Biochemical Engineering Journal (v.62, #C)

BEJ Keywords (IV).

Optimizing the removal of fluoride from water using new carbons obtained by modification of nut shell with a calcium solution from egg shell by V. Hernández-Montoya; L.A. Ramírez-Montoya; A. Bonilla-Petriciolet; M.A. Montes-Morán (1-7).
Carbons with specific calcium compounds were obtained from pecan nut shells modified with a calcium solution extracted from egg shells and they were efficient for the fluoride removal.Display Omitted► The pecan shells (PS) are an abundant waste derived of nut processing. ► The PS was used as precursor of activated carbon. ► The carbon was activated with a calcium solution extracted from egg shells. ► The activated carbon with calcium moieties were used in the fluoride removal from water. ► The calcium moieties are playing an important role in the fluoride adsorption mechanism.Carbons loaded with specific chemical moieties were prepared from pecan nut shells employing a natural modifier agent obtained from egg shell, which is rich in calcium, for the selective adsorption of fluoride from water. A L4 orthogonal array of the Taguchi method was used to optimize the synthesis conditions for obtaining these selective carbons. The samples obtained were characterized and the elemental composition, textural parameters and morphology were determined. Fluoride adsorption experiments were performed in synthetic and real groundwater samples. Results showed that carbons obtained from pecan nut shells modified with a calcium solution extracted from egg shells (CMPNS) were more effective for fluoride removal than those using the nut shell precursor as such. The calcium chemical species on the carbon surfaces were more important in the fluoride adsorption process than the carbon textural parameters. In addition, hydrogencarbonate was found to be the main competitor for the active sites of CMPNS during the fluoride removal process.
Keywords: Activated carbon; Adsorption; Calcium; Egg shell; Fluoride; Pecan nut shell;

► Biofilms growing on anodes and cathodes posed two-side effects on power generation. ► Thick biofilms reduced the power generation in a long-term operation. ► The MFCs with Pt-free cathodes achieved similar power generation as that with the Pt-based cathodes. ► The low-cost and stable operation of Pt-free biocathodes is promising for the real-world application.The effects of biofilm growth on anode and cathode surfaces on the power generation from wastewater in single chamber microbial fuel cells (SCMFCs) were investigated in this paper. SCMFCs with the clean/pre-colonized anodes and the platinum-based/platinum-free cathodes were operated for 26 weeks. The pre-colonized (4-week colonization) anodes were tested with three areas (2, 10 and 40 cm2) and compared with the MFCs started with clean and sterilized anodes. The power generation of MFCs increased with the anode areas (2–10 cm2), but kept a plateau for the anode area of 40 cm2. The MFCs with the clean anodes had lower power generation (268 mW/m2) than those with the pre-colonized anodes (801 mW/m2) in the first week of operation. With the operation proceeding to 4–5 weeks, the power generation of the clean anodes and pre-colonized anodes became similar and stabilized at 470 mW/m2. In terms of cathode performance, platinum-free cathodes (carbon cloth, surface area: 5 cm2) and platinum-based cathodes (Pt loading: 0.5 mgPt/cm2, surface area: 5 cm2) were compared. The Pt-based cathodes had higher power generation (330 mW/m2) than those of the Pt-free cathodes (253 mW/m2) at the startup period, but the difference quickly vanished after a few weeks of operation. This study demonstrated that the advantage of the MFCs with the pre-colonized anodes and platinum-based cathodes disappeared after 3–5 weeks of operation due to the thick biofilm growth on anodes and the aerobic biofilm formation on cathodes. Pt-free cathodes with controlled biofilm growth are promising for low-cost materials, stable power generation and long-term operation of MFCs.
Keywords: Microbial fuel cell; Platinum-free anode; Anode area; Pre-colonized anode; Power generation;

