Biochemical Engineering Journal (v.48, #2)

BEJ Keywords (II).

With the unique physio-chemical and biological properties, hyaluronic acid (HA) finds a wide range of applications in biomedical, cosmetic and healthcare fields. The microbial HA production is limited by poor mixing performance and low oxygen mass transfer rate. In this work, a novel cavern volume controlled culture model was proposed and applied to microbial HA production by batch culture of Streptococcus zooepidemicus. In the cavern model-controlled culture process, the complete mixing was achieved via the dynamic control of agitation speed in the range from 50 rpm to 470 rpm, and HA yield increased from 5.5 ± 0.1 g/L of the control to 6.2 ± 0.1 g/L. There was not significant influence of the model-controlled culture on the molecular weight of HA compared with the control, in which the agitation speed was kept at 294 rpm. With better mixing performance and improved oxygen mass transfer efficiency, the cavern volume controlled culture model may be helpful for the production of the other microbial biopolymers like xanthan gum, curdlan and poly(glutamic acid).
Keywords: Hyaluronic acid; Streptococcus zooepidemicus; Rheology; Mixing; Batch processing; Oxygen transfer;

Hyaluronic acid (HA) is a linear high molecular weight glycosaminoglycan polymer with its molecular weight determining its physiological role, rheological properties and applications. The commercially used microbial source—Streptococcus zooepidemicus is incapable of synthesizing very high molecular weight polymer with low polydispersity, an essential niche in the HA market. In this organism, three important metabolic processes—glycolysis, hyaluronic acid synthesis and biomass formation compete for carbon source, nitrogen source, energy and precursors to determine the molecular weight of the synthesized polymer. After studying the role of culture conditions on these competing processes, it was found that the best strategy for enhancing molecular weight was to weaken the glycolytic process, strengthen HA synthesis process and maintain nitrogen under limiting condition to reduce the rate of biomass formation. Based on this newly developed strategy—temperature switches, addition of precursor N-acetylglucosamine and addition of pyruvate experiments enhanced the molecular weight of the polymer by 23%, 74% and 64%, respectively. Improving precursor levels, particularly UDP-N-acetylglucosamine and thereby strengthening the HA flux plays a direct role in improving molecular weight of hyaluronic acid.
Keywords: Hyaluronic acid; Streptococcus zooepidemicus; Metabolic flux analysis; Biopolymer; Kinetics; Stirred tank;

The fermentation of syngas (CO, CO2, and H2) produced from biomass gasification for the production of ethanol has received increased attention due to the low cost and abundance of cellulosic feedstocks. Since CO plays a critical role in the available reducing equivalents and carbon conversion, this work assessed the effects of constant CO partial pressure (P CO), ranging from 0.35 to 2.0 atm, on cell growth, acetic acid production, and ethanol production using Clostridium carboxidivorans P7T. Several key findings included: (a) the maximum cell concentration increased with increasing P CO, increasing 440% with a P CO increase from 0.35 to 2.0 atm, (b) ethanol production changed from non-growth-associated to growth-associated with increasing P CO, (c) acetic acid production (gram acetic acid per gram cells) decreased for P CO  ≥ 1.05 atm relative to P CO  ≤ 0.70 atm, and (d) acetic acid appeared to be converted in the latter growth stages for P CO of 1.35 and 2.0 atm. Several explanations point to the potential importance of P CO and the P CO to P C O 2 ratio on electron and ATP production. Since gasification processes that generate syngas could result in differing gas partial pressures, process variations could significantly change growth and product formation as evidenced by metabolic changes observed in this work due to changing P CO and/or the P CO to P C O 2 ratio.
Keywords: Biomass; Biofuels; Bioresources; Syngas; Carbon monoxide; Ethanol;

