Biochemical Engineering Journal (v.97, #C)


The causative agents of brucellosis from genus Brucella can infect a variety of animal species and human. However, conventional methods for the detection of Brucella are time-consuming and the handling of microorganism is hazardous for laboratory personnel. In this study, a nanobiosensor based on gold nanoparticles (AuNPs) and oligonucleotide probe was designed for the visual detection of Brucella spp.A specific oligonucleotide probe from IS711 gene region was functionalized with AuNPs (AuNP-probe). Then, AuNP-probe was exposed to target (complementary) and non-target (non-complementary) DNA for the hybridization. Also, hybridization conditions were analyzed in the presence of Brucella genomic DNA and extracted DNA from clinical samples.Upon acid addition, a red color for the samples containing complementary DNA was observed, whereas in the samples without complementary DNA, AuNP-probe turned blue-purple. The results were investigated visually and also by UV–vis spectroscopic measurements. This method detected up to 1.09 pg μL−1 of unamplified Brucella genomic DNA. Since Brucella is a facultative intracellular bacteria, clinical samples were amplified by PCR, and colorimetric detections were performed. The results showed high sensitivity and selectivity for the amplified clinical samples.To the best of our knowledge, this developed assay is highly specific, and this is the first assay that can be used with low cost, and as a method of rapid, direct and visual detection of Brucella spp.
Keywords: Gold nanoparticles; Brucella spp.; Biosensors; Immobilization; DNA; Aggregation;

Fabrication of a novel immobilization system and its application for removal of anthracene from soil by Han Xie; Hongying Liu; Yuqin Xie; Minjia Yang; Shanshan Guo; Zhiren Zhou; Heng Xu (8-16).
Display OmittedThe remediation of anthracene (ANT) polluted soil has become a concern due to its difficult diffusion from soil particles and adverse impacts on environmental and human health. To remove ANT from soil efficiently, Ganoderma lucidum (G. lucidum) mycelia pellets combined with corn-cobs were encapsulated in hydrophobically modified Ca-alginate (CA) by poly-ϵ-caprolactone (PCL), without the need for enzyme purification. The optimum PCL concentration in CA/PCL/corn-cobs bead was 12%, which provided enzymes secreted by the G. lucidum mycelia with a good storage stability and a similar activity to the immobilized laccase. Scanning Electron Microscope and Brunauer–Emmett–Teller method were applied to characterize the bead. CA/PCL/corn-cobs ensured the highest ANT removal efficiency regardless of different pH values and temperatures. The ANT removal from soil followed third-order reaction model. Removal of ANT reached 96.2% ± 2.0% from soil after 20 days of incubation at pH 5.0 and 45 °C, which is attributed to the fast secretion of enzymes stimulated by lignocellulosic substances of the corn-cobs, the protection of enzymes by matrix capsules, and the strong adsorption of ANT on the relatively hydrophobic surface of bead. The efficient ANT removal was also observed in aged soil samples simulated by addition of soil organic matter.
Keywords: Diffusion; Biodegradation; Mass transfer; Dynamic modeling; Ganoderma lucidum; Immobilization;

IgG-dependent aggregation of Staphylococcus aureus inhibits bacteriophage attack by Yasunori Tanji; Airi Tanaka; Kaori Tani; Miki Kurimoto; Kazuhiko Miyanaga (17-24).
Staphylococcus aureus causes a variety of diseases, including bovine mastitis. The standard treatment regimen is treating it with antibiotics. However this approach has drawbacks, including high cost and the incidence of antibiotic-resistant bacteria. As a result, there has been renewed interest in the use of bacteriophages (phages) to control S. aureus. However, aggregation facilitates this bacterium’s persistence in the host, and the ability to aggregate has been increasingly recognized as an important Staphylococcus virulence factor. This study revealed that polyclonal bovine IgG enhanced aggregation of S. aureus (SA003), isolated from raw-milk samples from cows with mastitis. IgG-dependent aggregation delayed lysis of SA003 by the specific bacteriophage (ΦSA012), originally isolated from sewage influent. Addition of IgG also lowered the phage–host adsorption rate constant (k a). The k a value of the IgG-containing condition was approximately 1/8 of that of the IgG-free condition. In addition to the decrease of k a value, increase of the minimum inhibitory concentration (MIC) of ΦSA012 toward SA003 was observed. Fluorescence microscopical and confocal laser microscopical observations revealed that phages were mainly localized on the surfaces of cell aggregates. IgG-dependent S. aureus aggregation in crude milk is a problem that must be solved before phage therapy can be successfully used to treat bovine mastitis caused by this bacterium.
Keywords: Aggregation; Antibiotic; Biofilms; Biomedical; Bacteriophage; IgG; Staphylococcus aureus;