Hydrolysis kinetics in anaerobic digestion of waste activated sludge enhanced by α-amylase by Kun Luo; Qi Yang; Xiao-ming Li; Guo-jing Yang; Yang Liu; Dong-bo Wang; Wei Zheng; Guang-ming Zeng (17-21).
► The thermodynamics of WAS hydrolysis enhanced by α-amylase was investigated. ► The reaction activation energy was obtained. ► The substances conversion during WAS hydrolysis at the temperature of 50 °C was analyzed. ► The conversion coefficients of VSS to SCOD, carbohydrates and NH4 +–N were obtained. ► ANOVA was used to analyze the effect of enzyme dosage and temperature on WAS hydrolysis.The thermodynamics of waste activated sludge (WAS) hydrolysis process enhanced by additional α-amylase were evaluated in this paper. The effects of enzyme dosage and temperature on WAS hydrolysis were further discussed based on the analysis of variance (ANOVA). The results showed that the rate constant of α-amylase hydrolysis process (α-amylase = 0.06 g/g dry sludge (DS)) increased from 0.106 to 0.215 h−1 with the temperature increasing from 40 to 70 °C, and the reaction activation energy for VSS hydrolysis reduced from 62.72 kJ/mol (control test) to 20.19 kJ/mol (α-amylase treatment). Kinetics analysis indicated that the enzymatic hydrolysis process well fitted the first-order kinetics model at 50 °C, and the conversion coefficients (α) of VSS to soluble chemical oxygen demand (SCOD), carbohydrates and NH4 +–N was found to be 0.266, 0.043 and 0.038, respectively. The model could certainly provide a dynamical description of mechanism and would be benefit to analysis, design, optimization and control of enzymatic hydrolysis process of WAS.
Keywords: α-Amylase; Anaerobic processes; Biodegradation; Dynamic modeling; Hydrolysis; Waste activated sludge (WAS);

Hybrid modeling of counterion mass transfer in a membrane-supported biofilm reactor by Ana R. Ricardo; Rui Oliveira; Svetlozar Velizarov; Maria A.M. Reis; João G. Crespo (22-33).
► Counterion transport in a membrane-supported biofilm reactor was modeled. ► Model was based on combining mechanistic and multivariate techniques. ► Two hybrid modeling strategies were tested: cooperative and competitive mixture-of-experts (MOE). ► MOE structure proved to be better: combines model elements in a mediated network. ► MOE characterized the bioprocess without the need of a complex kinetic model.This paper presents a hybrid mechanistic/statistical model for predicting counterion fluxes across an ion-exchange membrane in a membrane-supported biofilm reactor. The model was calibrated with operating data for the removal of nitrate and perchlorate from a simulated contaminated drinking water stream. Two different modeling strategies were tested: a cooperative parallel hybrid model and a competitive mixture-of-experts (MOE) structure both joining a mechanistic Donnan-dialytic transport model and a multivariate projection to latent structures (PLS) model. The MOE structure proved to be a better predictive tool since it combines the two hybrid model elements in a mediated network. The PLS model was used to identify the process variables that are responsible for the mechanistic model inaccuracy. The results showed that biocompartment physicochemical data need to be considered in the modeling of the transport of counterions across the membrane, especially in situations in which the target counterion (e.g., perchlorate or nitrate) is metabolically reduced in the biocompartment. By using this strategy, the complex biofilm contribution to the transport was accounted for, without the need of developing mechanistic models built on simplified and/or inaccurate assumptions.
Keywords: Modeling; Ion exchange membrane; Membrane bioreactors; Biofilms; Biodegradation; Water treatment;

A novel conductometric creatinine biosensor based on solid-state contact ammonium sensitive PVC–NH2 membrane by Ibrahim Isildak; Osman Cubuk; Melda Altikatoglu; Murat Yolcu; Vildan Erci; Nihat Tinkilic (34-38).
► This study reports a novel, highly sensitive and stable conductometric biosensor. ► The biosensor was based on solid-state contact ammonium-sensitive membrane sensor chip. ► Creatininase was chemically immobilized on the surface of the ammonium sensitive membrane. ► The biosensor exhibited reproducible and stable response at least for 10 h. ► It also showed excellent operational and storage stability for at least 4 weeks.A novel, highly sensitive and stable conductometric biosensor for creatinine determination is developed. The biosensor is based on solid-state contact ammonium-sensitive sensor. Creatininase is chemically immobilized on the surface of the solid-state contact ammonium-sensitive membrane via glutaraldehyde covalent attachment method. The developed conductometric creatinine biosensors demonstrate high sensitivity and short response time toward creatinine. The detection limit of the biosensor was about 2 × 10−6  M and the response time was shorter than 10 s in phophate buffer solution at pH 7.20. The linear dynamic range of the biosensor was between 1 × 10−1 and 9 × 10−6  M creatinine concentration in phosphate buffer solution at pH 7.20. The biosensor exhibited good operational and storage stability for at least 4 weeks kept in dry at 4–6 °C. It had a reproducible and stable response during continuous work at least for 10 h with the relative standard deviation of 0.5% (n  = 48) for creatinine of 1 × 10−3  M in phosphate buffer solution.
Keywords: Conductometric biosensor chip; Creatinine; Biosensor; Creatininase immobilization; Solid-state biosensor;

