Biochemical Engineering Journal (v.42, #3)

BEJ Keywords (II).

A combined promoter expression vector pBV–PAL for high-level expression of phenylalanine ammonia lyase gene of Rhodosporidium toruloides was constructed. Pal gene was cloned and inserted into the region between SalI and PstI restriction sites of expression vector pBV220 (containing P L P R promoter) to obtain recombinant expression vector pBV220–PAL. The tac promoter obtained from the plasmid pKtac was inserted into the expression vector pBV220–PAL to construct expression vector pBV–PAL. The recombinant plasmid pBV220–PAL and pBV–PAL were introduced into Escherichia coli JM109 by transformation. The result showed that the transformant E. coli JM109 (pBV–PAL) gave a much higher PAL activity than that transformant E. coli JM109 (pBV220–PAL). Recombinant PAL expression level of the transformant JM109 (pBV–PAL) was about 9.6% of total cellular protein, specific enzyme activity was 2.3-fold higher than that of the transformant JM109 (pBV220–PAL), reached 35 U/g (dry cells weight, DCW). PAL specific activity of 123 U/g (DCW) could be achieved in a 5-l fermentor. 80.5% conversion rate of trans-cinnamic acid to l-phenylalanine and 5.12 g/l l-phenylalanine were obtained after 3 h bioconversion using the transformant JM109 (pBV–PAL). The recombinant strain JM109 containing the combined promoter expression vector pBV–PAL was shown to be effective and practical to product l-phenylalanine.
Keywords: Phenylalanine ammonia-lyase; Recombinant DNA; Combine promoter; Enzyme activity; Fermentation; Bioconversion;

The simultaneous formation of mevinolinic acid (lovastatin; antihypercholesterolemia drug) and (+)-geodin (by-product) by Aspergillus terreus ATCC 20542 in the batch and fed-batch cultivation in the stirred tank bioreactor was investigated and described in this paper. The main factors influencing the formation of these two secondary metabolites were the initial nitrogen concentration and the aeration rate of the medium. The experiments aimed at achieving as high as possible lovastatin titre accompanied by as low as possible (+)-geodin concentration. The application of lactose-fed discontinuous fed-batch process allowed increasing lovastatin formation, in comparison with the batch process. Nevertheless (+)-geodin titre increased too. But the control of pH at the levels of 7.6 and 7.8 was successfully applied to repress the formation of the by-product both in batch and fed-batch experiments. Additionally, apart from pH control, the supplementation of the medium with nicotinamide and calcium pantothenate was used to facilitate the formation of lovastatin. The simultaneous pH control and B-group vitamin supplementation allowed achieving the best results in the batch cultivation.
Keywords: Lovastatin; Mevinolinic acid; (+)-Geodin; Kinetics; Batch; Fed-batch; Bioreactor;

The effect of the presence of n-dodecane as a potential oxygen vector during oxygen-limited continuous cultures of a Bacillus strain was studied, under extreme nutrient supply conditions: glucose excess, limitation and starvation. The addition of n-dodecane to the aqueous phase of a mechanically agitated and aerated fermentation increased the k L a by up to 35%. The n-dodecane additions to Bacillus licheniformis cells during starvation (oxygen limitation with concomitant glucose starvation) caused a severe detrimental progressive change in cell physiological state with respect to cytoplasmic membrane polarisation and permeability which was mitigated against by alleviating either the oxygen limitation (by increasing the mean energy dissipation rate or by the addition of n-dodecane as an oxygen vector) or by alleviating the carbon limitation (by resuming the carbon feed or by the addition of a glucose pulse). Further that during periods of excess glucose (glucose pulse) a much higher k L a was required to prevent the onset of anaerobic mixed acid fermentation than could be provided by the addition of n-dodecane alone. n-Dodecane can be used to increase the k L a when added in sufficient quantities to the aqueous phase of a mechanically agitated and aerated bioreactor but the magnitude of this increase is process and vessel geometry specific.
Keywords: Bacillus licheniformis; n-Dodecane; Multi-parameter flow cytometry; Oxygen vector; Steady-state; Termophilic wastewater treatment;

