Mn‐peroxidase production by Panus tigrinus CBS 577.79: response surface optimisation and bioreactor comparison

This study reports the statistical optimisation through response surface methodology of the growth medium for Panus tigrinus manganese‐dependent peroxidase (MnP) production in shaken culture. Three crucial variables, including carbon source, malonic acid and Mn²⁺, were optimised in a nitrogen‐limited medium. Sucrose was the best carbon source for MnP production. Mn²⁺ ions and malonic acid significantly stimulated MnP production at an optimal concentration of 53 mg dm^?3 and 8.2 mmol dm^?3, respectively, resulting in 0.83 U cm^?3. Further experiments were performed in lab‐scale stirred tank (STR) and bubble‐column (BCR) reactors using the previously optimised liquid medium. BCR proved to be more adequate than STR in supporting MnP production, leading to 3700 U dm^?3 after 144 h with a productivity of 25.7 U dm^?3 h^?1. On a comparative basis with other production data in lab‐scale reactors, these results appear to be compatible with scale transfer. Copyright © 2006 Society of Chemical Industry     

BTEX removal from contaminated groundwater by a co‐culture of Pseudomonas putida and Pseudomonas fluorescens immobilized in a continuous fibrous‐bed bioreactor

A fibrous‐bed bioreactor with immobilized cells of Pseudomonas putida and Pseudomonas fluorescens was used to treat groundwater contaminated with benzene, toluene, ethylbenzene, and xylenes (collectively know as BTEX). The kinetics of BTEX biodegradation in the fibrous‐bed bioreactor operated under continuous well‐mixed conditions was studied at room temperature. Aeration was not used in the process fed with groundwater samples with an average total BTEX concentration of 2.75 mg dm^?3. All BTEX compounds present in the groundwater feed were concurrently and completely biodegraded even under oxygen‐limited or hypoxic conditions. Nearly 100% removal efficiency was obtained when the retention time was greater than 1 h. BTEX removal efficiency decreased with decreasing the retention time, with p‐ and o‐xylenes showed up first in the treated groundwater, followed by benzene and then other BTEX compounds. Biodegradation rates of BTEX generally increased with increasing BTEX concentration and loading rate. The maximum BTEX biodegradation rate was 5.76 mg h^?1 dm^?3 at the loading rate of 6.54 mg dm^?3 h^?1. The bioreactor had a stable performance, maintaining its ability for efficient BTEX degradation without requiring additional nutrients for more than 1 month. The good performance of the fibrous‐bed bioreactor was attributed to the high cell density (～15 g dm^?3 reactor volume) in the fibrous matrix. © 2002 Society of Chemical Industry     

Optimization of inulinase production by solid‐state fermentation in a packed‐bed bioreactor

BACKGROUND: This work is focused on inulinase production by solid‐sate fermentation (SSF) using sugarcane bagasse, corn steep liquor (CSL), pre‐treated cane molasses, and soybean bran as substrates in a 3‐kg (dry basis) packed‐bed bioreactor. SSF was carried out by the yeast Kluyveromyces marxianus NRRL Y‐7571 and response surface methodology was used to optimize the temperature, air flow rate and initial mass of cells. RESULTS: The optimum inulinase activity (436.7 ± 36.3 U g^?1 dry substrate) was obtained at 24 h at an inlet air temperature of 30 °C, air flow rate 2.2 m³ h^?1 and 22 g of cells for fermentation. Inulinase productivity at these conditions was 18.2 U gds^?1 h^?1. Kinetic evaluation at the optimized conditions showed that the maximum inulinase production was verified at 24 h of fermentation. The carbon dioxide and the metabolic heat generation are directly associated with the consumption of total reducing sugars present in the medium. CONCLUSION: The high productivity achieved in this work shows the technical viability of inulinase production by SSF in a packed‐bed bioreactor. Copyright © 2009 Society of Chemical Industry     

High‐level production of recombinant His‐tagged rhamnulose 1‐phosphate aldolase in Escherichia coli

