Biochemical Engineering Journal (v.42, #1)

BEJ Keywords (II).

An enzyme-coupled system, containing a ketoreductase for prochiral ketones reduction and a mannitol dehydrogenase for NADPH regeneration, was discovered from the cell-free extract of Rhodotorula sp. AS2.2241 and used successfully for the asymmetric reduction of 2-acetylpyridine, giving (S)-1-(2-pyridine)ethanol in >99% ee with recycling of NADPH for 4220 times. Moreover, the endogenously coupled enzymes system could also reduce various aromatic ketones such as 3- and 4-acetylpyridines with 96–99% ee.
Keywords: Biocatalysis; Biotransformations; Chromatography; Optimization; NADPH regeneration; Rhodotorula sp. AS2.2241 ketoreductase;

Enzymatic synthesis of citronellol laurate in organic media and in supercritical carbon dioxide by Maja Habulin; Saša Šabeder; Miguel Acebes Sampedro; Željko Knez (6-12).
Short-chain esters of carboxylic acids and monoterpene alcohols are very important compounds in food, cosmetic and pharmaceutical industries. Lipase-catalyzed esterification of β-citronellol and lauric acid was performed in organic solvents and in supercritical carbon dioxide (SC CO2). The optimal conditions found in organic solvents at atmospheric pressure were: an equimolar ratio of substrates, a temperature of 60 °C, an agitation speed of 500 rpm, 12% (w/w of substrates) enzyme preparation of lipase B from Candida antarctica. The highest conversion in SC CO2 was obtained at 60 °C and 10 MPa with ethyl methylketone serving as a co-solvent.
Keywords: Citronellol laurate; Biocatalysis; Immobilized enzymes; Lipase; Food engineering; Supercritical carbon dioxide;

In Escherichia coli, there are two major pathways, i.e. Cpx and σ E, for dealing with the extracytoplasmic stress in the cell envelope. Due to the unique periplasmic processing steps and the tendency to form periplasmic inclusion bodies, penicillin acylase (PAC) offers a model system for studying the induction of extracytoplasmic stress associated with recombinant proteins overproduction in the periplasm of E. coli. In this study, E. coli strains carrying the lacZ reporter gene fusion with the promoters of three stress-responsive genes, i.e. degP, cpxP, and rpoH, were constructed in the JM109 background for characterization. We demonstrate that pac overexpression induced the extracytoplasmic stress primarily via the Cpx pathway. The upregulated cpxP promoter activity can be a suitable sensor for in vivo monitoring of the extracytoplasmic stress upon pac overexpression. However, such physiological challenge was not observed and all the three promoter activities were reduced when arabinose was used to induce pac overexpression. This result suggests that the physiological impact observed for the IPTG (isopropyl-β-d-thiogalactopyranoside)-induced cultures could be overcome by the use of arabinose for induction. The extracytoplasmic stress response associated with pac overexpression could be significantly alleviated by the exogenous presence of DegP, but only partially alleviated by its mutant derivative of DegPS210A.
Keywords: Chaperone; Inclusion body; Penicillin acylase; Periplasm; Physiology; Stress regulons;

Cellular structure in an N-acetyl-chitosan membrane regulate water permeability by Tomoki Takahashi; Masanao Imai; Isao Suzuki (20-27).
A novel model of the water permeation mechanism in an N-acetyl-chitosan membrane with a cellular structure is proposed. Although the entire membrane structure has a hydrophilic character, the cellular structure incorporates junction zones that practically prevent water permeation. Chitosan membranes with a controlled degree of deacetylation (DD) were prepared using a casting method. Changes in the water flux and total water content of the membrane were observed with a change in DD. The membrane properties were analyzed and evaluated using water permeability measurements, scanning electron microscopy (SEM), X-ray diffraction (XRD), and differential scanning calorimetry (DSC). SEM observations indicated that the membrane structure was an individual cellular structure and that this cellular structure grew with decreasing DD. XRD measurements indicated the crystal structure of the membrane was amorphous regardless of the DD in the experimental range. The free water content (W f), the freezable bound water (W fb), and the bound water not able to freeze (W b) were evaluated by DSC. The free water mainly contained inside the cellular structure, and resulted in swelling the chitosan membrane. Water flux was measured using ultrafiltration apparatus; it was dependent on the operational pressure, membrane thickness, and the feed solution viscosity, and obeyed the Hagen–Poiseuille flow. At a higher DD, water permeation proceeds due to degradation of the cellular structure; the amount of water in permeation channels was greater than that for lower DD membranes even though the total water content in the membrane was less. The water flux of the chitosan membrane was determined by the water content constructing channels through the membrane and not on the total water content in the membrane.
Keywords: N-Acetyl-chitosan; Membrane; Deacetylation degree; Cellular structure; Water permeability; Novel permeation mechanism;