Influence of temperature on protein hydrolysis in a cyclic batch enzyme membrane reactor by Carlos A. Prieto; Emilia M. Guadix; Antonio Guadix (217-223).
A cyclic batch enzyme membrane reactor (CBEMR) incorporating a 8000-Da polyethersulphone membrane was intended for enhancing the enzyme (Protex 6L from Bacillus licheniformis, EC. use in the production of a whey protein hydrolysate. A mechanistic mathematical model comprising zero-order kinetics for the hydrolysis and second-order deactivation for the enzyme was proposed and validated through experiments. The influence of reaction temperature was studied and process optimisation (given the production requirements) was performed in terms of number of batch reactions that minimise the total amount of enzyme used. The optimal operation of the CBEMR allowed savings of up to 44 and 32% of enzyme compared to the single batch operation mode at 50 and 60 °C, respectively. No enzyme savings were detected when temperature was fixed at 70 °C. In general, the optimal operation temperature was 60 °C, yielding lower enzyme consumption for all productivities of the reactor.
Keywords: Membrane bioreactors; Proteolysis; Enzyme bioreactors; Optimisation; Whey protein hydrolysates; Enzyme reuse;

The catabolite repressor/activator protein Cra (FruR) is known to repress the glycolysis pathway genes and activate the gluconeogenic pathway genes. Disruption of cra gene may, therefore, activate glycolysis and thus improve the glucose consumption rate, which may in turn lead to the increase of the specific metabolite production rate. However, it was found in our previous investigation that the substrate consumption rate could not be improved for the aerobic cultivation of cra mutant due to activation of Entner–Doudoroff (ED) pathway, and the repression of the TCA cycle caused by the down-regulation of icdA and aceBAK genes. To overcome this problem, we investigated the effects of edd and arcA genes knockout on the glucose consumption rate. The cra.edd mutant showed the higher glucose uptake rate as compared with the wild type under microaerobic condition. Since arcA gene knockout mutant repressed pfl operon under microaerobic condition, the glucose consumption rate was not improved for cra.edd.arcA mutant. In order to show that the increase in the glucose consumption rate can lead to the increase of the metabolite production rate, we considered the lactate production by pflA mutant. It was shown that the substrate consumption rate could be significantly improved for cra.pflA mutant as compared with pflA mutant, and thus the lactate production rate could be improved by this double mutant if fructose was used as a carbon source.
Keywords: Escherichia coli; Cra mutant; Cra.pflA mutant; Cra.edd double mutant; Cra.edd.arcA mutant; Glucose consumption rate; Lactate fermentation;

The Arc system is a two-component regulatory system composed of ArcA and ArcB in Escherichia coli. In the present study, the effects of arcA and arcB genes knockout on the TCA cycle activation in E. coli were investigated for the anaerobic and microaerobic conditions. Under anaerobic condition, the TCA cycle was up-regulated along with high lactate production, together with up-regulation of LDH for arcB mutant as compared with the parent strain. Due to down-regulation of aceE, aceF and lpdA genes which code for PDHc and low activity of Pfl in arcB mutant, the glycolysis as well as oxidative pentose phosphate pathway was down-regulated under anaerobic condition. The TCA cycle enzymes were further up-regulated when nitrate was added by modifying the redox state along with lower lactate production for arcB mutant. Different from the case of anaerobic condition, the glycolysis was activated under microaerobic condition, which may be partly due to the increased activity of PDHc encoded by aceE, F and lpdA genes. Under microaerobic condition, the TCA cycle genes together with their corresponding enzymes were up-regulated for arcB mutant as compared with the parent strain. These characteristics were further enhanced in arcA mutant as compared with the case of arcB mutant. The up-regulation of the TCA cycle together with down-regulation of cydB gene expression caused higher redox state in the arcA/B mutants, which in turn repressed the TCA cycle. Then the TCA cycle could be further increased by the addition of nicotinic acid (NA).
Keywords: Escherichia coli; arcA; arcB; Microaerobic condition;