Display OmittedFeruloyl glycerols (FGs), water-soluble glycerides (monoferuloyl glycerol (MFG) and diferuloyl glycerol (DFG)) of ferulic acid, can be used as natural ultraviolet (UV) filters and antioxidants in chemical, food, pharmaceuticals, and drug industries. In order to promote the synthesis of FGs, the effect of process parameters, optimization, thermodynamic and kinetic properties on the enzymatic transesterification of ethyl ferulate (EF) with glycerol in [BMIM]PF6 were investigated. The maximum yields of MFG (63.72 ± 1.26%) and DFG (78.80 ± 2.09%) were achieved at low glycerol concentrations in [BMIM]PF6. The activation energies for EF conversion and transesterification to form MFG and DFG were calculated as 40.16, 31.43 and 85.38 kJ/mol, respectively, based on the Arrhenius law. Reaction kinetics agreed with the Ping–Pong Bi–Bi mechanism with the inhibitions of EF and glycerol. The enzymatic mechanism of the transesterification of EF with glycerol in [BMIM]PF6 was also proposed.
Keywords: Monoferuloyl glycerol; Diferuloyl glycerol; Enzymatic transesterification; Ethyl ferulate; Kinetics; Reaction mechanism; [BMIM]PF6;

The high cost of enzymes is one of the main bottlenecks affecting the industrial production of cellulosic ethanol, which therefore requires the development of improved bioprocesses for the manufacture of cellulases. The present work concerns the selection of operating parameters for enzyme production in three-phasic bioreactors, using sugarcane bagasse as substrate. The parameters considered included cultivation method, substrate particle size and pretreatment, agitation speed, and pH. For both shake flask and stirred tank bioreactor (STB), a new sequential cultivation method employing steam explosion pretreated sugarcane bagasse significantly improved enzyme production, compared to conventional submerged fermentation. Larger substrate particle size provided a better support for fungal growth in shake flasks, while smaller particles resulted in greater homogeneity in stirred tank bioreactors. Maximum endoglucanase and xylanase production in the STB were 1599 ± 66 and 4212 ± 133 IU L−1, respectively, under sequential cultivation using pretreated bagasse particles smaller than 0.5 mm, agitation speed of 700 rpm, and pH 5.0. The findings provide useful information concerning the influence of operational variables on (hemi) cellulases production in STB three-phasic cultivations, which should contribute to the development of bioprocesses using lignocellulosic materials in large-scale bioreactors.
Keywords: Enzyme production; Cellulase; Filamentous fungi; Submerged culture; Xylanase; Sugarcane bagasse;

Characteristics of a novel low density cell-immobilized magnetic supports in liquid magnetically stabilized beds by Zakaria Al-Qodah; Mohammad Al-Shannag; Eman Assirey; Wasim Orfali; Khalid Bani-Melhem; Kholoud Alananbeh; Nahla Bouqellah (40-49).
The adsorptive and hydrodynamic characteristics of low density non-porous magnetic supports used for biocatalyst immobilization have been investigated. The magnetic particles consist of a sand core have been covered with a magnetite layer followed by a layer of nano size activated carbon or fly ash with the aid of epoxy resin. The resulted particles showed good adsorbing behavior toward resting cells of Escherichia coli from batch culture of different cell concentrations. The maximum adsorption capacity for both particles were calculated using Langmuir isotherm model as 18 (1.22 million cells) and 14 (0.95 million cells) mg/g, respectively. The hydrodynamic study in a liquid magnetically stabilized beds confirms that these particles have lower fluidizing velocity and attain a higher expanded stabilized bed than particles of magnetic dense core. These results confirm the applicability of these particles to immobilize microorganisms for different applications. It was found that the conversion using these particles was 30% higher than that using traditional dense magnetic particles due to the longer residence times in the bed.
Keywords: Magnetic supports; Cell immobilization; Epoxy resin; Magnetic stabilized beds;