On the control of molecular weight distribution of polyhydroxybutyrate in Azohydromonas lata cultures by Giannis Penloglou; Eirini Kretza; Christos Chatzidoukas; Sophia Parouti; Costas Kiparissides (39-47).
► High-polymer-content microbial production of PHB with tailor-made molecular properties. ► Effect of key fermentation process variables on the molecular weight distribution (MWD) of PHB. ► Effect of post-treatment process conditions on PHB recovery efficiency and degradation. ► Effect of the MWD of PHB on its recovery efficiency of PHB.The microbial production of polyhydroxybutyrate (PHB) has been reported by several studies dealing with the identification and optimisation of key-process variables and fermentation strategies for the maximisation of polymer yield and productivity. However, little work has been done on the control of the molecular weight distribution (MWD) of PHB, as a product quality variable that significantly affects its end-use properties and processing characteristics. In this study, batch and fed-batch cultures of Azohydromonas lata, as a PHB host bacterium, are conducted in flasks and in a lab-scale bioreactor, where a number of culture nutritional and operational variables (i.e., medium composition, dissolved oxygen concentration, harvesting time, feeding policy and post-treatment conditions) are examined in a comprehensive way for their effect on the time evolution of the molecular properties of the produced PHB. Under specific nutritional conditions, batch or fed-batch cultivation mode and pulse or continuous nutrients addition policies, a wide range of number and weight average molecular weights of PHB was attained. Moreover, the harvested culture post-treatment conditions were assessed and found to greatly affect the MWD of PHB. Finally, the optimal nutritional and operational (i.e., feeding policy) conditions were established for the maximisation of the intracellular PHB content and productivity. In general, the PHB is efficiently produced in A. lata bacteria with relatively narrow and unimodal MWD, however, under extreme conditions favouring the enzymic or mechanical degradation of PHB, a broad and bimodal MWD can be effected
Keywords: Azohydromonas lata; Molecular weight distribution; Fermentation; Polyhydroxybutyrate; Polyhydroxyalkanoates; Polymer recovery efficiency;

The effects of fluoride and aluminum ions on ferrous-iron oxidation and copper sulfide bioleaching with Sulfobacillus thermosulfidooxidans by Tácia C. Veloso; Lázaro C. Sicupira; Isabel C.B. Rodrigues; Larissa A.M. Silva; Versiane A. Leão (48-55).
► Fluoride-containing gangue minerals are soluble in bioleaching liquors. ► Fluoride at 10 mg L−1 completely inhibits S. thermosulfidooxidans growth. ► Al/F molar ratios of 2 and higher provides efficient fluoride detoxification. ► Fe(II) oxidation is increased at sub-lethal fluoride concentrations. ► Bioleaching in the presence of fluoride containing minerals is accomplished with aluminum.Microorganisms that grow at high temperatures can improve Fe(II) bio-oxidation and thereby its technological applications, such as bioleaching and H2S removal. Conversely, elements present in industrial growth media, such as fluoride, can inhibit bacterial growth and iron bio-oxidation. In this work, the influence of fluoride on the kinetics of ferrous-iron bio-oxidation with Sulfobacillus thermosulfidooxidans was investigated. The effects of fluoride concentrations (0–0.5 mmol L−1) on both iron oxidation and bacterial growth rates were assessed. In addition, the effect of the addition of aluminum, which was intended to complex free fluoride and reduce the concentration of HF through the formation of aluminum–fluoride complexes, was also investigated. The results show that 0.5 mmol L−1 NaF completely inhibited bacterial growth within 60 h. Nevertheless, fluoride toxicity to S. thermosulfidooxidans was minimized by control of the aluminum–fluoride ratio in the system because, at a 2:1 aluminum–fluoride molar ratio, bacterial growth was similar to that observed in the absence of fluoride ions. Despite a slower bacterial growth rate, fluoride concentrations less than the inhibitory concentration increased the Fe(II) oxidation rate. Successful copper bioleaching (80–100%) from fluoride-containing sulfide ores (1% total fluoride) was demonstrated in the presence of aluminum.
Keywords: Batch processing; Thermophiles; Growth kinetics; Fluoride toxicity; Aluminum complexes; Waste treatment;