Adsorption and elution behaviors of bovine serum albumin in metal-chelated affinity cryogel beds by Lianghua Wang; Shaochuan Shen; Xingjiao He; Junxian Yun; Kejian Yao; Shan-Jing Yao (237-242).
Metal-chelated supermacroporous cryogels are effective supports for affinity chromatographic separation of biomolecules in downstream processes. In this work, polyacrylamide cryogel beds were prepared in glass columns (16 mm inner diameter) and coupled with iminodiacetic acid (IDA). These cryogels were loaded with Zn2+ and Ni2+ and the so-called Zn2+-IDA-cryogels and Ni2+-IDA-cryogels were obtained. Permeabilities and height equivalent to theoretical plates (HETPs) of these cryogel beds were measured and the cryogel structure was analyzed using scanning electron microscopy (SEM). Bovine serum albumin (BSA) was employed as a model protein to elucidate the adsorption and elution behaviors of these cryogels under various conditions, such as different flow rate, solution pH, and composition of the eluents. The results showed that the Zn2+-IDA-cryogels and Ni2+-IDA-cryogels in this study had interconnected supermacropores and high water permeabilities (∼10−11  m2). The loading flow velocity had a weak influence on the breakthrough curves and binding capacities for BSA, while the solution pH had an evident effect on the binding capacities for BSA in these cryogels. Maximum binding capacity for BSA was observed near the isoelectric point of BSA. The bound BSA can be eluted effectively using an imidazole solution. A low-eluting flow rate was found to be beneficial to the elution process. Possible mechanisms were proposed and discussed.
Keywords: Supermacroporous cryogels; Immobilized metal affinity chromatography; Bovine serum albumin; Protein adsorption; Binding capacity; Breakthrough; Elution;

Pulping of rice straw with high-boiling point organosolv solvents by A. Rodríguez; L. Serrano; A. Moral; L. Jiménez (243-247).
Rice straw was used as an alternative raw material to obtain paper pulp. Pulping was done by using high-boiling point organic solvents of decreased polluting power relative to classical (Kraft, sulphite) solvents but affording operation at similar pressure levels.The holocellulose, α-cellulose and lignin contents of rice straw (viz. 60.7, 41.2 and 21.9%, respectively) are similar to those of some woody raw materials such as pine and eucalyptus, and various non-wood materials including olive tree prunings, wheat straw and sunflower stalks.Pulping tests were conducted by using ethyleneglycol, diethyleneglycol, ethanolamine and diethanolamine under next operating conditions: 80% solvent, 180 °C and 150 min. The solid/liquid ratio was 6. The amine solvents were found to provide pulp with better properties than did the glycol solvents. Diethanolamine pulp exhibited the best viscosity, Kappa index, drainage index and burst index (viz. 956 mL/g, 17.0, 21°SR and 1.23 kN/g, respectively). Paper made from ethanolamine pulp exhibited the best stretch (1.99%), tear index (0.44 mNm2/g) and brightness (41.7%).
Keywords: Non-wood; Organosolv; Paper; Pulp; Rice straw;

Liquid membrane extraction of bio-active amphiphilic substances: Recovery of surfactin by Krasimir Dimitrov; Frédérique Gancel; Ludovic Montastruc; Iordan Nikov (248-253).
The interest of application of liquid membrane (pertraction) processes for recovery of biosurfactants from aqueous media was demonstrated. Transport of pure surfactin in three-liquid-phase system was studied. Surfactin was successfully extracted from slightly acid media (pH 5.65–6.05) applying batch pertraction in a rotating discs contactor and using n-heptane as liquid membrane. The process efficiency was found to be strongly affected by the feed solution acidity (83% at pHF 6.05 and 97% at pHF 5.65 after 4 h pertraction).An atypical pH effect was observed when the behaviour of surfactin extraction from aqueous media by non-polar solvents (n-heptane and n-octane) was studied. The obtained high extraction degrees from both acid and basic media and the clearly reduced degree of extraction from neutral media could be attributed to the different conformations of surfactin in these media.
Keywords: Surfactin; Extraction; Liquid membranes; Biosurfactants; Pertraction; Process integration;

