase-activity and -stability remain unchanged at concentrations <200 mM but at 200 mM, 35°C, after 70 h process, 90% irreversible inactivation occurs as no AMase-activity on benzamide revives.

Synthetic zeolites as carrier for enzyme immobilization in laboratory-scale fixedbed columns

The physical adsorption of a model enzyme, acid phosphatase from potato, on the commercial 13X zeolite was investigated. The highest reached enzyme recovery was 51% at 20°C and 0.01 mg/mL of enzyme in the solution. The formation of multilayers of protein on the support was demonstrated. The immobilization of protein on exchanged forms of the zeolite (replacing Na with Li and K) is less effective. The rate of substrate hydrolysis is controlled by surface kinetics and both interphase and intraphase mass transfer resistances can be neglected. Substrate affinity remains unchanged while operational half-life of the immobilized enzyme significantly increases (up to 1200 min in comparison with 60 min for the free enzyme at 30° c).

Hydrothermal carbonization of Biomass: New experimental procedures for improving the industrial Processes

This study aims to introduce new experimental methods, not yet described in the literature, to be adopted in hydrothermal carbonization processes. Silver fir was selected as model biomass in batch experiments in the range 200-300°C, up to 120min of reaction time, and at a 7:1 water to solid ratio. Simple equations were proposed for modeling the evolution of the process variables during the reaction, particularly the electrical conductivity of the liquid phase, correctly described by a simple two-step first order mechanism, regardless of the reaction temperature. At 200°C, a perfect correspondence (R2=0.9992) exists between liquid phase electrical conductivity and solid phase carbon content. The authors propose to monitor the industrial process withdrawing from the reactor the liquid and sampling its conductivity. The benefits of a flash expansion step between the reactor and the hydrochar drying units were discussed, and experiments demonstrated the usefulness of this process innovation.