An expression system based on Escherichia coli and the T5 promoter allowed the overproduction of a his‐tagged rhamnulose‐1‐phosphate aldolase (RhuA; EC 4.1.2.19), an enzyme with applications in the production of deoxyazasugars and deoxysugars compounds. Shake flask and bioreactor cultivation with E coli M15 (pQErham) were performed under different media and inducing conditions for RhuA expression. A Defined Medium (DM) with glucose as carbon source gave a high volumetric and enzyme productivity (3460 AU dm^?3 and 288 AU dm^?3 h^?1 respectively) compared with Luria–Bertoni (LB) medium (2292 AU dm^? 3 and 255 AU dm^?3 h^?1). The minimum quantity of (isopropyl‐β‐D ‐thiogalactoside) IPTG for optimal induction was estimated in 18–20 µmol IPTG gDCW^?1. The highest volumetric production of RhuA (8333 AU dm^?3) was obtained when IPTG was added in the late log‐phase. No significant differences were found in specific RhuA activity for induction temperatures of 30 and 37 °C. An effective two‐step purification process comprising affinity chromatography and gel permeation has been developed (overall recovery 66.5%). These studies provide the basis for the further development of an integrated process for recombinant RhuA production suitable for biotransformation applications. Copyright © 2003 Society of Chemical Industry     

Effect of inducer addition on production of 4,5‐dihydroxyphthalate by Pseudomonas testosteroni

The effect of the addition method of an inducer (o‐phthalate) for the enhancement of 4,5‐dihydroxyphthalate (DHP) production was examined using Pseudomonas testosteroni. The reduction of the lag phase seen when inducer was added in the first 10 h of growth culture did not affect the maximum production rate of DHP. The optimal inducer concentration was about 2 g dm⁻³ and the rate of DHP production was about four times higher that achieved in the absence of inducer. The fed‐batch production of DHP was also carried out and 8 g dm⁻³ of DHP was obtained. © 2000 Society of Chemical Industry     

Comparative bioparticle and hydrodynamic characteristics of conventional and tapered anaerobic fluidized‐bed bioreactors

By maintaining the same operational conditions of one conventional fluidized‐bed bioreactor (CFB) and two tapered fluidized‐bed bioreactors (TFBs), the performance of the TFBs with taper angles of 5 ° and 2.5 ° were found to be superior to that of the CFB with a taper angle of 0 °. Experimental results together with statistical analyses showed that the bioparticle and hydrodynamic characteristics of the TFBs were significantly different from those of the CFB. Also, bioparticle stratification occurred in the three bioreactors. The biofilm thickness (δ) and the specific biomass (β) of the three bioreactors varied in the following decreasing order 5 ° > 2.5 ° > 0 ° under the same volumetric loading. Meanwhile, the specific energy dissipation rate (ω) and the bioparticle washout rates (W = 0.214 ± 0.219; 0.537 ± 0.493 g BAC dm⁻³day⁻¹) of the two TFBs were considerably lower than that of the CFB (W = 1.086 ± 0.916 g BAC dm⁻³ day⁻¹). A lower ω value results in increases in δ and β, and a lower dry density of the biofilm (ρ_d). Accordingly, the performance enhancement with TFBs should be related to their lower ω and W, thicker δ and larger β values. © 2000 Society of Chemical Industry     

Zinc uptake by Streptomyces rimosus biomass using a packed‐bed column

The ability of Streptomyces rimosus biomass to bind zinc ions in batch mode was shown recently. The aim of this study was to determine the zinc uptake capacity by Streptomyces rimosus biomass in continuous mode. Bacterial biomass was able to bind more Zn(II) after pretreatment with sodium hydroxide (1 mol dm⁻³) than without treatment. The maximum adsorption capacity and the adsorption capacity at the saturation point calculated by means of both the exchange zone model and the Thomas model were practically identical of about 2.9 mg_Zn(II) g⁻¹_biomass. This result was lower than the batch adsorption capacity of Streptomyces rimosus, indicating that the packed‐bed is not the most appropriate process to exploit the bacterial biomass adsorption capacity. The effect of zinc concentration in the range of 10 to 200 mg_Zn(II) dm⁻³ on the biosorption capacity of the packed‐bed was not significant. Biomass regeneration with 0.1 mol dm⁻³ HCl gave a 90% recovery of the adsorbed Zn(II). © 1999 Society of Chemical Industry     

