High level expression of Glomerella cingulata cutinase in dense cultures of Pichia pastoris grown under fed-batch conditions.

A Pichia pastoris transformant carrying the cutinase cDNA of Glomerella cingulata was over-expressed in a 5L bioreactor (2.0L working volume) under fed-batch conditions. Bioreactor experiments rely on varying selected parameters in repeated rounds of optimisation: here these included duration of induction, pH and temperature. Highest cell densities (320gL(-1) wet cell weight) with a cutinase production of 3800mgL(-1) and an activity of 434UmL(-1) were achieved 24h after induction with methanol in basal salt medium (at pH 5 and 28°C). Characterisation of the cutinase showed that it was stable between pH 6 and pH 11, had an optimum pH of 8.0 and retained activity for 30min at 50°C (optimum temperature 25°C).The preferred substrates of G. cingulata cutinase were the medium- to long-chain ρ-nitrophenyl esters of ρ-nitrophenylcaprylate (C8), ρ-nitrophenyllaurate (C12) and ρ-nitrophenylmyristate (C14), with the highest catalytic efficiency, kcat/Km of 7.7±0.7mM(-1)s(-1) for ρ-nitrophenylcaprylate. Microscopic analyses showed that the G. cingulata cutinase was also capable of depolymerising the high molecular weight synthetic polyester, polyethylene terephthalate.

Applications of the Box-Wilson design model for bio-hydrogen production using Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564).

Box-Wilson design (BWD) model was applied to determine the optimum values of influencing parameters in anaerobic fermentation to produce hydrogen using Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564). The main focus of the study was to find the optimal relationship between the hydrogen yield and three variables including initial substrate concentration, initial medium pH and reaction temperature. Microbial growth kinetic parameters for hydrogen production under anaerobic conditions were determined using the Monod model with incorporation of a substrate inhibition term. The values of micro(max) (maximum specific growth rate) and K, (saturation constant) were 0.398 h(-1) and 5.509 g L(-1), respectively, using glucose as the substrate. The experimental substrate and biomass-concentration profiles were in good agreement with those obtained by the kinetic-model predictions. By varying the conditions of the initial substrate concentration (1-40 g L(-1)), reaction temperature (25-40 degrees C) and initial medium pH (4-8), the model predicted a maximum hydrogen yield of 3.24 mol H2 (mol glucose)(-1). The experimental data collected utilising this design was successfully fitted to a second-order polynomial model. An optimum operating condition of 10 g L(-1) initial substrate concentration, 37 degrees C reaction temperature and 6.0 +/- 0.2 initial medium pH gave 80% of the predicted maximum yield of hydrogen where as the experimental yield obtained in this study was 77.75% exhibiting a close accuracy between estimated and experimental values. This is the first report to predict bio-hydrogen yield by applying Box-Wilson Design in anaerobic fermentation while optimizing the effects of environmental factors prevailing there by investigating the effects of environmental factors.

Bio-hydrogen production using a two-stage fermentation process.

A two-stage fermentation process consisting of dark and photo-fermentation periods was carried out in a batch reactor. In the first stage, glucose was fermented in the dark stage using Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564; CSN1-4) to produce acetate, CO2 and H2. The acetate produced in the first stage is fermented to H2 and CO2 by Rhodobacter sphaeroides NCIMB 8253 for further hydrogen production in the second, illuminated stage. The yield of hydrogen in the first stage was about 3.10 mol H2 (mol glucose)(-1) at a glucose concentration of 10 g L(-1), pH 6 +/- 0.2 and 37 degrees C and the second stage yield was about 1.10-1.25 mol H2 (mol acetic acid)(-1) at pH 6.8 +/- 0.2 and 32 degrees C, without removal of the Clostridium CSN1-4. The overall yield of hydrogen in the two-stage process, with glucose as the main substrate was higher than single-stage fermentation.