Kinetics of aerobic phenol biodegradation by the acidophilic and hyperthermophilic archaeon Sulfolobus solfataricus 98/2 by Pierre Christen; Armando Vega; Laurence Casalot; Gwenola Simon; Richard Auria (56-61).
► Kinetics of phenol degradation at high temperature and low pH with an archaeon. ► Growth (μ) and specific degradation (q S ) are inhibited by phenol. ► True μ max is half of the fitting parameter (μ*) given by the Haldane model. ► μ max and q Smax are low but inhibition constants (K i , K ′ i ) and optimal phenol concentrations for growth and degradation rates (S m , S ′ m ) are fairly good. ► The yield factor (Y X/S ) depends on phenol concentration.Biodegradation of 51–745 mg l−1 of phenol by a well-acclimatized strain of Sulfolobus solfataricus, a thermoacidophilic archaeon, was studied in batch experiments at 80 °C and pH 3.2. Phenol inhibited growth and specific degradation rates (μ and q S ). Fitting the experimental growth data with the Haldane model gave the following kinetic parameters: μ*  = 0.094 h−1,  K S  = 77.7 mg l−1, K i  = 319.4 mg l−1 (R 2  = 0.950). The true μ max, calculated from μ*, was 0.047 h−1. A volumetric degradation rate (V max) was calculated by fitting the phenol consumption data with the Gompertz model. The value of V max increased with initial phenol concentration (S i ) up to 14.4 mg l−1  h−1. The q S values, calculated from V max, were fitted with the Haldane equation, yielding q Smax of 0.110 g g−1  h−1. The yield factor (Y X/S ) depends on S i and reached a maximum of 0.83 g g−1 at S i  = 93 mg l−1. S. solfataricus 98/2 displayed low μ max and q Smax but a good tolerance to phenol (fairly high K i , K ′ i , high Y X/S ). This ability to grow on and degrade phenol (93 mg l−1  < optimal S i  < 175 mg l−1) at high temperature and low pH is unique and may be useful for removing phenol from hot acidic contaminated effluents. Other possible application could lie in the production of the enzymes involved in the key steps of phenol degradation provided the cloning of the enzymes-related genes in fast-growing mesophiles.
Keywords: Phenol; Biodegradation; Hyperthermophiles; Batch culture; Growth kinetics; Haldane model;

Co-metabolic models of Streptococcus thermophilus in co-culture with Lactobacillus bulgaricus or Lactobacillus acidophilus by Ricardo Pinheiro de Souza Oliveira; Beatriz Rivas Torres; Patrizia Perego; Maricê Nogueira de Oliveira; Attilio Converti (62-69).
To shed light on the interactions occurring in fermented milks when using co-cultures of Streptococcus thermophilus with Lactobacillus bulgaricus (StLb) or Lactobacillus acidophilus (StLa), a new co-metabolic model was proposed and checked either in the presence of inulin as a prebiotic or not. For this purpose, the experimental data of concentrations of substrates and fermented products were utilized in balances of carbon, reduction degree and ATP. S. thermophilus exhibited always quicker growth compared to the other two microorganisms, while the percentage of lactose fermented to lactic acid, that of galactose metabolized, and the levels of diacetyl and acetoin formed strongly depended on the type of co-culture and the presence of inulin. The StLb co-culture led to higher acetoin and lower diacetyl levels compared to StLa, probably because of more reducing conditions or limited acetoin dehydrogenation. Inulin addition to StLa suppressed acetoin accumulation and hindered that of diacetyl, suggesting catabolite repression of α-acetolactate synthase expression in S. thermophilus. Both co-cultures showed the highest ATP requirements for biomass growth and maintenance at the beginning of fermentation, consistently with the high energy demand of enzyme induction during lag phase. Inulin reduced these requirements making biomass synthesis and maintenance less energy-consuming. Only a fraction of galactose was released from lactose, consistently with the galactose-positive phenotype of most dairy strains. The galactose fraction metabolized without inulin was about twice that in its presence, which suggests inhibition of the galactose transport system of S. thermophilus by fructose released from partial inulin hydrolysis.
Keywords: Streptococcus thermophilus; Lactic acid bacteria; Co-culture; Co-metabolic models;