Enhanced production of biosurfactant by a marine bacterium on statistical screening of nutritional parameters by Soumen Mukherjee; Palashpriya Das; C. Sivapathasekaran; Ramkrishna Sen (254-260).
Marine microorganisms can serve as rich sources for novel biosurfactants with diverse biological activities. In the present investigation, the nutritional requirement for the growth and biosurfactant production by a marine bacterium was determined using a Plackett–Burman-based statistical screening procedure. Six out of the eleven factors of a reported production medium were found to be critically affecting the biosurfactant metabolism. Glucose, the carbon substrate of the medium was the most influential factor with an effect contribution of 78.13% and a very low p-value of <0.001. Glucose, NH4NO3 and FeSO4·7H2O had a direct proportional correlation with biosurfactant production while, K2HPO4, KH2PO4 and MgSO4·7H2O showed inversely proportional relationship with biosurfactant production in the selected experimental range. A simpler modified medium (MM) was formulated based on the statistical screening results. Modified medium combination (MM-1) having the following composition in g l−1: glucose 30; NH4NO3 6.0; K2HPO4 1.1; MgSO4·7H2O 0.3; KH2PO4 2.8 × 10−2 and FeSO4. 7H2O 6 × 10−2 showed 84.7% increase in biosurfactant yield over the reported medium (SM). Fourier transform infrared spectroscopy and thin layer chromatography studies showed that the biosurfactants produced in the modified medium (MM) were similar to those produced in the reported medium (SM).
Keywords: Fermentation; Submerged culture; Chromatography; Optimization; Plackett–Burman design; Biosurfactant;

Three kinetic models of the transesterification of palm oil fatty acids to ethanol using an immobilized lipase were developed. The models differ from one another with respect to the rate-limiting step and the point at which the ethanol molecule becomes involved in the reaction. The kinetic parameters were estimated by fitting experimental data of the transesterification of palm oil with various ethanol concentrations. The models are able to account for the effects of substrates and products involved in the transesterification throughout the entire reaction. There was a good agreement between experimental results and those predicted by the proposed model equations in which ethanol was assumed to be involved directly in an alcoholysis reaction with palm oil. Furthermore, the calculated results show that the rate constants for alcoholysis of palm oil with ethanol are much higher than those for the hydrolysis reaction. From the proposed model equations, the effects of ethanol concentration on the initial production rates and yields of fatty acid ethyl ester and free fatty acids were simulated. The simulation results show that increasing the initial ethanol concentration produces an increase in the initial production rate and yield of fatty acid ethyl ester and lowers the final concentration of free fatty acid whereas lower ethanol concentration led to a higher final concentration of free fatty acid.
Keywords: Fatty acid ethyl ester; Immobilized lipase; Kinetic; Modeling; Palm oil; Transesterification;

Enhancement of poly(arginyl-histidine) production by Verticillium kibiense E18 by Ikumi Kurihara; Yoshitaka Ishii; Kohtaro Kirimura; Kuniki Kino (270-275).
An ergot fungus Verticillium kibiense E18 produced two cationic peptides, ɛ-poly-l-lysine (ePL) and poly(l-arginyl-d-histidine) (PRH). The ePL was used as a food preservative, and it was expected that PRH would be used as a novel material, such as cationic and antimicrobial peptide. To enhance PRH production of strain E18, various culture conditions were investigated. Glucose was a suitable carbon source for PRH production, although glycerol was a suitable carbon source for growth. The cultivation temperature significantly influenced both cell growth and PRH production. The optimal temperatures for cell growth and PRH production were 28 and 30 °C, respectively. Moreover, strain E18 produced more PRH when an additional 5.0 μg/L FeSO4·7H2O was added to the production medium. Under optimal conditions, strain E18 enhanced PRH production, while suppressing ePL production. The maximum PRH production was 183.9 mg/L, which is approximately 60-fold higher than that of the initial culture condition.
Keywords: Poly(arginyl-histidine); Culture conditions; Verticillium kibiense; Cationic peptide; Antimicrobial peptide; Poly(amino acid);

Cadmium uptake by algal biomass in batch and continuous (CSTR and packed bed column) adsorbers by Vítor J.P. Vilar; Sílvia C.R. Santos; Ramiro J.E. Martins; Cidália M.S. Botelho; Rui A.R. Boaventura (276-289).
In this work, the properties of marine algae Gelidium, algal waste from agar extraction industry and a composite material were investigated for cadmium(II) biosorption. Equilibrium experiments were performed at three pH values (4, 5.3 and 6.5). Equilibrium data were well described by the Langmuir and Langmuir–Freundlich isotherms. Two models predicting the pH influence in the cadmium biosorption (discrete and continuous models) have been developed in order to better describe the equilibrium. The continuous model also considers a heterogeneous distribution of carboxylic groups, determined by potentiometric titration. The results of batch kinetic experiments performed at different pH values were well fitted by two mass transfer models and the homogeneous diffusion coefficients for the cadmium ions inside the biosorbent were obtained. Continuous stirred tank reactor (CSTR) and packed bed column configurations were also examined for the biosorption of cadmium ions. A strong acid (0.1 M HNO3) was used as eluant to regenerate the biosorbents in the column. Several mass transfer models were applied with success to describe the biosorption process in batch mode, CSTR and fixed bed column.
Keywords: Biosorption; Algal biomass; Batch; CSTR; Packed bed column;