In-situ microbial degumming technology with Bacillus sp. HG-28 for industrial production of ramie fibers by Pei Fan; Feng He; Ying Yang; Mingzhang Ao; Jie Ouyang; Yi Liu; Longjiang Yu (50-58).
Display OmittedIndustrial production of ramie fibers by chemical degumming results in problems such as environmental pollution and inferior fiber quality, which are unsustainable and hinder the development of relevant industries. For these reasons, an in-situ microbial degumming process with direct application of Bacillus sp. HG-28 was developed and presented here to solve these problems. This strain was selected for its high degumming ability and low cellulose damage. In the degumming process, the gum in ramie bast fibers induced the strain to secrete high activities of pectinase and xylanase, both of which were proven to be essential for degumming. After 16 h of degumming, gum content reduction was measured to be 76.92% while cellulose loss could not be evidently detected. This is more efficient than other reported microbial degumming methods that do not use microorganisms directly. According to the results of in-situ microbial degumming in industrial scale, the residual gum content of degummed ramie fibers decreased to 1.81%, the bundle breaking tenacity reached 5.09 cN/dtex and, additionally, the consumption of chemicals, water and energy were significantly reduced, proving superior to chemical process. This study provides a sustainable alternative for the conventional chemical degumming industry.
Keywords: Ramie fibers; Microbial degumming; Cellulose; Biodegradation; Environmental preservation; Enzyme activity;

In this study, a novel chitinase-producing bacterium Chitinibacter sp. GC72 was isolated and investigated for N-acetyl-D-glucosamine production from crayfish shell enzymatic degradation. A dimeric chitinase was purified, which had an optimal activity at a pH of 6.8 and 40 °C. The metal ions Al3+, Cu2+, and Zn2+ inhibited the chitinase activity, whereas Ca2+, Mn2+, and Mg2+ promoted the activity. The chitinase was active on p-NP-GlcNAc with apparent Km value of 152.83 μmol/L and Vm value of 49.12 μmol/L min at 37 °C. Based on the hydrolysate formed, the chitinase was characterized as an exo-hydrolytic N-acetyl glucosaminidase. Furthermore, combined treatment of ultra-micro grinding and ultrasonic with direct enzymatic hydrolysis of crayfish shell for GlcNAc production was investigated, which resulted in 15.2 g of GlcNAc production from 100 g of crayfish shell following chitinase degradation for 36 h.
Keywords: Chitinase; Chitinibacter; Purification; Biodegradation; Waste treatment; Enzyme biocatalysis;

Line monitoring by near-infrared chemometric technique for potential ethanol production from hydrothermally treated Eucalyptus globulus by Yoshiki Horikawa; Makiko Imai; Keiko Kanai; Tomoya Imai; Takashi Watanabe; Keiji Takabe; Yoshinori Kobayashi; Junji Sugiyama (65-72).
This study reports a method that combines near-infrared (NIR) measurements with multivariate analysis to predict the saccharification efficiency of hydrothermally pretreated Eucalyptus globulus during ethanol conversion. Optimization of the NIR data with or without spectral treatment determined the best calibration model in the region 10000–4000 cm−1 of the original spectra, with an RMSEP of 2.08% and R p 2 of 0.99. By investigating the regression coefficient to understand the key regions and chemical components, for original and multiplicative scatter correction (MSC)-treated spectra, the water absorption and higher wavenumber regions were important. For the second derivative spectra, the regression model was constructed based on the CH overtone vibrations (6050–5500 cm−1). The regression coefficient demonstrated that the removal of hemicellulose resulted in higher lignin content, which might affect the biomass properties in terms of water absorption and enhanced enzymatic hydrolysis evaluated by dinitrosalicylic acid (DNS) method. For a higher throughput system, aqueous sample analysis was performed using an immersion probe equipped with an InGaAs detector, which generated an acceptable calibration model having RMSEP of 4.25% and R p 2 of 0.94. These results show the great potential of NIR spectroscopy for achieving fast, accurate, and nondestructive analysis, and its highly adaptability for maintaining an ethanol bioconversion system.TGS, triglycine sulfate
Keywords: NIR spectroscopy; Pretreatment; Cellulase; Cellulose; Lignin; Bioprocess monitoring;