The less active Gentamicin C1 have been selectively removed from the biosynthetic mixture containing Gentamicins C1, C1a, C2 and C2a by facilitated pertraction with D2EHPA dissolved in dichloromethane as the liquid membrane U-shaped pertraction cell. The pertraction has been analyzed by means of initial and final mass flows, permeability and selectivity factors. The main factors, which control the pertraction selectivity, were identified to be the pH-gradient between the feed and stripping phase, mixing intensity of the aqueous phases and carrier concentration in the membrane phase. The maximum selectivity factor has been recorded for pH 8 of feed phase, pH 3 of stripping phase, rotation speed lower than 100 rpm, and carrier concentration of 10 g/l.
Keywords: Gentamicin; Antibiotic; Bioseparations; Liquid membranes; Liquid–liquid extraction; Pertraction;

Optimisation of olive oil extraction by means of enzyme processing aids using response surface methodology by Bahar Aliakbarian; Danilo De Faveri; Attilio Converti; Patrizia Perego (34-40).
The aim of the present work was to optimise the oil extraction process of the Italian olive fruits of Coratina cultivar using a previously selected enzyme formulation. To this purpose, the combined effects of the malaxation time (t) and the concentration of the enzyme formulation (C E), added at the beginning of the malaxation step, were investigated by a 32 full factorial design combined with response surface methodology, selecting the concentration of total polyphenols (TP) and o-diphenols (OD), the oil extraction yield (Y) and the antiradical power (ARP) as the response variables. The model enabled us to identify the optimum operating conditions (t  = 91.64 min and C E  = 25.00 mL/kgpaste), under which it predicted TP = 844.30 μgCAE/goil; OD = 120.80 μgCAE/goil; ARP = 24.93 μgDPPH/μLextract and Y  = 15.72 goil/100 gpaste. These results, which were confirmed by additional tests performed within the selected ranges of variables, suggest that the use of the proposed enzyme treatment could enhance quantity and quality levels of the product.
Keywords: Olive oil; Phenolics; Enzymes; Optimisation; Modelling;

For the complete hydrolysis of cellulose, the cellulolytic fungi produce a whole set of commercially important enzymes called cellulases. The aim of this work was to investigate an approach to enhance the production of these enzymes by co-culturing Trichoderma reesei and Aspergillus niger in a bioreactor to convert cellulose substrate into soluble sugars through a synergetic action of enzyme complex simultaneously produced by these two fungi. The experiments were conducted as fed batch growth on a Cellulose–Yeast extract medium. A mixture of lactose and lactobionic acid was added into the bioreactor as cellulase inducers. The results of mixed culture experiments exhibited a highly significant increase in the production of volumetric enzyme activity (98.4 U L−1  h−1), filter paper activity (7.1 U mL−1), carboxymethyl cellulase activity (4.7 U mL−1), soluble proteins (2.1 mg mL−1), dry biomass (21.4 g−1  L−1), and percentage of utilized cellulose (89.4%) as compared with A. niger monocultures.
Keywords: Cellulose; Lactose; T. reesei; A. niger; Cellulase enzymes; Mixed culture;

The aim of this study was to determine if preliminary treatment of melanoidin based wastewater by an advanced oxidation process (AOP) made the associated DOC and DON more biodegradable. UV-C irradiation of H2O2 was used to create the hydroxyl radical for chemical oxidation, while aerobic batch bioassays were used to assess the subsequent biodegradability of the DOC and DON associated with melanoidin. State-of-the-art natural organic matter identification techniques were used to identify melanoidin and study the subsequent degradation products throughout the chemical and biological oxidation processes.Melanoidins are large (50–70 kDa), coloured, nitrogenous organic compounds that are refractory to sewage treatment plant (STP) biodegradation, but are susceptible to degradation by sunlight in receiving environments. This study showed that over an extended chemical oxidation period (72 h, which was sufficient to reach maximum chemical degradability) DOC and DON removal from a synthetic melanoidin wastewater were 92% and 48%, respectively. In comparison, the DOC and DON removal after 4 h of chemical oxidation followed by 7 days of aerobic biodegradation were 82% and 86%, respectively. Thus, the removal of organic bound nitrogen was drastically improved by using a combination of chemical and biological oxidation in place of the hydroxyl radical AOP alone.Molecular weight fractionation results showed that the cleavage of the large nitrogenous melanoidin molecules, during chemical oxidation, produced small DON molecular weight compounds (<1 kDa) and ammonia, which were readily biodegradable. It was apparent that the small molecular weight DOC formed during the hydroxyl radical oxidation of melanoidin was biodegradable, but unlike the DON could also be chemically oxidised given sufficient exposure time to the hydroxyl radical.
Keywords: Aerobic process; Waste-water treatment; Biodegradation; Colour; Filtration; Chemical treatment;