An alternative for pre‐treatment of high‐strength raw whey wastewaters: submerged membrane bioreactors

The direct treatment of whey wastewater at various sludge ages (10–75 days) and high biomass concentration (above 50 g mixed liquor suspended solid (MLSS) dm^?3) in a submerged membrane bioreactor (sMBR) is described. The chemical oxygen demand (COD) of raw whey varied in the range of 60 and 90 g dm^?3. After feeding the sMBR with raw whey, effluent COD reduced to about 20 g dm^?3. The effluent was free of suspended solids and total coliform bacteria. Total phosphorus (TP) and orthophosphate (Ortho‐P) in the influent varied between 204 and 880 mg dm^?3 and between 180 and 620 mg dm^?3, and effluent TP and Ortho‐P reduced to 113 and 109 mg dm^?3, respectively. The ammonium and nitrate concentrations in the influent were in the ranges of 3.4 and 120 mg dm^?3 and 10 and 503 mg dm^?3, respectively. The effluent ammonium concentration varied between 17.6 and 198 mg dm^?3 and nitrate concentrations varied between 0.9 and 69 mg dm^?3. Effluent turbidity varied between 23 and 111 FAU (Formazin Attenuation Unit). The results show that sMBR is an effective pre‐treatment system for high‐strength agro‐wastewaters because of its ability to reduce the pollution load. Copyright © 2004 Society of Chemical Industry     

Improvement of heat removal in solid‐state fermentation tray bioreactors by forced air convection

BACKGROUND: Heat removal is one of the major constraints in large‐scale solid‐state fermentation (SSF) processes. The effect of internal air circulation by forced convection on heat and water transfer has not been studied in SSF tray bioreactors. Formulation of a mathematical model for SSF requires a good estimation of the mass and heat transfer coefficients. RESULTS: A stainless steel tray bioreactor (80.6 L capacity) was used. Aspergillus niger C28B25 was cultivated under SSF conditions on an inert support. Temperature, moisture content, biomass and substrate concentrations were measured. Water and energy integral balances were used to estimate the heat and mass transfer coefficients involved in the process. The Reynolds number (N_Re) in the headspace of the tray bioreactor ranged from 2.5 to 2839, which increased the global heat transfer coefficient from 4.2 to 6.9 (W m^?2 K^?1) and the mass transfer coefficient from 1.0 to 2.1 (g m^?2 s^?1). Mathematical model predictions of the temperature and moisture content of the fermentation bed showed a high goodness‐of‐fit with the experimental results. CONCLUSIONS: This is the first report describing the effect of N_Re of air in the headspace of a SSF tray bioreactor on the heat and mass transfer coefficients and temperature regulation in SSF. Copyright © 2011 Society of Chemical Industry     

Detoxification of mercury by immobilized mercuric reductase

Mercuric reductase which originated from a recombinant Escherichia coli PWS1 was purified and immobilized on a chemically modified diatomaceous earth support. The mercury reduction kinetics, pH dependence, storage stability, and reusability of the immobilized enzyme were investigated. Four dyes were examined for their electron transfer efficiency with the soluble and bound mercuric reductase. Continuous mercury detoxification by the immobilized mercuric reductase was also performed in fixed‐bed processes. The effects of bed‐length, mercury loading rate, and electron donor on the performance of the fixed beds were assessed. Immobilized mercuric reductase exhibited substrate‐inhibition‐type kinetics with a maximal activity (1.2 nmol Hg mg⁻¹ protein s⁻¹) occurring at an initial Hg²⁺ concentration of 50 µmol dm⁻³. The optimal pH was 7.0 for the soluble and immobilized mercuric reductase, but the immobilized enzyme maintained higher relative activity for less favorable pH values. Immobilization of the enzyme appeared to significantly enhance its storage stability and reusability. Of four artificial electron donors tested, azure A (5 mmol dm⁻³) demonstrated the highest relative activity (78%) for soluble mercuric reductase. For the immobilized enzyme, neutral red (5 mmol dm⁻³) gave a relative activity of nearly 82%. With a fixed‐bed, the mercury‐reducing efficiency of using neutral red was only 30–40% of that obtained using NADPH. Fixed‐bed operations also showed that increased bed length facilitated mercury reduction rate, and the optimal performance of the beds was achieved at a flow rate of approximately 100–200 cm³ h⁻¹. © 1999 Society of Chemical Industry     

