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     

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     

Effects of culture pH on the production of bioactive polysaccharides by Agaricus blazei in batch cultures

The effects of culture pH on the production, molecular weight distribution and the bioactivity of polysaccharides produced by Agaricus blazei were evaluated by four pH‐controlled batch cultures. As the culture pH of each batch was controlled from 4.0 to 7.0, the maximum polysaccharide concentration increased from 561 to 1252 mg dm^?3, but the average molecular weight of the polysaccharides decreased monotonically from 1080 kDa to 600 kDa, the relative amount of β‐glucan from 56% to 33%, and the TNF‐α release by macrophage cells from 1440 to 760 pg per 5 × 10⁴ cells. Polysaccharides with higher molecular weights, β‐glucan contents and bioactivities were obtained in the submerged cultures of Agaricus blazei at lower culture pH but with lower yields. Copyright © 2004 Society of Chemical Industry     

Selection and isolation of glucose-tolerant amylolytic Aspergillus by cyclic fed batch culture process

A simple cyclic fed batch culture system was developed for selection and subsequent isolation of glucose-tolerant Aspergillus niger strain. A culture medium which contained 1.2 g dm⁻³ of glucose was inoculated with a non-glucose-tolerant A. niger (K_i =20.25 g dm⁻³). A culture medium of higher glucose concentration (100 g dm⁻³ and 200 g dm⁻³) was fed at a rate equal to the rate of HN₄⁺ consumption by means of a pH control system. The maximum and minimum liquid levels in the fed batch culture vessel were determined by two liquid level detectors which activated and deactivated a harvest pump. The novelty of the selection system is that the frequency and pressure of selection increase gradually but continuously, and they are determined by the intrinsic potential of the culture. The process was fully automatic. An Aspergillus mutant which had a glucose inhibition constant of 3200 g dm⁻³ was isolated after six generations. The process should be particularly useful for screening filamentous microorganisms growing on novel substrates or tolerating inhibitors.     

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     

Removal of linuron from water by natural and activated bentonite

The sorption of linuron on bentonite desiccated at 110°C untreated, and acid‐treated with H₂SO₄ solutions over a concentration range between 0.25 M and 1.00 M from aqueous solution at 25°C has been studied by using batch experiments. In addition, column experiments were carried out with the bentonite sample treated with the 1.00 M H ₂SO₄ solution [B‐A(1.00)] by using two aqueous solutions of linuron of different concentrations (C=4.97 mg dm⁻³ and C=7.63 mg dm⁻³ ). The experimental data points have been fitted to the Langmuir equation in order to calculate the sorption capacities (X_m) of the samples; X_m values range from 0.02 g kg⁻¹ for the untreated bentonite [B‐N] up to 0.20 g kg⁻¹ for the sample acid‐treated with the 1.00 M H₂ SO₄ solution. The removal efficiency (R ) has also been calculated; R values ranging from 15.86% for the [B‐N] sample up to 41.54% for [B‐A(1.00)]. The batch experiments show that the acid‐treated bentonite is more effective than the natural bentonite in relation to sorption of linuron. The column experiments show that the B‐A(1.00) sample might be reasonably used in removing linuron, the column efficiency increasing from 61.8% for the C=7.63 mg dm⁻³ aqueous solution of linuron up to 77.6% for the C=4.97 mg dm⁻³ one. © 1999 Society of Chemical Industry     

Effect of yeast extract on the expression of thioredoxin–human parathyroid hormone from recombinant Escherichia coli

Terrific broth, a complex medium containing a high content of yeast extract, was chosen to cultivate recombinant Escherichia coli with the plasmid encoding the fusion protein gene of thioredoxin (Trx) and human parathyroid hormone (hPTH). The volumetric yield of Trx–hPTH fusion protein in the culture with Terrific broth reached about 800 mg L^?1 after optimization. It was found that high content of yeast extract in Terrific broth promoted cell growth and Trx–hPTH fusion protein production. However, the more interesting fact was confirmed that high content of yeast extract was also responsible for the high‐level expression of Trx–hPTH fusion protein without specific inducer addition. Further studies indicated that the expression levels of Trx–hPTH fusion protein without specific inducer addition varied greatly with the content and the source of yeast extract contained in the media. Considering that some proteins are toxic to the host and their continuous expression may result in decreasing plasmid stability and protein yields, one should be cautious in selecting yeast extract in media for cultivating E. coli with plasmids carrying toxic genes under T7 control. Copyright © 2006 Society of Chemical Industry     