Batch cultivation of Catharanthus roseus hairy roots was attempted in a statistically optimized nutrient medium. This resulted in a biomass growth of 5.12 ± 0.46 g/l and ajmalicine content of 24.9 ± 1.2 mg/l on day 30 beyond which the product concentration declined. Using the observed batch kinetics data a mathematical model was developed which could describe the experimental observations to the extent of 99% accuracy as demonstrated by the statistical validity test. The model was thereafter extrapolated to fed-batch cultivation to identify suitable nutrient feeding strategies (by off-line simulations) for the overproduction of ajmalicine. Eventually the experimental implementation of three such feeding strategies and comparison with the model predictions further reinforced the efficacy of the mathematical model under dynamic fed batch conditions. A 60% increase in ajmalicine overall productivity and a 2.5 fold increase in volumetric yield was obtained by the “increasing feed rate strategy” as opposed to batch cultivation. To further improve the alkaloid accumulation a combination of statistically optimized mixture of elicitor(s) (jasmonic acid, methyl jasmonate and KCl) was added after the termination of model based increasing fed batch cultivation which resulted in a significantly high ajmalicine concentration of 123.2 ± 8.63 mg/l (a 4 fold increase compared to batch). This is the first report of such a strategy for overproduction of ajmalicine in rather complex hairy root cultivation.
Keywords: Ajmalicine; Modelling; Fed batch culture; Kinetic parameters; Elicitation; Plant cell culture;

Display OmittedAn amperometric glucose sensor with excellent sensitivity, very low detection limit and low glucose oxidation potential was prepared by casting of suspension of copper oxide (CuO) nanoparticles/multi-walled carbon nanotubes (MWCNTs) on the surface of a glassy carbon electrode (GCE). Dimethyl formamide (DMF)/H2O (9:1) was used as a new suspending media. Electrochemical activity of the electrode toward the oxidation of glucose was studied using differential pulse voltammetry, cyclic voltammetry and electrochemical impedance spectroscopy. A synergistic effect was substantiated between the MWCNTs and nano-CuO for glucose electroxidation. The effects of nanocomposite layer thickness and MWCNTs/CuO mass ratio on the sensor performance were investigated and optimized. The detection limit, and linear range of the sensor were identified as 0.07 (±0.03) μmol L−1 (S/N = 3) and 0.5–2000.0 μmol L−1, respectively, under applied oxidation potential of 0.35 V (vs. Ag/AgCl). The sensor exhibited excellent sensitivity of 3968.42 (±0.84) μA L mmol−1  cm−2 to glucose, being higher than those of the previously reported similar sensors. The determination of glucose (5.0 μmol L−1, n  = 5) with the sensor resulted in RSD% of 3.4. Furthermore, the reproducibility of the sensor was equal to 5.7%. The developed sensor was compared with other enzyme-less glucose sensors and its superiority over them was demonstrated reasonably. The optimized sensor was applied to glucose determination in blood samples. The efficiency of the sensor for glucose determination was comparable with that of a commercial enzymatic sensor.
Keywords: Biosensors; Glucose; Sensors; Heterogeneous reaction; Cupric oxide; Carbon nanotube;