Schisandrin B (Sch B), consisting of a mixture of its stereoisomers, namely (−)Sch B and (±)γ-schisandrin, is the most abundant and biologically active dibenzocyclooctadiene lignan present in Fructus Schisandrae (FS). The objective of this study is to develop a process for large-scale separation and purification of a single stereoisomer of Sch B, (−)Sch B, which offers the highest desirable bioactivities. To this end, a crystallization-based separation and purification process has been conceptualized. Bench-scale crystallization experiments guided by experimental solid–liquid equilibrium phase diagrams were performed to verify process feasibility. A (−)Sch B product with a purity of 98.5 wt% and a (±)γ-schisandrin-enriched product with a purity of 65.0 wt% were obtained. The (−)Sch B product caused a 32% increase in cellular glutathione level and the (±)γ-schisandrin-enriched product a 26% increase, indicating a potentially more efficacious pharmaceutical preparation.
Keywords: Separation; Purification; Stereoisomers; Crystallization; Process integration; Bioprocess design;

Trametes versicolor pellets production: Low-cost medium and scale-up by Eduard Borràs; Paqui Blánquez; Montserrat Sarrà; Glòria Caminal; Teresa Vicent (61-66).
A cost analysis of a decolourization process using the ligninolytic fungus Trametes versicolor in pellet form was carried out. It established that the key to making the process cost-efficient was to reduce the cost of the culture medium for pellet production, which accounts for over 95% of the total cost of the process, due to the high price of malt extract. A cheaper defined medium was formulated in order to obtain spherical pellets approximately 3 mm in diameter. The pH of the medium played an important role in pellet production. Experiments were therefore conducted to allow the control of the pH. A stirred-tank bioreactor (1 L) was ruled out, since problems related to oxygen transfer and types of agitation have been shown to exert a great influence on pellet formation. Use of air-pulsed bioreactors solved these problems, allowing both pellet production and scale-up of the process in a 10 L air-pulsed bioreactor. The new pellet production process reduced the total cost by up to 94.4% per unit volume of wastewater treated. In a decolourization test the pellets obtained from the new process showed an outstanding performance.
Keywords: Bioreactor; Scale-up; White-rot; Filamentous fungi; Wastewater treatment; Economic study;

Application of Brazilian-pine fruit coat as a biosorbent to removal of Cr(VI) from aqueous solution—Kinetics and equilibrium study by Julio C.P. Vaghetti; Eder C. Lima; Betina Royer; Jorge L. Brasil; Bruna M. da Cunha; Nathalia M. Simon; Natali F. Cardoso; Caciano P. Zapata Noreña (67-76).
In the present study we reported the feasibility of the Brazilian-pine fruit coat, named piñon wastes (PW; Araucaria angustifolia) as biosorbent to remove Cr(VI) from aqueous solutions.The PW biosorbent was characterized by N2 adsorption–desorption isotherms, FTIR spectroscopy, scanning electron microscopy, elemental analysis, mineral composition determination, and functional groups detection.The ability of PW to adsorb Cr(VI) was investigated by using batch adsorption procedure. The effects such as pH, contact time and biosorbent dosages on the adsorption capacity were studied. The adsorption kinetics followed the Elovich chemisorption kinetic model, obtaining the following the initial adsorption rate, 284.9, 396.9 and 461.5 mg g−1  h−1 using a 500.0, 700.0 and 1000.0 mg L−1 initial concentration of Cr(VI), respectively. The maximum adsorption capacity of PW was 240.0 mg g−1 for Cr(VI), using Sips isotherm model. This high adsorption capacity of PW places this biosorbent as one of the best adsorbents for removal of Cr(VI) from aqueous effluents.
Keywords: Brazilian-pine fruit wastes; Biosorbent; Cr(VI); Adsorption; Nonlinear isotherm fittings; Adsorption kinetics;

Monitoring of sorbitol in Pichia pastoris cultivation applying sequential injection analysis by Burkhard Horstkotte; Carolina Arnau; Francisco Valero; Olaf Elsholz; Víctor Cerdà (77-83).
A simple and robust analyser system based on sequential injection analysis for the determination of sorbitol in Pichia pastoris MutS phenotype batch fermentation is described. Applicability for the bioprocess monitoring of sorbitol in batch culture medium in a concentration range of 8–1.5 g l−1 with satisfying sorbitol recovery was demonstrated during 60 h. Optimization of physical and chemical parameters is further described. The method proved to be reproducible (R.S.D. < 3%), robust, and fast (17 injections per hour). The SIA sorbitol analyzer showed to be applicable for the monitoring of sorbitol concentration in a mixed substrate P. pastoris fermentation strategy. No interference of methanol was observed. The method is suited for the implementation of sorbitol concentration closed-loop control strategies and for the study of the effect of sorbitol concentration in heterologous protein productivity.
Keywords: Sequential injection analysis; Sorbitol; Batch cultivation; Pichia pastoris; Mixed substrates;