Synthesis, characterization and MRI application of dextran-coated Fe3O4 magnetic nanoparticles by R.Y. Hong; B. Feng; L.L. Chen; G.H. Liu; H.Z. Li; Y. Zheng; D.G. Wei (290-300).
Biocompatible ferrofluid based on dextran-coated Fe3O4 magnetic nanoparticles (MNPs) was prepared through a one-step method. In contrast to the conventional co-precipitation method, hydrazine hydrate was added as reducing agent and precipitator in the present investigation. The effects of hydrazine hydrate, the weight ratio of dextran to MNPs and the molecular weight of dextran on the dispersibility of MNPs in water were investigated. Also, the particles size of modified MNP and coating efficiency of dextran on MNPs were measured. In addition, biocompatible ferrofluid was intravenously injected into rabbits, the iron content in blood and organs at different times were measured by atomic absorption spectrometer, and the bio-distribution and the bio-transportation of ferrofluid in organs was examined. Then, the magnetic resonance (MR) images of liver, marrow and lymph were acquired by MRI experiments before and after intravenous injection of ferrofluid. Image analysis revealed that the MR signal intensity of these organs notably decreased after intensified by ferrofluid. However, when there existed tumors in organs, the signal intensity of tumor did not change after injection. From that the tumor can easily be identified, which indicated a potential application of the as-prepared MNP in functional molecular imaging for biomedical research and clinical diagnosis.
Keywords: Magnetic nanoparticles; Dextran; Bioprocess monitoring; Rheology; Animal cell culture; MRI;

Immobilized metal ion affinity chromatography (IMAC) in expanded bed mode is used for purifying recombinant green fluorescent protein (GFP) overexpressed in Escherichia coli. The purification is carried out on two different matrices, i.e. Ni2+ Streamline™ and Ni2+ cross-linked alginate beads. The binding isotherms to both IMAC media followed the Langmuir model. The maximum binding capacity (q max) of Ni2+ Streamline™ and Ni2+ cross-linked alginate for the GFP was 1,42,860 FU ml−1 and 18,000 FU ml−1, respectively. The expanded bed column chromatography using Ni2+ Streamline™ gave 2.7-fold purification with 89% of GFP recovery, while Ni2+ alginate gave 3.1-fold purification with 91% of GFP recovery. SDS-PAGE of purified GFP in both cases showed single band. The results obtained in the expanded bed chromatography are compared with those obtained in packed bed chromatography.
Keywords: Adsorption; Bioseparation; Fluidization; Purification; Green fluorescent protein; Immobilized metal affinity chromatography;

Biofilm and granular systems to improve Anammox biomass retention by I. Fernández; J.R. Vázquez-Padín; A. Mosquera-Corral; J.L. Campos; R. Méndez (308-313).
Appropriate biomass retention in reactors is a crucial factor for the accurate operation of the anaerobic ammonium oxidation (Anammox) process due to the slow growth rate of this bacterial population. In the present work two different approaches were studied and compared to improve Anammox biomass retention minimizing wash-out events: (1) formation of granular biomass using influents with high inorganic salts concentrations by production of saline precipitates acting as promoters for biomass aggregation (reactor SBR1); (2) use of zeolite particles as carrier material for Anammox biofilm formation (reactor SBR2). Both alternatives allowed the reduction of biomass wash-out in the effluent to values as low as 18 mg VSS L−1 (SBR1) and 3 mg VSS L−1 (SBR2). As a consequence the biomass concentration increased significantly inside each reactor. In the case of the SBR2 the specific Anammox activity (SAA) of the biomass was also enhanced increasing from 0.35 up to 0.5 g N (g VSS d)−1. Both approaches allow the improvement of the biomass retention, the first option indicating the suitability of the Anammox process to treat wastewaters with high salt content. The second one with zeolite particles could be a good strategy to apply the Anammox process to low salinity wastewaters.
Keywords: Adsorption; Anaerobic processes; Anammox; Chemoautotrophs; Salinity; Wastewater treatment;

