Agata Spera

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.

Operational stability of Brevibacterium imperialis CBS 489-74 nitrile hydratase

Brevibacterium imperialis CBS 489-74 was grown in broths prepared with yeast and malt extract, bacteriological peptone and 2% glucose or differently modified with the addition of Na-phosphate buffer, FeSO 4 , MgSO4 and CoCl 2 . The peak production of nitrile hydratase (NHase) did not change significantly. At the stationary growth phase, the units per milliliter of broth (60 units ml -1 ) were more important than those at the exponential growth phase. The NHase operational stability of whole resting cells was monitored following the bioconversion of acrylonitrile to acrylamide in continuous and stirred UF-membrane reactors. The rate of inactivation was independent on buffer molarity from 25 to 75 mM and on pH from 5.8 to 7.4. Enzyme stability and activity remained unchanged in distilled water. The initial reaction rate increased from 12.8 to 23.8 g acrylamide/g dry cell/h, but NHase half-life dropped from 33 to roughly 7 h when temperature was varied from 4°C to 10°C. The addition of butyric acid up to 20 mM did not improve enzyme operational stability, and largely reduced (94%) enzyme activity. Acrylonitrile caused an irreversible damage to NHase activity. High acrylonitrile conversion (86%) was attained using 0.23 mg cells/ml in a continuously operating reactor.

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.

Influence of initial glucose concentration on nitrile hydratase production in Brevibacterium imperialis CBS 498-74

The growth of Brevibacterium imperialis CBS 498-74 (new classification, Microbacterium imperiale), with glucose, acrylonitrile, acrylamide and methacrylamide as the C-source, was studied in a shake flask at 28 °C for culture periods of up to 150 h. The optimum initial glucose concentration for nitrile hydratase (NHase) production (131 U ml - 1 broth) was 5 g l - 1 . Higher concentrations were found to depress the volumetric enzyme production. Acrylonitrile, acrylamide and methacrylamide cannot be used as the sole C-source. At all tested growth conditions, the highest NHase productivity (U ml - 1 broth h - 1 ) was reached after 24 h of incubation. Specific activities (U mg - 1 DWC) in the cell were found to be: 31 with 5 g l - 1 glucose, 43 with 5 g l - 1 glucose plus 20 mM acrylonitrile, 47 with 5 g l - 1 glucose plus 20 mM acrylamide. The addition of methacrylamide was found to be detrimental under all tested concentrations. Yield coefficient increased progressively with initial glucose concentration until 3.5 g l - 1 and then decreased. Maintenance energy requirement was continuously increasing function of the initial glucose concentration. NHase activity in the whole cell suspension was tested following the biotransformation of acrylonitrile (50 mM) into acrylamide at 20 °C in 50 mM sodium phosphate buffer, pH 7.0. The differently induced NHase had very close K M (from 9.35 to 9.80 mM). The enzyme in cells grown using glucose as the sole C-source had a V m a x of 41.86 μmol min - 1 mg - 1 DWC, whereas the acrylonitrile and acrylamide induced NHase had a V m a x of 51.04 and 56.19 μmol min - 1 mg - 1 DWC, respectively. The measured activation energy, 28.6 KJ mol - 1 , indicated a partial control by mass transport through the cell wall.

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.

Biodegradation of bromoxynil using the cascade enzymatic system nitrile hydratase/amidase from Microbacterium imperiale CBS 498-74. Comparison between free enzymes and resting cells

This work investigates the biodegradation of bromoxynil to the corresponding acid to reduce its acute toxicity. Sequential reactions catalysed by nitrile hydratase (NHase) and amidase (AMase), naturally present in Microbacterium imperiale CBS 498-74, have been used. The kinetic behaviour of the crude extract (CE) and of the resting cell (RC) confined enzymes (NHase and AMase) is compared. In both preparations the same NHase/AMase ratio has been measured. The study was performed using batch and continuous UF-membrane reactor configurations. This paper highlighted a different pH-optimum for each enzyme; a high acid to amide ratio at pH 5.5; and exponential temperature dependence for both enzymes. The halved activation energy indicated the presence of diffusional limitations for the RC-enzymes. However, the higher stability at pH 7.0 for RC-NHase (half-life = 1386 h) and the correct choice of operational conditions allowed the driving to completeness of the bromoxynil biotransformation into the corresponding acid in the batch reactor.