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This work aims to study kinetic data on biomass hydrothermal carbonization by a new point of view. The time course of essential properties signaling hydrochar evolution is found to be sigmoidal ubiquitously. The logistic curve here proposed, attributable to the Hill equation, furnished near-perfect regressions (R2 as high as 0.999) in all cases studied. The broad applicability and the excellent correlations stimulated the research heuristically and routed process optimization. The evidence is reported that the success of fittings bases on profound mechanistic reasons. Arguments are discussed supporting the hypothesis that hydrochar formation is a stochastic phenomenon. The method deduces the Hill equation by applying statistical methods to the hydrothermal carbonization, modeled as a discrete Markov-chain process. The role of the model equation parameters is also discussed. The potentiality of the method proposed in this pioneering work could pave the way for a new paradigm in the modeling of hydrothermal carbonization.

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 120 min 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.

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.

The bioconversion of benzonitrile to benzamide and benzoic acid catalysed by resting cells of Microbacterium imperiale CBS 498-74 was investigated in batch and UF-membrane reactors. The microorganism converts nitriles through a two-step reaction, catalysed by a nitrile hydratase (NHase)–amidase (AMase) system. The kinetic parameters, Kmappand Vmapp, tested in 50 mM sodium phosphate buffer, pH 7.0, for benzonitrile and benzamide bioconversion were evaluated in batch reactor at 20 °C. Kmapp resulted 1.34 and 0.042 (mM), and Vmapp 1.1 and 0.072 (μmol min−1 mgDCW−1) for NHase and AMase, respectively. Batch and UF-membrane reactors were used to study the effect of operating variables such as enzyme and substrate concentration, temperature and residence time. The appropriate choice of operating conditions allowed to selectively control the NHase/AMase system and consequently the reactor output. A UF-membrane bioreactor operating at 20 °C and a τ of 10.3 h, allowed 96.9% conversion into benzoic acid; in contrast, when operating at 5 °C and a τ of 22.5 h 70.5% benzamide accumulated in the bioreactor.

The biohydration of acrylonitrile, propionitrile and benzonitrile catalysed by the NHase activity contained in resting cells of Microbacterium imperiale CBS 498-74 was operated at 5, 10 and 20 degrees C in laboratory-scale batch and membrane bioreactors. The bioreactions were conducted in buffered medium (50 mM Na(2)HPO(4)/NaH(2)PO(4), pH 7.0) in the presence of distilled water or tap-water, to simulate a possible end-pipe biotreatment process. The integral bioreactor performances were studied with a cell loading (dry cell weight; DCW) varying from 0.1 mg(DCW) per reactor to 16 mg(DCW) per reactor, in order to realize near 100% bioconversion of acrylonitrile, propionitrile and benzonitrile without consistent loss of NHase activity.

A new experimental technique, employing a continuous stirred-tank reactor, for studying enzyme kinetics in the presence of inhibitor-contaminated substrate is described. The proposed method is simulated mathematically for competitive, uncompetitive, and mixed-type noncompetitive inhibition. The step-by-step experimental procedure is described, as is the necessary data analysis for determining the kinetic parameters. Differences in system response for enzyme inhibition by excess substrate and by an impurity are illustrated, and a stability analysis of the system is performed.

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.

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.

Conversion of cephalosporin C into glutaryl 7-aminocephalosporanic acid was catalysed by D-aminoacid oxidase from Trigonopsis variabilis, covalently immobilized on the polystyrenic resin Duolite A365. Spontaneous degradation of substrates was limited without depressing enzymatic activity at the optimum reaction pH 8.0. The highest product yield was 1.77 mmol per g of biocatalyst, attained at 15iC in both batch stirred and fluidized-bed reactors.

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

3-Cyanopyridine (3-cnp) modulates via inactivation and/or inhibition, the amidase activity of the resting cells of Microbacterium imperiale CBS 489-74 which contained an in situ nitrile hydratase-amidase (NHase/AMase) cascade system suitable for catalyzing the bio-production of nicotinic acid form 3-cnp. The combined effect of 3-cnp concentration and temperature is investigated on the sole AMase activity by selectively inactivating the NHase activity. The study was performed in continuous stirred membrane reactors (CSMRs) fed with 100 mM nicotinamide solutions containing 25–200 mM 3-cnp at various temperatures. At 25–40 °C, the inactivation constant is negligible, even at high 3-cnp concentration, and the decrease in AMase activity is reversible. At 50 °C, a synergistic effect of temperature and 3-cnp inactivates AMase irreversibly, thus preventing a high yield being reached.