The operational strategy of intermittent cycle extended aeration system in treating sewage

An Intermittent Cycle Extended Aeration System (ICEAS) offers advantages for treating sewage; such as easy operation, process flexibility, and low capital cost. A laboratory‐scale study was made with synthetic‐domestic wastewater (COD = 300 mg dm⁻³; BOD = 210 mg dm⁻³) to investigate appropriate conditions for reduced operating cost. The results from this study indicated that the maximum hydraulic loading and organic loading were 3.5 m³ m⁻³ d⁻¹ and 0.735 kg BOD m⁻³d⁻¹ respectively. The BOD and COD of effluent were 15.5 mg dm⁻³ and 29.6 mg dm⁻³ for the cycle time and aeration time of 3.4 h and 2.65 h. It was not necessary to supply external artificial substrates in the reactor to deal with low wastewater flow that caused the starvation of sludge. Specific oxygen uptake rate (SOUR) was used as the index of microbial activity. The study indicated that the microbial activity could be restored (SOUR = 20.5 mg g⁻¹ MLVSS h⁻¹) after 5–6 days of cultivation when the sludge was deprived of substrate for 17 days. © 1999 Society of Chemical Industry     

Sequential Degradation of Phenol and Cyanide by a Commensal Interaction Between Two Microorganisms

Sequential degradation of phenol and cyanide was achieved by a commensal interaction between phenol-degrading and cyanide-degrading bacteria. Phenol-degrading bacteria degraded the phenol rapidly to nontoxic compounds which cyanide-degrading bacteria could successively metabolize as a carbon source for growth. By UV spectroscopy, the phenol-degrading bacteria were presumed to act via a meta-cleavage pathway to yield an intermediate which was metabolized as a carbon source by cyanide-degrading bacteria. By this commensal interaction, 500 mg dm⁻³ of phenol and 26 mg dm⁻³ of cyanide were completely degraded within 40 h in the batch culture. Using a two-stage reactor system, continuous degradation of phenol and cyanide was also realized. © 1997 SCI.     

Plasmid stability in a recombinant S cerevisiae strain secreting a bifunctional fusion protein

The recombinant Saccharomyces cerevisiae strain, YPB‐G, producing and secreting Bacillus subtilis α‐amylase and Aspergillus awamori glucoamylase as a fusion protein yielded efficient utilisation of starch. A segregated population balance model has been used to determine the probability of plasmid loss and plasmid copy number. The kinetics of cell growth and product (fusion protein) formation were based on a genetically structured model. The predictions were compared with the experimental observations obtained for the unstable recombinant S cerevisiae cells in a 1.5 dm^?3 batch bioreactor with 30 g dm³ initial starch under non‐aerated conditions. The main advantage of the present model is that three different genetic classes were defined on the basis of the existence of plasmid and of the expression of the enzymes, ie cells containing plasmids and expressing the gene product, x₁; cells containing plasmids and but not expressing the gene product, x₂; and cells without plasmids, x₃. It is confirmed by this model that the cells without plasmids outgrow and dominate in the fermentation medium (2.27 g dm^?3 vs 0.51 g dm^?3) as more and more glucose becomes available by the degradation of starch. © 2001 Society of Chemical Industry     