Dilute sulfuric acid pretreatment of cardoon for ethanol production by Ignacio Ballesteros; Mercedes Ballesteros; Paloma Manzanares; M. José Negro; J. Miguel Oliva; Felicia Sáez (84-91).
Cardoon (Cynara cardunculus L.) is an herbaceous species originating in the Mediterranean area that can be considered as a potential lignocellulosic feedstock for biofuels and high added value products.In this work, cardoon biomass (stems and leaves) was pretreated with dilute sulfuric acid in a batch reactor. The effect of temperature (160–200 °C), acid loading (0–0.2%, w/w) and solid/liquid ratio (5–10%, w/v) on overall sugar yields was studied using a response surface method according to a Box–Behnken experimental design. Overall xylose yield reaches a maximum of 90% of the content in raw material at 180 °C and 0.1% (w/v) acid addition and 7.5% (w/v) solid concentration. Regarding overall glucose yield, best results were achieved at 200 °C, 0.2% (w/w) acid loading and 7.5% (w/v) solid concentration. According to statistical optimization used optima conditions to maximize both overall glucose and xylose yield were 184.5 °C and 0.15% (w/v) acid concentration. At these conditions, 70% and 93% of glucose and xylose, respectively, could be recovered. The highest simultaneous saccharification and fermentation (SSF) yields are obtained when biomass was pretreated at 200 °C with 0.2% sulfuric acid reaching values close to 65% of theoretical.
Keywords: Bioconversion; Ethanol; Simultaneous saccharification and fermentation; Pretreatment; Cynara cardunculus;

Bioactivity of solanesol extracted from tobacco leaves with carbon dioxide–ethanol fluids by Wen Huang; Zhenshan Li; Hai Niu; Jiawen Wang; Yuan Qin (92-96).
The central composite design combined with response surface methodology was used to optimal operating parameters of ethanol-modified supercritical CO2 extraction and evaluate the effects of operating parameters on crude-solanesol yield and extract anti-free-radical activity (DPPH radical scavenging activity). The results show that pressure, temperature and time appeared independent and interactive effects on the crude solanesol yield as well as the extract anti-free-radical activity. The optimal conditions for crude-solanesol yield were 45 °C, 38 MPa and 2.6 h, while that for the extract anti-free-radical activity 47 °C, 36 MPa and 2.5 h. Under the optimal conditions, the highest solanesol extracted could arrived at 3.74 wt% with 52.3 wt% purity (96.8% extraction ratio), and the maximal extract anti-free-radical activity 57.49%. Meanwhile, it was found that the extract anti-free-radical activity was correlated very well with the crude solanesol yield, resulting in model with high coefficient of determination (R 2  = 0.946). The research demonstrates that ethanol-modified supercritical CO2 extraction conducted by the central composite design combined with response surface methodology, which could produce high crude solanesol yield with high quality as well high level of bioactivity, is a prospective alternative to the traditional heat-reflux extraction.
Keywords: Solanesol; Ethanol-modify; Supercritical CO2 extraction; Tobacco leaves; Bioactivity;

Ultrasonic extraction of waste solid residues from the Salvia sp. essential oil hydrodistillation by Dragan T. Veličković; Dragan M. Milenović; Mihailo S. Ristić; Vlada B. Veljković (97-104).
Enormous quantities of aromatic plants such as Salvia sp. are used in the production of essential oils all over the world, thereby creating a huge waste solid residue. Since the spent plant material (SPM) may contain biological compounds, it could be exploited as secondary raw material for obtaining different bioactivities using solvent extraction, with possible technological, economical and ecological justification. A novel extraction technique, namely the ultrasonic extraction, and two extracting solvents of distinct polarity (petroleum ether and 70% (v/v) aqueous ethanol solution) were used to obtain extractive substances (ES) from the SPM of two Salvia sp. The investigation was focused on the ES yield, the extraction kinetics and the composition of the final extracts obtained. The maximum concentration of ES in the liquid extracts was reached after about 20–30 min of sonication. The kinetics of ultrasonic extraction was described by the unsteady-state diffusion through the plant material and the film theory. The chemical composition of the extracts depended on the extraction conditions and the type of the plant material. Many biologically active compounds were detected in the extracts. So far the “waste” SPM from the Salvia sp. essential oil hydrodistillation may be considered as a possible source of the exploitable natural products. The benefit of the ultrasound action was related to shortening of the extraction time (60 min) compared to the classical extraction (6 h).
Keywords: Sage; Salvia sp.; Spent plant material; Ultrasonic extraction;