The bioconversion of 3-cyanopyridine using the in situ nitrile hydratase–amidase cascade system of resting Microbacterium imperiale CBS 498-74 cells was investigated in an ultrafiltration-membrane reactor, operated in either batch or continuous mode. The effects of operating conditions such as the amount of biocatalyst, substrate concentration, substrate feeding rate, mean residence time, and enzyme-to-substrate ratio, were investigated with the aim of achieving almost 100% substrate conversion and high reactor productivity. As a result, it was found that the NHase–AMase cascade system could be adequately exploited in a continuous reactor configuration. The differing temperature dependence of nitrile hydratase and amidase kinetics enabled the operational parameters to be module d to ensure (i) nitrile hydratase operational stability (at 5 °C), and (ii) 100% conversion of 3-cyanopyridine into nicotinic acid, or, alternatively, (iii) enrichment of the effluent stream with the intermediate nicotinamide (up to 80% conversion). It was possible to select operating conditions that allowed long periods of operation (at least 100 h) at a constant flow-rate without enzyme activity loss.

The aim of the present study is to explore the potential use of Microbacterium imperiale CBS 498–74 resting cells as a catalyst for the bioconversion of nicotinamide into nicotinic acid. This strain converts nitrile into the corresponding acid following a two-step reaction catalysed by nitrile hydratase and amidase via an amide as intermediate. The effect of temperature, cell load and substrate feeding strategy were investigated with controlled continuous stirred membrane bioreactors (CSMR) in an attempt to improve reaction conversion as well as reactor performances. The temperature dependence of amidase activity was investigated in both batch reactors and CSMR. An activation energy of about 52.6/53.5 kJ mol−1 was determined, indicating absence of mass-transport phenomena. Amidase operated under mild conditions suitable for the synthesis of labile organic molecules and it was stable up to 50 °C. Nicotinamide (substrate) at concentrations of ≥300 mM partially inhibited the enzyme. In the batch reactor, total conversion was achieved. In CSMR, the residence time was optimized to attain high conversion (up to 88%). These data indicate the potentiality of the continuous bioprocess for industrial application.

The bioconversion of propionitrile to propionamide was catalysed by nitrile hydratase (NHase) using resting cells of Microbacterium imperiale CBS 498-74 (formerly, Brevibacterium imperiale). This microorganism, cultivated in a shake flask, at 28 °C, presented a specific NHase activity of 34.4 U mgDCW−1 (dry cell weight). The kinetic parameters, Km and Vmax, tested in 50 mM sodium phosphate buffer, pH 7.0, in the propionitrile bioconversion was evaluated in batch reactor at 10 °C and resulted 21.6 mM and 11.04 μmol min−1 mgDCW−1, respectively. The measured apparent activation energy, 25.54 kJ mol−1, indicated a partial control by mass transport, more likely through the cell wall. UF-membrane reactors were used for kinetic characterisation of the NHase catalysed reaction. The time dependence of enzyme deactivation on reaction temperature (from 5 to 25 °C), on substrate concentrations (from 100 to 800 mM), and on resting cell loading (from 1.5 to 200 μg DCW ml−1) indicated: lower diffusional control (Ea=37.73 kJ mol−1); and NHase irreversible damage caused by high substrate concentration. Finally, it is noteworthy that in an integral reactor continuously operating for 30 h, at 10 °C, 100% conversion of propionitrile (200 mM) was attained using 200 μg DCW ml−1 of resting cells, with a maximum volumetric productivity of 0.5 g l−1 h−1.

The hydrolysis of N -glutaryl- l -phenylalanine p -nitroanilide catalysed by α-chymotrypsin (α-CT) was studied in the presence of the following quaternary ammonium salts: tetrapentyl ammonium bromide (TPeABr), tetrabutyl ammonium bromide (TBABr), tetrapropyl ammonium bromide (TPABr), tetraethyl ammonium bromide (TEABr) and tetramethyl ammonium bromide (TMABr). The activity of the enzyme is strongly affected by the salts that act as activators. Superactivity has been detected in the presence of TPeABr, TBABr, TPABr and TEABr. The enzyme activity seems to depend on the molecular structure of the salts; the higher the molecular weight of the alkyl residues in the cationic ammonium group, the higher the superactivity. In the whole investigated range, the enzyme hydrolysis rate resulted to be a monotonic increasing function of the salt concentration. The model of a non-essential activator was adopted to describe the effect of the salts on the hydrolysis activity and good agreement was found between the experimental results and the model predictions. The dependence of the enzyme activity on the substrate concentration was also studied to further verify the applicability of the model. Finally, a preliminary study about the effect of these additives on α-chymotrypsin thermal stability was performed.

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).

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.