Influence of growth medium composition on the lipolytic enzyme activity of Ophiostoma piliferum (CartapipTM)

Production of esterases (carboxyl esterase EC 3.1.1.1) and lipases (glycerol ester hydrolase EC 3.1.1.3) by Ophiostoma piliferum (Cartapip^TM), a fungus commercialized to decrease fatty acyl esters in wood, is described. The influence of various combinations of carbon and nitrogen sources, in the growth medium, was examined. Medium containing yeast extract as a nitrogen source and olive oil as a carbon source was found to be optimal for extracellular esterase (221 U dm⁻³) and lipase (152 U dm ⁻³) activities. Further increases in those enzyme activities were achieved by decreasing medium pH from 6.5 to 5.5 (esterase 508 U dm⁻³; lipase 415 U dm ⁻³) and increasing medium calcium content from 8 m mol dm⁻³ to 160 m mol dm⁻³ (esterase 4084 U dm⁻³; lipase 508 U dm ⁻³) © 1999 Society of Chemical Industry     

Decolorization of Direct Blue GLL with enhanced lignin peroxidase enzyme production in Comamonas sp UVS

BACKGROUND: The present work aims to study the production of lignin peroxidase (LiP) enzyme by Comamonas sp UVS using various media, and lignocellulosic waste materials, and its effect on decolorization of Direct Blue GLL (DBGLL). RESULTS: Yeast extract medium was found to be more effective for the production of LiP and also for the decolorization of DBGLL. The bagasse powder along with yeast extract induced LiP activity. Comamonas sp UVS decolorized DBGLL dye (50 mg L^?1) within 13 h at static condition in YE broth. It could degrade up to 300 mg L^?1 of dye within 55 h. The maximum rate (Vmax) of decolorization was 12.41 ± 0.55 mg dye g cell^?1 h^?1 with the Michaelis constant (Km) value as 6.20 ± 0.27 mg L^?1. The biodegradation was monitored by UV‐Vis, GC‐MS and HPLC. CONCLUSION: The use of agricultural by‐products for the activity enhancement of the ligninolytic enzymes is a cost effective process. It also resolves the problem of the disposal of agro‐residues. This system can be applied for the degradation of different recalcitrant compounds. Copyright © 2008 Society of Chemical Industry     

Interaction of transport resistances with biochemical reaction in packed bed solid state fermenters: The effect of gaseous concentration gradients

Mass transfer plays an important role in solid state fermentation (SSF) systems. Earlier work on SSF in tray bioreactors⁷ indicated that steep gaseous concentration gradients developed within the substrate bed, owing to mass transfer resistances, which may adversely affect the bioreactor performance. For all practical purposes these gradients have been eliminated using a packed bed column bioreactor with forced aeration. Gaseous concentrations (oxygen and carbon dioxide) and enzyme activities were measured at various bed heights for various air flow rates during the course of fermentation. The results indicated that concentration gradients were decreased effectively by increasing air flow rate. For example, the actual oxygen and carbon dioxide concentration gradients reduced from 0.07% (v/v) cm^?1 and 0.023% (v/v) cm^?1 to 0.007% (v/v) cm^?1 and 0.0032% (v/v) cm^?1 respectively when the air flow rate was increased from 5 dm³ min^?1 to 25 dm³ min^?1. This resulted in an overall improvement in the performance of the bioreactor in terms of enzyme production.     

Kinetic modelling of lactic acid production from whey by Lactobacillus casei (NRRL B‐441)

The biomass growth, lactic acid production and lactose utilisation kinetics of lactic acid production from whey by Lactobacillus casei was studied. Batch fermentation experiments were performed at controlled pH and temperature with six different initial whey lactose concentrations (9‐77 g dm^?3) in a 3 dm³ working volume bioreactor. Biomass growth was well described by the logistic equation with a product inhibition term. In addition, biomass and product inhibition effects were defined with corresponding power terms, which enabled adjustment of the model for low‐ and high‐substrate conditions. The Luedeking‐Piret equation defined the product formation kinetics. Substrate consumption was explained by production rate and maintenance requirements. A maximum productivity of 2.5 g dm^?3 h^?1 was attained with an initial lactose concentration of 35.5 g dm^?3. Copyright © 2006 Society of Chemical Industry     