Ethanol production from paper sludge by immobilized Zymomonas mobilis by Yuya Yamashita; Akihiro Kurosumi; Chizuru Sasaki; Yoshitoshi Nakamura (314-319).
During past decades, the considerable efforts have been made to utilize lignocellulose as biomass feedstock for the optimal production of bio-ethanol as an alternative source of fuel. In this work, the effective bioconversion of paper sludge to ethanol was investigated by using Zymomonas mobilis NBRC 13756. Major components of the raw paper sludge were carbohydrates and ash. Fermentation utilizing strains of Z. mobilis instead of traditional yeasts and the use of simultaneous saccharification and fermentation (SSF) process have been proposed due to their high ethanol yields with cost-effectiveness. Free cells of Z. mobilis resulted in no ethanol production even after 24 h of incubation because cells’ growth was inhibited by metal ions contained in the paper sludge. The application of SSF with Ca-alginate-immobilized cells of Z. mobilis was performed and 18 g/L of ethanol was obtained after 48 h of incubation at an initial paper sludge concentration of 200 g/L. In addition, the repeated batch fermentation of Ca-alginate-immobilized Z. mobilis cells was attempted for producing ethanol up to run 4. Our work suggests that Ca-alginate-immobilized cells of Z. mobilis could effectively produce ethanol from paper sludge not only under the batch fermentation but also under the repeated batch fermentation.
Keywords: Zymomonas mobilis; Paper sludge; Bio-ethanol; Metal ions; SSF; Immobilization;

Effect of hydrodynamic cavitation on zooplankton: A tool for disinfection by Subhash Shivram Sawant; Arga Chandrashekar Anil; Venkat Krishnamurthy; Chetan Gaonkar; Janhavi Kolwalkar; Lidita Khandeparker; Dattesh Desai; Amit Vinod Mahulkar; Vivek Vinayak Ranade; Aniruddha Balchandra Pandit (320-328).
Application of hydrodynamic cavitation for disinfection of water is gaining momentum, as it provides environmentally and economically sound options. In this effort, the effect of cavitating conditions created by differential pump valve opening and that created by flowing through a cavitating element (orifice plates) on the microbes (zooplankton in sea water) is described. The experimental results are compared with modelling of cavitating conditions that includes cavity dynamics, turbulence generated by individual oscillating cavity, cell wall strength and geometrical and operating parameters of cavitation device. Theoretical model for quantifying the cavitationally generated turbulent shear and extent of microbial disinfection has been developed. Experimental results indicated that cavitation and/or turbulent fluid shear dominantly originating from cavitation are effective tools for sea water disinfection as more than 80% of the zooplankton present in the sea water were killed. It was also observed that shock waves generated due to cavitation is not the sole cause for zooplankton disruption. A correct physical mechanism accounting fluid turbulence and shear, generated from stable oscillation of cavity, significantly contribute towards the disruption. Further refinement of the model presented will serve as a basis for higher degree of disinfection and provide a practical tool for sea water disinfection.
Keywords: Cell disruption; Hydrodynamic cavitation; Zooplankton; Modelling; Heat transfer; Wastewater treatment;

Simultaneous modeling of enzyme production and biomass growth in recombinant Escherichia coli using artificial neural networks by M. Erdem Günay; I. Emrah Nikerel; Ebru Toksoy Oner; Betül Kirdar; Ramazan Yildirim (329-335).
In this work, the biomass growth and the TaqI endonuclease production by recombinant Esherichia coli were studied using artificial neural networks. The effects of the medium components on biomass growth and enzyme yield were modeled by various networks. After the most successful networks were statistically determined, they were used to extract additional knowledge such as the possible correlations between the biomass growth and the enzyme yield, and the relative significance of the medium components. It was found that the change of the biomass growth and the enzyme yield with the change of KH2PO4 concentration was strongly correlated with an R-value of −0.954. Some mild correlations were also observed for the other components. It was also found that the relative significances of the medium components were in the same order for both outputs; (NH4)2HPO4 concentration was determined as the most important parameter followed by the glucose, KH2PO4 and MgSO4 concentrations.
Keywords: TaqI endonuclease; Enzyme production; Modeling; Optimization; Artificial intelligence; Neural networks;