Biodegradation kinetics and modeling of whey lactose by bacterial hemoglobin VHb-expressing Escherichia coli strain by Muayad M. Abboud; Isam H. Aljundi; Khaled M. Khleifat; Saif Dmour (166-172).
The batch fermentation of cheese whey lactose was achieved using Escherichia coli:pUC8:16 recombinant strain that was transformed with Vitreoscilla hemoglobin gene(vgb). In this process, 70% of the initial whey lactose was biodegraded during 24 h of incubation time. Biodegradation was accompanied with a turnover of glucose intermediate and a production of lactic acid. Total lactic acid produced by this recombinant strain was 57.8 mmol/L compared with a reference lactic acid producing strain, Lactobacillus acidophilus, that yielded only 55.3 mmol/L of lactic acid from the same initial whey lactose concentration. The engineering of vgb gene transformation in E. coli strain has led to increase in bacterial biomass and boosted lactic acid production, relative to other strains that lack the vgb gene like E. coli:pUC9 or E. coli wild type or Enterobacter aerogenes. Contrary to Monod's, Haldane's model gave a good fit to the growth kinetics data. Kinetic constants of the Haldane equation were μ m  = 0.5573 h−1, K s  = 4.8812 g/L, K I  = 53.897 g/L. Biomass growth was well described by the logistic equation while Luedeking–Piret equation defined the product formation kinetics. Substrate consumption was explained by production rate and maintenance requirements. In simulation studies including the Haldane model, an evident agreement was observed between measured and calculated biomass, product, and substrate concentrations.
Keywords: Whey lactose biodegradation; E. coli strain VHb; Fermentation; Kinetic models; Haldane equation;

Optimum protease production of 518 U by Bacillus subtilis DM-04 in submerged fermentation was attained by response surface method. An alkaline protease, exists as zwitterionic form at pH 7.0 was purified to 23.5-fold by a combination of cation and anion exchange chromatography, ethanol precipitation followed by reverse-phase HPLC. The purified protease (Alzwiprase) contributes 29.0% of overall extracellular proteases of B. subtilis DM-04, has a subunit molecular mass of 16.9 kDa and exists as a monomer. It shows optimum activity at 45 °C and pH 10.0, respectively. The K m and V max values of Alzwiprase towards casein were determined as 59 μM and 336 μg min−1, respectively. Irreversible inhibition of enzyme activity of Alzwiprase with serine protease inhibitors demonstrates that it belongs to serine protease family, more particularly endopeptidase K and/or subtilisin-like protease. The significant stability and compatibility towards organic solvents, urea, surfactants, commercial laundry detergents as well as excellent stain removal and dehairing properties of Alzwiprase hold a tremendous promise for its industrial application.
Keywords: Enzyme; Submerged; Microbial; Bacillus subtilis; Chromatography; Protease; Kinetics; Solid-state fermentation; Optimization;

Adsorptive removal of cobalt from aqueous solution by utilizing lemon peel as biosorbent by Amit Bhatnagar; A.K. Minocha; Mika Sillanpää (181-186).
The present study was undertaken to evaluate the feasibility of lemon peel waste for the removal of cobalt ions from aqueous solutions. Batch experiments were performed to study the adsorption of cobalt on lemon peel adsorbent. The maximum adsorption capacity of lemon peel adsorbent for cobalt removal was ca. 22 mg g−1. Three simplified kinetic models viz. pseudo-first-order, pseudo-second-order, and Weber and Morris intraparticle diffusion models were tested to describe the adsorption process. Kinetic parameters, rate constants, equilibrium sorption capacities, and related correlation coefficients for kinetic models were determined. It was found that the present system of cobalt adsorption on lemon peel adsorbent could be described more favorably by the pseudo-second-order kinetic model. The adsorption process has been found to be exothermic. The results of the present study suggest that lemon peel waste can be used beneficially in treating industrial effluents containing heavy metal ions.
Keywords: Waste treatment; Heavy metals; Cobalt removal; Lemon peel waste; Adsorption; Biosorption;

Preparation and biological characterization of cellulose graft copolymers by Wassila Dahou; Djamila Ghemati; Atika Oudia; Djamel Aliouche (187-194).
Acrylic acid (AA) and acrylonitrile (AN) were graft polymerized onto cellulose fluff pulp using ceric ammonium nitrate as initiator. The resulting copolymers were saponified with dilute sodium hydroxide and characterized by FT-IR, SEM and TGA. The potential value of the modified cellulose was assessed through measurements of absorbency properties. A fibre-hydrogel was prepared by an addition of a bifunctional monomer, ethyleneglycol dimethacrylate (EDMA) used for grafting. In second approach, biocide cellulose carbamate was prepared by impregnating the fibres in aqueous thiourea solution and subsequent grafting with acrylonitrile. Antimicrobial activity of the treated cellulose sample was studied against Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis according to AATCC test method 100–1999. The results show that the treated fibre gives higher antimicrobial activity. The strong antimicrobial functions achieved on modified fibres, proved that the synthesized biomaterial was effective, very simple and practical to the textile finishing industry.
Keywords: Cellulose carbamate; Graft polymerization; Acrylic acid; Acrylonitrile; Absorbency; Antimicrobial activity;