Production of L‐methionine by immobilized pellets of Aspergillus oryzae in a packed bed reactor

Production of L ‐methionine by immobilized pellets of Aspergillus oryzae in a packed bed reactor was investigated. Based on the determination of relative enzymatic activity in the immobilized pellets, the optimum pH and temperature for the resolution reaction were 8.0 and 60 °C, respectively. The effects of substrate concentration on the resolution reaction were also investigated and the kinetic constants (K_m and V_m) of immobilized pellets were found to be 7.99 mmol dm^?3 and 1.38 mmol dm^?3 h^?1, respectively. The maximum substrate concentration for the resolution reaction without inhibition was 0.2 mol dm^?3. The L ‐methionine conversion rate reached 94% and 78% when substrate concentrations were 0.2 and 0.4 mol dm^?3, respectively, at a flow rate of 7.5 cm³ h^?1 using the small‐scale packed bed reactor developed. The half‐life of the L ‐aminoacylase in immobilized pellets was 70 days in continuous operation. All the results obtained in this paper exhibit a practical potential of using immobilized pellets of Aspergillus oryzae in the production of L ‐methionine. © 2002 Society of Chemical Industry     

Lecithin‐enhanced biotransformation of cholesterol to androsta‐1,4‐diene‐3,17‐dione and androsta‐4‐ene‐3,17‐dione

A biotransformation process using Mycobacterium sp was studied for androsta‐1, 4‐diene‐3,17‐dione (ADD) and androsta‐4‐ene‐3,17‐dione (AD) production from cholesterol. Cholesterol has a poor solubility in water (～1.8 mg dm^?3 at 25 °C), which makes it difficult to use as the substrate for biotransformation. Lecithin is a mixture of phospholipids of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), which behave like surfactants and can form planar bi‐layer structures in an aqueous medium. Therefore, a small amount of lecithin (<1 g dm^?3) can be used to form stable colloids with cholesterol at a relatively high concentration (20 g dm^?3) in water. In this work, an energy density of 1000 J cm^?3 from sonication was provided to overcome the self‐association of cholesterol and to generate a stable lecithin–cholesterol suspension that could be used for enhanced biotransformation. The lecithin–cholesterol suspension was stable and could withstand typical autoclaving conditions (121 °C, 15 psig, 20 min). In contrast to conventional surfactants, such as Tween 80, that are commonly used to help solubilize cholesterol, lecithin did not change the surface tension of the aqueous solution nor cause any significant foaming problem. Lecithin was also biocompatible and showed no adverse effect on cell growth. Compared with the medium with Tween 80 as the cholesterol‐solubilizing agent, lecithin greatly improved the biotransformation process in regard to its final product yield (～59% w/w), productivity (0.127–0.346 g dm^?3 day^?1), ADD/AD ratio (6.7–8), as well as the long‐term process stability. Cells can be reused in repeated batch fermentations for up to seven consecutive batches, but then lose their bioactivity due to aging problems, possibly caused by product inhibition and nutrient depletion. © 2002 Society of Chemical Industry     

Phenol degradation in a fixed‐bed bioreactor using micro‐cellular polymer‐immobilized Pseudomonas syringae

Highly porous (85% void volume) polymer beads with interconnecting micro‐pores were prepared for the immobilization of Pseudomonas syringae for the degradation of phenol in a fixed‐bed column bioreactor. The internal architecture of this support material (also known as PolyHIPE Polymer) could be controlled through processing before the polymerization stage. The transient and steady state phenol utilization rates were measured as a function of substrate solution flow rate and initial substrate concentration. The spatial concentration of the bacteria on the micro‐porous support particles as well as within them was studied using scanning electron microscopy at various time intervals during the continuous operation of the bioreactor. It was found that although bacterial penetration into the porous support was present after 20 days, bacterial viability however, was compromised after 120 days as a result of the formation of a biofilm on the support particles. The steady state phenol utilization at an initial phenol concentration of 200 mg cm^?3 was 100% provided that the flow rate was less than 7 cm³ min^?1. Substrate inhibition at a constant flow rate of 4.5 cm³ min^?1 was found to begin at 720 mg dm^?3. The critical dilution rate for bacteria washout was high as a result of the highly hydrophobic nature of the support and the reduction of pore interconnect size due to bacterial growth within the pores in the vicinity of the surface of the support. Copyright © 2004 Society of Chemical Industry     