.Display Omitted► The interactions between insulin and EGCG are found always enthalpically favorable. ► Hydrogen bonding is essential in the interactions. ► Both ΔH and ΔS for the binding increase with the increase of EGCG concentration. ► The binding of EGCG only slightly changes the secondary structure of insulin.Isothermal titration calorimetry (ITC), circular dichroism (CD) spectroscopy, fluorescence spectroscopy and dynamic light scattering were utilized to investigate the molecular interactions between human insulin and EGCG. The ITC results suggest that the binding stoichiometry is independent of pH, salt concentration and temperature, but increases with the increase of EGCG concentration. The interactions between insulin and EGCG are always enthalpically favorable, indicating that hydrogen bonding is always involved in the binding process. However, with the increase of EGCG concentration, both ΔH and ΔS increase gradually, indicating an enhanced contribution of hydrophobic interaction at the physiological condition. The binding of EGCG to insulin can be promoted by increasing temperature at about 120 mmol/L NaCl and pH values away from insulin's isoelectric point. The CD results indicate that the binding of EGCG slightly changes the secondary structure of insulin. The fluorescence spectra reveal that EGCG binding gives little effect on the polarity of the environments in which tyrosine residues exist and the solvent-accessible hydrophobic surfaces of insulin. The addition of EGCG decreases the hydrodynamic radius of insulin, indicating that EGCG directly binds to the protein
Keywords: Biophysical chemistry; Protein; Aggregation; Protein denaturation; (−)-Epigallocatechin-3-gallate; Molecular interactions;

Optimisation of olive oil extraction and minor compounds content of Tunisian olive oil using enzymatic formulations during malaxation by Noomen Hadj-Taieb; Naziha Grati; Mohamed Ayadi; Imen Attia; Houda Bensalem; Ali Gargouri (79-85).
► We tested the effect of enzymatic formulation on Tunisian olive oil extraction yields. ► We tested fungal pectinolytic and cellulolytic enzymes. ► We enhanced the oil extracted by 1.5% in the presence of added enzymes. ► We improved the extraction of minors’ compound such as polyphenols and pigments.The aim of this work was to investigate and evaluate culture filtrates of different enzymatic formulations in terms of their individual and synergistic effects with regard to the quality and extraction yields of Tunisian olive oil from the Chemlali Sfax variety. The formulations, which contained a number of hydrolytic enzymes, particularly pectinases, xylanases and cellulases were tested both separately and in combination. The results demonstrated that when compared to those of the control, the oil extraction yields of both types of olives (green and black) treated by the enzymatic formulations were enhanced by 1.5%. Interestingly, the synergistic effect of different activities (pectinase, cellulase and xylanase) was able to improve the extraction of polyphenols to 10% and of pigment compounds, namely chlorophylls and carotenoids, by 25 and 30%, respectively. The olive oil stability, particularly the one from green olives, which was treated by the enzymatic mixture, was observed to increase up to 3.5 h. Overall, the findings presented in the current study demonstrate that the enzymatic formulations under investigation exhibited promising properties and attributes which make them potential strong candidates for future application in the oil industry.
Keywords: Olive oil; Pectinase; Cellulase and xylanase; Polyphenols; Pigments;