Production of polyhydroxyalkanoates from dephenolised and fermented olive mill wastewaters by employing a pure culture of Cupriavidus necator by Gonzalo Agustín Martinez; Lorenzo Bertin; Alberto Scoma; Stefano Rebecchi; Gerhart Braunegg; Fabio Fava (92-100).
The feasibility of producing polyhydroxyalkanoates (PHAs) by feeding a pure culture of Cupriavidus necator with a pre-treated olive mill wastewater (OMW) was demonstrated at 500 mL shaken flask scale. The OMW was previously dephenolised and then fermented to produce an effluent rich in volatile fatty acids (VFAs). The latter stream (OMWAcid) was then employed as the carbon source for PHAs production.Firstly, pre-grown cells were fed with different dilutions of OMWAcid, namely: 25, 50, 75 and 100% (v/v). Significant inhibitory effects were observed when OMWAcid concentration was 75 and 100%. Thereafter, experiments with laboratory prepared solutions, simulating the OMWAcid, allowed to demonstrate that polyphenols significantly contributed to the observed inhibition. Furthermore, The copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (poly(HB-co-HV)), containing 11% of hydroxyvalerate, was accumulated up to 55% of the cells dry weight when two consecutive accumulation batch processes were carried out with 25% of OMWAcid and without adding any exogenous carbon source.The obtained results are promising in the perspective of continuing the production study at a bench-top bioreactor scale and thereafter analysing the possibility of developing a biotechnological PHAs production process as a part of an integrated OMW valorization process.
Keywords: Polyhydroxyalkanoates; Waste treatment; Cupriavidus necator; Anaerobic processes; Biotransformation; Substrate inhibition;

Significant variables for production of xylanase and β-mannanase including moisture content, initial pH and inoculum size and significant variables for enzymes extraction including volume of solvent, extraction time and agitation speed were screened and optimized, respectively. With the use of mixture of corn stover powder and wheat bran as substrates and double distilled water as solvent for enzymes extraction, maximum xylanase activity (19613.25 U/g) and β-mannanase activity (8479.82 U/g) could be obtained under the optimized conditions [moisture content 74.0%, initial pH 4.5, inoculum size 11.6% (v/w), volume of solvent 16.0 ml/g dry substrate, extraction time 108 min and agitation speed 172 rpm]. Cellulases activities including endoglucanase activity (195.13 U/g), filter paper activity (41.87 U/g) and β-glucosidase activity (132.63 U/g) were obtained concomitantly. Compared with xylanase activity (1734.78 U/g) and β-mannanase activity (928.41 U/g) under initial conditions, optimization resulted in 10.31-fold increase for xylanase activity and 8.13-fold increase for β-mannanase activity, respectively. The crude enzymes solution was suitable for saccharification of aqueous ammonia solution pretreated corn cob powder and maximum yields of xylose (236.63 mg/g) and reducing sugar (553.94 mg/g) were obtained.
Keywords: Xylanase; β-mannanase; Optimization; Production; Extraction; Saccharification;

Degradation of MXC by host/guest-type immobilized laccase on magnetic tubular mesoporous silica by Yuxiang Yang; Qinmei Wei; Jianbo Zhang; Yanjie Xi; Hongming Yuan; Cheng Chen; Xiangnong Liu (111-118).
Display OmittedIn this paper, magnetic host/guest-type immobilized laccase was prepared by co-adsorption of super-paramagnetic particles (SPMNPs) and laccase into the pore channels of tubular mesoporous SiO2 by the “size-matching effect”. After immobilization, the thermal stability and repetition usage-ratio of immobilized laccase were improved significantly, the loading amount of enzyme and SPMNPs was determined by Lowry’s method and EDS (Energy Dispersive Spectroscopy), respectively. Under the optimal conditions, the removal efficiency of MXC (Methoxychlor) by immobilized laccase reached 69.4% and the removal efficiency still remained 45.4% after six cycles of operations. The studies on degradation kinetics of MXC by free and immobilized laccase were also carried out, respectively. In virtue of GC–MS, 1H NMR and 13C NMR analysis, (CH3OC6H5)2C=CHCl and 1,1-diphenylethylene were identified as intermediate and final degradation products of MXC, respectively, further demonstrating the degradation mechanism of MXC by the immobilized laccase.
Keywords: Tubular mesoporous SiO2; Immobilized enzymes; Biocatalysis; MXC; Kinetic parameters; Biodegradation;