A model-based investigation of the potential advantages of multi-layer packed beds in solid-state fermentation by David Alexander Mitchell; Lara Elize Nascimento Cunha; Alex Vinicius Lopes Machado; Luiz Fernando de Lima Luz; Nadia Krieger (195-203).
We use a mathematical model, based on the N-tanks-in-series approach, to evaluate the potential advantages that can be obtained in solid-state fermentation processes by operating packed-bed bioreactors as multi-layer beds. We explore classical operation, in which air is blown unidirectionally through a substrate bed that remains immobile, with two strategies that involve movement of the layers. The first strategy involves batch operation, in which the positions of the layers are changed at 1 h intervals, in a cycling motion. The second strategy involves continuous plug-flow of the layers, with the regular addition of new layers at the air outlet and removal of spent layers at the air inlet. Under the conditions of the simulation, the rate of metabolic heat generation during the steady state of the continuous plug-flow process is only 60% of the peak value predicted for classical operation. As a result, the maximum bed temperature in the continuous plug-flow process is 4.5 °C lower than that predicted for classical operation. We conclude that the operation of multi-layer packed beds in the continuous plug-flow mode can improve bioreactor performance significantly.
Keywords: Bioreactor systems; Solid-state; Heat transfer; Mass transfer; Packed-bed bioreactors; Mathematical modeling;

Recombinant Saccharomyces cerevisiae strains constructed by metabolic engineering approaches can ferment xylose but with low efficiency. We constructed a S. cerevisiae strain via combined approaches of recombinant DNA technology, chemical mutagenesis and evolutionary adaptation for an efficient xylose utilization and ethanol fermentation. A haploid derivative of an industrial ethanol-fermenting S. cerevisiae strain was first engineered to express the XYL1 and XYL2 genes from Pichia stipitis, encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, and the endogenous XKS1 gene, encoding xylulokinase (XK). This recombinant strain, LEK122, was then subjected to EMS mutagenesis followed by adaptive evolution, resulting in a single isolate, LEK513, which displayed significantly improved xylose-utilizing property. The specific growth rate of the LEK513 strain was 0.225 h−1 under aerobic condition (0.205 h−1 under oxygen-limited condition) with xylose as the sole carbon source, while that of the LEK122 was 0.055 h−1. During 100 h batch cultivation, the optical density of LEK513 reached 60, while LEK122 only grew to 7.5. In the same time period, LEK513 consumed 95% of the xylose in the medium, while LEK122 only consumed 20% of that. The LEK513 strain produced 11% more ethanol in oxygen-limited fermentation than it did in aerobic fermentation.
Keywords: Ethanol; Evolutionary adaptation; Mutagenesis; Saccharomyces cerevisiae; Xylose;

Characterization of a glucosyltransferase from Erwinia sp. D12 and the conversion of sucrose into isomaltulose by immobilized cells by Haroldo Y. Kawaguti; Érica M. Celestino; Ana L.L. Moraes; Dong K. Yim; Larissa K. Yamamoto; Hélia H. Sato (211-217).
Erwinia sp. D12 is able to produce an isomaltulose synthase (EC 5.4.99.11) that converts sucrose into isomaltulose. The enzyme was partially purified using a two-step chromatographic process on DEAE-Sephadex A-50 and DEAE-Sepharose CL-6B. The molecular mass of 63 kDa was estimated by Sephadex G-200 gel filtration, and the K m and V max values determined for the enzyme were 138 mM and 9.81 μmol/min/mg protein with sucrose as the substrate, respectively. Enzyme activity was optimal at pH 6.0 and 40 °C. The glucosyltransferase was completely inhibited by Hg2+ and Ag+. An experimental design and response surface methodology were used to evaluate the influences of temperature, pH and substrate concentration on isomaltulose production from cells immobilized in chitosan. With the aid of a two-level full factorial design (23-FFD), the statistical analysis of the results showed that, in the range studied, the factors had a significant (p  < 0.05) effect on isomaltulose production. The conditions that improved isomaltulose production were: temperature around 35 °C, pH 6.0 and sucrose concentration lower than 40%.
Keywords: Enzyme; Chitosan; Immobilized cell; Isomaltulose; Microbial; Shake-flask;