Anaerobic and aerobic treatment of a simulated textile effluent

A simulated textile effluent (STE) was generated for use in laboratory biotreatment studies; this effluent contained one reactive azo dye, PROCION Red H‐E7B (1.5 g dm⁻³); sizing agent, Tissalys 150 (1.9 g dm⁻³); sodium chloride (1.5 g dm⁻³) and acetic acid (0.53 g dm⁻³) together with nutrients and trace elements, giving a mean COD of 3480 mg dm⁻³. An inclined tubular anaerobic digester (ITD) was operated for 9 months on the STE and a UASB reactor for 3 months. For a 57 day period anaerobic effluent from two reactors, a UASB and an ITD, was mixed and treated in an aerobic stage. In days 77–247 68% of the true colour of PROCION Red H‐E7B was removed by anaerobic treatment with no colour removal aerobically and up to 37% COD was removed anaerobically, with a corresponding BOD removal of 71%. For combined anaerobic and aerobic treatment a mean COD removal of 57% and BOD removal of 86% was achieved. Operation of the ITD at a 2.8 day HRT (volumetric loading rate (B _v) 1.24 g COD dm⁻³day⁻¹) and the UASB at a 2 day HRT (B _v 1.74 g COD dm⁻³day⁻¹) gave comparable COD removals but the UASB gave better true colour removal. Effluent from the combined process operating on this simulated waste still contained an average 1500 mg COD dm⁻³, and further treatment would be required to meet consent standards. © 1999 Society of Chemical Industry     

Design of different bioreactor configurations: application to ligninolytic enzyme production in semi‐solid‐state cultivation

The production of ligninolytic enzymes by Phanerochaete chrysosporium BKM‐F‐1767 (ATCC 24725) in laboratory‐scale bioreactors was studied. The cultivations were carried out in semi‐solid‐state conditions, employing corncob as carrier, which functioned both as a place of attachment and as a source of nutrients. Several bioreactor configurations were investigated in order to determine the most suitable one for ligninolytic enzyme production: a 1‐dm³‐static‐bed bioreactor, a 1‐dm³‐static‐bed bioreactor with air diffusers into the bed, a 0.5‐dm³‐static‐bed bioreactor with air diffusers into the bed and a tray bioreactor. Although the static‐bed configurations produced maximum individual lignin peroxidase (LiP) activities about 400 U dm⁻³ (1.0‐dm³ bioreactor) and about 700 U dm⁻³ (0.5‐dm³ bioreactor), manganese‐dependent peroxidase (MnP) was not detected throughout the cultures. Nevertheless, the tray configuration led to maximum individual MnP and LiP activities of about 200 U dm⁻³ and 300 U dm⁻³, respectively. Therefore, this configuration is the most adequate of the different bioreactor configurations tested in the present work, since the ligninolytic complex formed by MnP and LiP is more efficient for its application to bio‐processing systems. In addition, the results indicated the influence of the oxygen in ligninolytic enzyme production. © 2001 Society of Chemical Industry     

In‐line mixing for production of citric acid by Candida lipolytica grown on n‐paraffins

This study reports on the effects of internal fermenter and external in‐line agitation and fed‐batch mode of operation on citric acid production from Candida lipolytica using n‐paraffin as the carbon source. An optimum range of fermenter agitation speeds in the range 800–1000 rpm corresponding to Reynolds numbers of 50433–62947 (based on initial batch conditions) seemed to give the best balance between substrate utilization for biomass growth and citric acid production. Proof of concept evidence is presented that indicates that an external in‐line agitator could be used in place of high speed internal agitation to increase citric acid production. However, more work is required to optimize the external agitator concept. Application of multiple fed‐batch feedings can be used to extend the batch fermentation and increase final citric acid concentrations and product yield. Experiments were conducted implementing a three‐cycle fed‐batch process which increased overall citric acid yields to 0.8–1.0 g citric acid g^?1 n‐paraffin, approximately 200% improvement from those found in the normal batch process. The three‐cycle fed‐batch mode of operation also increased the final citric acid concentration to 42 g dm^?3 from about 6 g dm^?3 for normal batch operation. Increased citric acid concentrations in three‐cycle fed‐batch mode was achieved at longer fermentation times. Copyright © 2004 Society of Chemical Industry     

Production of lactic acid from beet molasses by calcium alginate immobilized Lactobacillus delbrueckii IFO 3202