► We expressed soluble form of human Fas ligand (shFasL) in Escherichia coli and Dictyostelium discoideum, respectively. ► We developed a newly completely synthetic medium for high cell density cultivation of D. discoideum. ► We used Ni-NTA affinity chromatography to purify the recombinant shFasLs expressed by the two different expression systems. ► The two kinds of recombinant shFasLs showed similar biological activities in inducing apoptosis in Fas-expressing cells.Human Fas ligand (hFasL) is a type II membrane protein that induces apoptosis in the Fas-bearing cells. Its special biological activity has the potential for the therapeutic use as an anti-cancer agent directed at enhancing apoptosis in tumor cells. In this study Escherichia coli and eukaryotic Dictyostelium discoideum were used to produce a soluble form of hFasL in large amounts. An expression vector for hFasL production in E. coli was constructed based on plasmid pET32a(+). By cultivation of the hFasL-producing E. coli clone on LB medium and induction with IPTG, a hFasL concentration of 1.0 mg L−1 was achieved. D. discoideum strain AX3-hFasL-H was cultured in a conventional stirred bioreactor on an improved synthetic medium using a simple fed-batch strategy, and cell densities of up to 8.3 × 107  cells/mL and a maximum hFasL concentration of 420 μg/L were obtained. Using Ni-NTA affinity chromatography purification, two kinds of recombinant hFasLs from E. coli and D. discoideum were purified with a purity of 94% and 90%, respectively. They showed similar biological activities in inducing apoptosis in Fas-expressing cells.
Keywords: Human Fas ligand; Escherichia coli; Dictyostelium discoideum; Soluble production; Affinity chromatography; Cytotoxic activity;

Particle shrinkage and morphology of milk powder made with a monodisperse spray dryer by Samuel Rogers; Winston Duo Wu; Sean Xu Qi Lin; Xiao Dong Chen (92-100).
► Using same-sized droplets in a small spray dryer, we produced uniform milk powder. ► Drying was accurately modelled with the Reaction Engineering Approach. ► The particle shrinkage is greater for feeds with low initial solids content. ► Particles generally buckle inwards, although they can inflate at higher drying temperatures.The drying of monodisperse droplets can produce uniform powders where every particle has experienced similar, predictable air conditions. For the first time, a single-stream dryer was used with monodisperse milk droplets having solids contents as high as those used in industrial spray dryers (over 40 wt% total solids). With size measurement of droplets and powders, the particle shrinkage was precisely determined. Changes in shrinkage and morphology were observed when varying feed solids contents and drying temperatures. The majority of particles had an inwardly buckled morphology and hollow, inflated particles were produced at higher temperatures due to boiling. Drying was modelled with a numerical simulation using the Reaction Engineering Approach (REA), which is a semi-empirical model of moisture removal rate. This model accurately predicted the moisture contents of the powders collected. Several sub-models were used for particle shrinkage. The most accurate shrinkage assumption was found to be isotropic shrinkage from the removal of water (known as perfect shrinkage). This may suggest that the surface crust (initially spherical) is viscoelastic and deforms in response to drying stresses.
Keywords: Food engineering; Monodisperse droplet drying; Mass transfer; Modelling; Reaction Engineering Approach; Image analysis;

Functionality of milk protein concentrate: Effect of spray drying temperature by Yuan Fang; Sam Rogers; Cordelia Selomulya; Xiao Dong Chen (101-105).
SEM images of relatively uniform Milk Protein Concentrate particles with distinct morphologies generated using a pilot scale microfluidic spray dryer at different inlet air temperatures of (a) 77 °C; (b) 178 °C.Display Omitted► Effects of spray drying conditions on functionality of Milk Protein Concentrate (MPC). ► Generation of monodisperse particles eliminates size variation and uneven drying conditions. ► Drying directly affects changes in particle morphology, protein denaturation and functionality. ► Effective drying at low temperature with proper contacts between liquid droplets and drying air.This study investigated the effect of spray-drying conditions on the resulting functionality of milk protein concentrate (MPC), utilizing specially produced mono-dispersed MPC particles. The particles were generated from a pilot microfluidic spray dryer at selected inlet air temperatures (77 °C, 107 °C, 155 °C and 178 °C). The solubility and extent of protein denaturation were characterized using focused beam reflectance measurement and polyacrylamide gel electrophoresis. Due to the controlled drying conditions and well-defined properties of the particles, a direct relationship between spray drying temperatures and the resultant particle functionality could be identified. The particle morphologies obtained from lower inlet air temperature appeared spherical whereas the one from higher inlet air temperature appeared deflated. The FBRM results indicated that the solubility of MPC particles deteriorated with increasing inlet air temperature with increasing protein denaturation. SDS-PAGE results suggested that the insoluble material were primarily casein rather than heat sensitive whey protein. These findings could be used to establish a better understanding of the relationship between drying conditions and MPC microstructures, and the corresponding influence on the functionality for non-traditional powder types.
Keywords: Spray drying; Drying air temperature; Mono-disperse particles; Solubility; Protein denaturation;