To investigate the characteristics and values of plant proteins for lignocellulose conversion, cellulase synergetic proteins for lignocellulose hydrolysis were separated from fresh corn stover. The synergetic proteins have strong hydrophobic characterization, yet no cellulose hydrolytic activity. Mass spectrometry analysis indicated that the hydrophobic protein with highest abundance might be cell membrane protein of Zea mays. Compared with steam-exploded corn stover hydrolysis with microbial cellulase mixture, the glucose production was improved by 31.88% with hydrophobic proteins addition. Synergetic mechanism analysis indicated that the hydrophobic proteins reduced unspecific cellulase adsorption onto non-cellulose component of lignocelluloses during hydrolysis. The effect is more significant for exoglucanase and β-glucosidase. The amount of hydrophobic proteins in fresh corn stover is large. Therefore, supplementing microbial cellulase complex with hydrophobic proteins of plant for lignocellulose hydrolysis is a promising way for cost reduction in lignocellulose conversion.
Keywords: Cellulase; Hydrophobic proteins; Corn stover; Synergism; Lignocellulose; Enzymatic hydrolysis;

The emergence of antibiotic-resistant mutants among pathogenic bacteria has re-focused interest in alternative antibacterial treatments such as “phage therapy”, where viruses are harnessed to infect and destroy bacteria included in their host range. The first stage in bacteriophage multiplication, its adsorption to the bacterial cell surface, has not been accurately resolved before. Previous studies focused on very low phage-to-bacteria concentration ratios. In this study, detailed kinetics of T4 adsorption to Escherichia coli B/r were obtained with high sampling frequency during the first 6 min, with suspensions of nearly the same initial number of phages and bacteria. The results were used to analyze several optional models and to choose the most suitable, based on simplicity and best fit to the data. It was found that simple, mono-attachment models adequately fit the experimental data.
Keywords: Phage therapy; Adsorption; Bacteria; Kinetics; Mathematical modeling; Dynamic simulation;

The effect of nitrogen (N)-limitation on the metabolism of the wild type Escherichia coli BW25113, its glnG, and zwf mutants was investigated based on the chemostat cultures at the dilution rate of 0.2 h−1 under both carbon (C)-limitation and N-limitation. It was shown that gdhA gene expression was down-regulated, and glutamine synthetase-glutamate synthase (GS/GOGAT) pathway was mainly utilized under N-limitation in E. coli BW25113. It was also shown that the specific glucose consumption rate and the specific CO2 evolution rate (CER) were higher in glnG mutant as compared to the wild type under C-limitation. It was also shown that GOGAT is the main pathway for N assimilation under C-limitation, while GDH is the main pathway under N-limitation in glnG mutant. In zwf mutant, glnA, glnG, and gltD gene expressions were significantly down-regulated, while malic enzyme was significantly activated for NADPH production under both C-limitation and N-limitation.
Keywords: Escherichia coli; glnG knockout; Glutamate synthesis pathway; Nitrogen assimilation; zwf knockout;

Improved determination of tramadol hydrochloride in biological fluids and pharmaceutical preparations utilizing a modified carbon paste electrode by Hazem M. Abu-Shawish; Nasser Abu Ghalwa; Faried R. Zaggout; Salman M. Saadeh; Ayoub R. Al-Dalou; Anwar A. Abou Assi (237-245).
A simple, rapid and sensitive method for the determination of tramadol hydrochloride in urine, milk and pharmaceutical preparations using two modified carbon paste electrodes was developed. One electrode (sensor A) is based on ion-association of tramadol hydrochloride with phosphotungstic acid (TD-PT) and the other (sensor B) with a mixture of phosphotungstic acid (TD-PT) and silicomolybdic acid (TD-SM). Among seven different solvent mediators tested, 2-nitrophenyl octyl ether (NPOE) exhibited a proper behavior including Nernstian slopes of the calibration curve at 57.8 ± 0.4 and 56.5 ± 0.8 mV per decade for sensors A and B. The response times were 8 and 5 s; detection limits 6.2 × 10−6 and 1.8 × 10−6  M; the concentration range 9.2 × 10−6 to 1.0 × 10−1  M and 5.5 × 10−6 to 1.0 × 10−1  M respectively. The present electrodes show good discrimination of tramadol hydrochloride from several inorganic, organic ions, sugars and some common drug excipients. The sensors were applied for the determination of tramadol hydrochloride in urine, milk and pharmaceutical preparations using potentiometric determination, standard addition and the calibration curve methods. The results obtained were satisfactory with excellent percentage recovery comparable and sometimes better than those obtained by other routine methods for the assay.
Keywords: Tramadol hydrochloride; Carbon paste electrode; Potentiometry; Ion-selective electrode;