Lactic acid was produced from pretreated beet molasses by the homofermentative organism Lactobacillus delbrueckii subsp delbrueckii IFO 3202 entrapped in calcium alginate gel using batch, repeated batch and continuous fermentation systems. In batch fermentation studies successful results were obtained with 2.0–2.4 mm diameter beads prepared from 2% sodium alginate solution. The highest effective yield (82.0%) and conversion yield (90.0%) were obtained from substrate concentrations of 52.1 and 78.2 g dm⁻³ respectively. The gel beads produced lactic acid for 14 consecutive batch fermentations without marked activity loss and deformation. In the continuous fermentation, the highest lactic acid (4.22%) was obtained at a dilution rate of 0.1 h⁻¹ while the highest productivity (13.92 g dm⁻³ h⁻¹) was obtained at a dilution rate of 0.4 h⁻¹. © 1999 Society of Chemical Industry     

Evaluation of biosorbents for Cu removal from wastewater in the presence of EDTA

BACKGROUND: This paper evaluates the use of several biosorbents for Cu removal from aqueous solutions in the absence and presence of ethylenediaminetetraacetic acid (EDTA). The objective was to determine the applicability of the sorption process after conventional physicochemical wastewater treatment, or as primary treatment, replacing the physicochemical process. RESULTS: Fixed‐bed experiments were performed at Cu influent concentrations of 2 and 20 mg dm^?3 and EDTA doses between 0 and 10 mg dm^?3. At low Cu concentration without EDTA, Cu uptake capacity followed the order Posidonia oceanica > chitosan > chitin > Scharlau AC > Darco AC, with a maximum, at C/C₀ = 0.2, of 23.2 mg g^?1. In the presence of EDTA, Cu was detected in the effluent from the beginning of the operation, except for the activated carbons and chitosan at low EDTA doses. At higher EDTA doses, the activated carbons showed the best performance. Uptakes at Cu concentration of 20 mg dm^?3 without EDTA were 51.6 (Posidonia oceanica) and 41.4 mg g^?1 (chitosan) at C/C₀ = 0.2. CONCLUSION: A sequence of one fixed bed with Posidonia oceanica followed by another with Scharlau AC should be an alternative to Cu precipitation, with Cu effluent concentration lower than 0.5 mg dm^?3 for more than 350 pore volumes. Copyright © 2007 Society of Chemical Industry     

Fermentation optimization for the production of lovastatin by Aspergillus terreus: use of response surface methodology

A Box–Behnken experimental design was used to investigate the effects of five factors—ie oxygen content in the gas phase; concentrations of C, N and P; and fermentation time—on the concentrations of biomass and lovastatin produced in batch cultures of Aspergillus terreus. The values of the various factors in the experiment ranged widely, as follows: 20–80% (v/v) oxygen in the aeration gas; 8–48 g dm^?3 C‐concentration; 0.2–0.6 g dm^?3 N‐concentration; 0.5–2.5 g dm^?3 phosphate‐concentration; and 7–11 days fermentation time. No previous work has used statistical analysis in documenting the interactions between oxygen supply and nutrient concentrations in lovastatin production. The Box–Behnken design identified the oxygen content in the gas phase as the principal factor influencing the production of lovastatin. Both a limitation and excess of oxygen reduced lovastatin titers. A medium containing 48 g dm^?3 C supplied as lactose, 0.46 g dm^?3 N supplied as soybean meal, and 0.79 g dm^?3 phosphate supplied as KH₂PO₄, was shown to support high titers (～230 mg dm^?3) of lovastatin in a 7‐day fermentation in oxygen‐rich conditions (80% v/v oxygen in the aeration gas). Under these conditions, the culture medium had excess carbon but limiting amounts of nitrogen. The optimized fermentation conditions raised the lovastatin titer by four‐fold compared with the worst‐case scenario within the range of factors investigated. Copyright © 2004 Society of Chemical Industry     