Effects of polyacrylamide‐co‐acrylic acid on cellulose production by Acetobacter xylinum

Polyacrylamide‐co‐acrylic acid (PA) added to shake flask cultures of Acetobacter xylinum at concentrations up to 3 g dm^?3 resulted in increased production of bacterial cellulose. For PA concentrations of 0–3 g dm^?3, 7‐day cellulose production rose monotonically from 2.7 ± 0.8 to 6.5 ± 0.5 g dm^?3 at a shaker speed of 175 rpm, and from 1.7 ± 0.01 to 3.7 ± 0.5 g dm^?3 at shaker speed of 375 rpm. Addition of PA also changed the morphology of the biomass from amorphous/stringy forms to spheroidal particles with diameters ≤2 mm. Similarly, bioreactor cultures grown in the absence of PA formed long fibrous masses which deposited on the internals, while those grown in the presence of 1–2 g dm^?3 PA formed small discrete particles with diameters ≤0.1 mm. Tests performed with 1 and 2 g dm^?3 PA, and stirrer speeds from 500 to 900 rpm, appeared to give the highest cellulose concentration of 5.3 ± 0.7 g dm^?3 in 64–68.5 h in the presence of 2 g dm^?3 PA at 700 rpm, although this value was statistically indistinguishable from that obtained at 1 g dm^?3 PA and 900 rpm. A qualitative model is proposed to describe the mechanisms by which PA affects biomass morphology, resulting in its advantageous formation as small, dispersed, spheroidal pellets. Quantitative analysis of the results gave inverse correlations between both the fraction of fructose carbon going to cellulose synthesis and the specific fructose consumption rate, and the maximum cellulose concentration and the fraction of fructose carbon going to by‐product formation. Since cellulose yield was almost universally improved by higher polyacrylamide concentration, it appears likely that increased viscosity reduces fructose uptake rate by limiting mass transfer. Copyright © 2003 Society of Chemical Industry     

A bioprocessing mode for simultaneous fungal biomass protein production and wastewater treatment using an external air‐lift bioreactor

A pilot plant investigation for bioprocessing has been undertaken to develop a simple, non‐aseptic, low‐cost single process for production of fungal biomass protein (FBP) and wastewater treatment using starch processing wastewater. It has been confirmed that the newly developed external air‐lift bioreactor was very suitable for bioconversion of starch materials and FBP production by the microfungi Aspergillus oryzae and Rhizopus arrhizus. Bioproduct yields of 8.5 g dm^?3 of FBP that contained 46–50% protein were obtained within a comparatively short retention time. A fungal biomass productivity in a range of 0.85–0.92 g dm^?3 h^?1 and removals of total suspended solids and 95% COD were achieved in batch, semi‐continuous and continuous processes. The operation modes of the semi‐continuous and continuous processes demonstrated a high biological dynamics in fungal biomass productivity and COD reduction. The semi‐continuous process appeared to be the most practical mode. © 2001 Society of Chemical Industry     

黄孢原毛平革菌产酶的简化结构动力学模型

In order to understand the behavior of ligninolytic enzyme production by white rot fungi Phanerochaete chrysosporium, study on time courses and a mathematical model for the production of lignin peroxidase (LiP) and manganese peroxidase (MnP) of the fungi was undertaken. Based on the Monod-Jacob operon model, the ligninolytic enzyme would be synthesized in the absence of a related repressor. The repressor is assumed to be active in the presence of ammonia nitrogen, and as combined as co-repressor, it causes the inhibition of enzyme synthesis. The model can explain the mechanism of extracellular ligninolytic enzyme production by white rot fungi. The results,as predicted by the model, correspond closely to those observed in experimental studies. In addition, some light is also shed on unmeasured variables, such as the concentrations of repressor and mRNA that are related to the enzyme synthesis.