Ethyl butyrate is a fruity flavor ester widely used in food and pharmaceutical products. The synthesis of ethyl butyrate in n-hexane, catalyzed by Candida rugosa lipase immobilized in two hydrophilic polyurethane foams (“HYPOL FHP 2002” and “HYPOL FHP 5000”) was performed. In this study, the effects of (i) the immobilization supports, (ii) the initial substrate concentrations and (iii) the water content of the system, on the activity and operational stability of C. rugosa lipase in both foams, during the esterification in continuous packed-bed reactor (PBR) and in repeated batches, were investigated. When low substrate concentrations were used, no deactivation was observed for both biocatalysts, along the continuous 30-d PBR operation. Conversely, under high substrate concentrations, a fast deactivation of the biocatalysts was observed. In consecutive batches, the deactivation was faster for the lipase in the less hydrophilic foam (“FHP 5000”) with a half-life of 53 h against 170.3 h for the other counterpart. Water molecules in the microenvironment did not present a deactivation effect on the biocatalysts. The low operational stability can be ascribed to the inhibitory effect of ethanol, which tends to accumulate inside the foams.
Keywords: Operational stability; Immobilized lipase; Polyurethane foams; Batch reactor; Continuous packed-bed reactor;

Binding activity of recombinant human L-selectin-Fcγ is modified by sialylation by Sven Enders; Sebastian B. Riese; Gesche Bernhard; Jens Dernedde; Werner Reutter; Rudolf Tauber (253-259).
The adhesion molecule L-selectin expressed on most leukocytes mediates tethering and rolling of leukocytes on activated endothelia and initiates the extravasation of leukocytes into inflamed tissues. Recombinant L-selectin-Fcγ is widely used both as a tool to study this key step of inflammation and as an anti-inflammatory compound in animal models of inflammation. Since previous studies on cellular L-selectin have indicated that glycosylation influences adhesive interactions of the adhesion molecule, we have examined whether the binding activity of L-selectin-Fcγ is affected by sialylation. Different forms of recombinant human L-selectin-Fcγ were expressed in CHO and K-562 cells and were purified by affinity chromatography using Protein A-Sepharose. A hypersialylated form of L-selectin-Fcγ was generated by culturing cells in the presence of 5 mM N-acetyl-beta-d-mannosamine, while a desialylated variant was obtained by treatment of purified L-selectin-Fcγ with neuraminidase. Binding activity to the synthetic biligand SiaLex-PAA-sTyr was measured by surface plasmon resonance (SPR) technology. While hypersialylated L-selectin-Fcγ showed decreased binding activity, desialylation elevated L-selectin-Fcγ binding to SiaLex-PAA-sTyr. The data show that sialylation of L-selectin-Fcγ reduces binding activity to ligand epitopes containing sialyl Lewis x and sulfated tyrosine residues. For the production of biologically active L-selectin-Fcγ conditions should be chosen that favor the generation of non-sialylated or of scarcely sialylated forms of the recombinant glycoprotein.
Keywords: L-selectin; Neuraminidase; Recombinant protein production; Sialic acid; Sialylation; Surface plasmon resonance;

A high amylase producing Bacillus subtilis WD 161 was used in a co-culture with Clostridium butylicum TISTR 1032 to enhance acetone–butanol–ethanol (ABE) production from starch. The mixed culture of C. butylicum TISTR 1032 and B. subtilis WD 161 without anaerobic pretreatment by reducing agent and N2 flushing increased amylase activity 10 folds and enhanced ABE production 5.4 and 6.5 folds from soluble starch and cassava starch, respectively, compared to those of the pure culture of Clostridium itself. The medium optimization for ABE production by the mixed culture without anaerobic pretreatment found that cassava starch concentration of 40 g/L, C/N ratio of 4.35 and the mixed nitrogen sources of 265 mM yeast extract and 100 mM NH4NO3 gave the highest ABE production in terms of final concentration and productivity. The benefits of using this high amylase producing aerobic Bacillus in a co-culture with anaerobic Clostridium were not only increasing substrate utilization and ABE production but there was also no requirement to add any costly reducing agent to the medium or flushing with N2 to ensure anaerobic condition. This thus makes the anaerobic fermentation more economical and cost effective. This co-culture system may contribute greatly to developing industrialized ABE production.
Keywords: Acetone–butanol–ethanol fermentation; Bacillus subtilis; Cassava starch; Clostridium butylicum; Mixed culture;