Emulsion formation and stability during reversed micelle extraction

The formation of emulsions in the reversed micelle extraction of proteins from filtered broth is a major problem which can significantly increase the cost of downstream separation. In this work the effect of some aqueous phase constituents such as buffer salts, other added cations, phospholipids and five molecular‐weight fractions (fractionated using ultrafiltration membranes) of a fermentation broth of Bacillus stearothermophilus on emulsion formation and stability of a water–AOT–isooctane reversed micelle extraction system were examined. The parameters used for the determination of emulsion stability were the settling time and interfacial tension, and this varied from 33 mN m⁻¹ for pure water–isooctane to around 1 mN m⁻¹ for high AOT concentrations (>100 mmol dm⁻³). K⁺ ions of the buffer solution, as well as K⁺ ions and Na⁺ ions from added salts had a destabilising effect on a water–AOT–isooctane emulsion. This effect was found to be greatest for the K⁺ ions, possibly due to their smaller hydrated radius compared with the other ions, and reduced the phase separation time with 50 mmol dm⁻³ AOT from 500 min to less than 5 min. The phospholipids phosphatidylcholine and phosphatidylinositol were also found to be effective demulsifiers, with phosphatidylcholine having a slightly greater effect. Albumin and β‐galactosidase both exhibited a strong emulsifying effect on a surfactant‐free water–isooctane system, but were capable of strongly reducing emulsification in the presence of AOT. High molecular weight fractions (>30 kDa) of the B stearothermophilus fermentation broth had a much stronger tendency to form emulsions than MW fractions <30 kDa, while the 30–50 kDa MW fraction had the best demulsifying effect, reducing the separation time to 75 min. Demulsifying properties of the components tested were attributed to the competition of these compounds with AOT for control of the aqueous–organic interface leading to a less stable mixed interfacial layer. © 2000 Society of Chemical Industry     

Maximum volumetric production of β‐galactosidase by Kluyveromyces fragilis in fed‐batch culture with automatic control

The production of β‐galactosidase by Kluyveromyces fragilis was studied in different culture systems, with dissolved oxygen concentration control and using defined media. An operating strategy of fed‐batch culture with automatic control of substrate addition regulated by dissolved oxygen concentration, consisting of the replacement of variable volumes of broth by fresh medium (once the fed‐batch culture has finished), was designed. The volumetric enzyme productivity (Q_p, 13 600 UI dm^?3 h^?1) obtained was 38% higher than that reached in continuous culture of K fragilis with dissolved oxygen concentration control and far higher than that obtained by batch culture of K fragilis under the same aeration conditions. © 2002 Society of Chemical Industry     

Production of Hexanoic Acid by Free and Immobilised Cells of Megasphaera elsdenii: Influence of in-situ Product Removal Using Ion Exchange Resin

The objective of this work was to improve the production of hexanoic acid by the anaerobic rumen bacterium, Megasphaera elsdenii, using product removal and immobilised cell approaches. Hexanoic acid, the major product of glucose metabolism by M. elsdenii strain ATCC25940, was produced at concentrations of 2–3 g dm⁻³ in stirred batch cultures. With pH controlled manually at 7, maximum concentrations of hexanoic acid increased to 6–8 g dm⁻³ with yields (g product per g glucose used) of approximately 30%. When an anion exchange resin, Amberlite IRA 400, was added during early stages of culture to minimise product inhibition, growth was not impaired and cell lysis, which was commonly seen during the stationary phase in control fermentations, was prevented. The presence of resin in pH-controlled, stirred batch fermentations increased the rate of glucose consumption and doubled hexanoic acid productivity: the equivalent of 11 g dm⁻³ of hexanoic acid was made with an estimated yield of up to 39%. Cells were immobilised successfully in κ-carrageenan and, when cell densities in inocula were sufficiently high, rates of glucose consumption and product formation were similar to free cells. Including resin in cultures of immobilised cells had effects similar to those above. Using a fed-batch mode with immobilised cells cultured in the presence of resin further increased final concentrations of hexanoic acid (up to 19 g dm⁻³) but yields were lower (20–30%) and productivity did not increase. These results show that production of volatile fatty acids can be improved significantly by product stripping onto an anion exchange resin. © 1997 SCI.     

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