Synthesis of fatty acids methyl esters (FAMEs) from Nannochloropsis gaditana microalga using heterogeneous acid catalysts by Alicia Carrero; Gemma Vicente; Rosalía Rodríguez; Gonzalo L. del Peso; Cleis Santos (119-124).
Oleaginous microorganisms like microalgae have emerged as a promising alternative feedstock in the production of fatty acid methyl esters (FAMEs) since they can accumulate high levels of lipids without competing with food production and having oil productivity values higher than oilseed crops. The lipids of Nannochloropsis gaditana microalga were extracted with methanol and analysed to determine its chemical composition. Since typical homogenous catalysis requires additional purification units and extracted oil presented many free fatty acids (FFAs) (∼22 wt%), FAMEs were synthesized using solid acid catalysts like ion-exchange resins (Amberlite-15, CT-275, CT-269), KSF clay and silica–alumina. Despite their high surface area, the lower acidity of silica–alumina led to a FAME yield lower than the ones obtained using KSF clay and ion-exchange resins. The good results obtained with these catalysts discard diffusion limitations when resins or KSF clay are used as catalysts. FAME synthesis through an indirect method with a previous lipid extraction was compared with the direct reaction of dry microalga biomass. Better results (FAME yields above 90 mol%) were obtained in the two-step method using ion-exchange resins. However, these catalysts lost their activity, so they were regenerated by washing with methanol and HCl.
Keywords: Fatty acid methyl esters; Nannochloropsis gaditana; Microalgae; Transesterification; Heterogeneous reaction; Solid acid catalysts;

Interaction of mercury and copper on papain and their combined inhibitive determination by Xue-Ying Liu; Hong-Yan Zeng; Meng-Chen Liao; Bo Feng; Bi Foua Claude Alain Gohi (125-131).
Influence and interaction of mercury ion (Hg2+) and copper ion (Cu2+) on papain activity in casein hydrolysis were investigated. Single Hg2+ or Cu2+ at low concentrations induced an increase in papain activity, but decreased it at high concentrations, confirming a typical hormesis phenomenon. The interaction of Hg2+ and Cu2+ at various concentration combinations showed that the binary interaction of 10−8  mol/L Cu2+ and 10−6  mol/L Hg2+ (Binary union S) buffer was of synergistic nature, while 10−4  mol/L Cu2+ and 10−4  mol/L Hg2+ (Binary union I) buffer was of competitive inhibition. The conformational changes in papain structure due to the interaction of binary metal ions were studied by ATR-FTIR, UV–vis and intrinsic fluorescence spectroscopies, also the changes of papain catalytic behavior were studied through kinetic analysis. Decreasing of α-helix content with increasing in intermolecular β-sheet aggregates content in Binary union I buffer resulted in an inactivation of papain activity by 57.2% and lower affinity for casein. On the contrary, papain activity increased with α-helix content increasing and intermolecular β-sheet aggregates content decreasing in Binary union S buffer. The competitive interaction between Cu2+ and Hg2+ on papain activity was found at higher concentrations (≥10−4  mol/L), and the inhibition of the binary metal ions on papain was of a noncompetitive type.
Keywords: Protease; Biocatalysis; Enzyme activity; Mercury ion; Copper ion; Kinetic parameters;

Anaerobic membrane bioreactors enable high rate treatment of slaughterhouse wastewater by P.D. Jensen; S.D. Yap; A. Boyle-Gotla; J. Janoschka; C. Carney; M. Pidou; D.J. Batstone (132-141).
Anaerobic membrane bioreactors (AnMBRs) enable high space loading by retaining solids selectively through microfiltration membranes. For organic industrial wastewaters, this offers an alternative to lagoons and granule based high-rate anaerobic treatment due to excellent effluent quality, high tolerance to load variations, and ability to produce a solids free effluent for the purposes of reuse. While there has been extensive work on low-strength and low solids effluent, there has been limited application in high-solids, high fats systems such as slaughterhouse wastewater, which are a key application. A 200 L AnMBR pilot plant operated at 2 Australian cattle slaughterhouses consistently removed over 95% of chemical oxygen demand (COD) from the wastewater. Virtually all degradable COD was converted to biogas, 78–90% of nitrogen and 74% of phosphorus in the wastewater were released to the treated permeate as ammonia and phosphate, respectively; which would enable subsequent nutrient capture. The mass loading rate limit of 3–3.5 g COD L−1  d−1 is imposed by the active biomass inventory, with this in turn limited to 40 g L−1 (TS) by the need to manage membrane fouling control.
Keywords: Anaerobic processes; Biogas; Membrane bioreactors; Slaughterhouse; Resource recovery; Waste-water treatment;