Biochemical Engineering Journal (v.28, #2)

BEJ Keywords (II).

IFC (CO2).

Kinetic study of fermentative biosurfactant production by Lactobacillus strains by Lígia Rodrigues; Ana Moldes; José Teixeira; Rosário Oliveira (109-116).
Screening of biosurfactant-producing ability of four Lactobacillus strains was performed, being shown that for all the tested strains biosurfactant production occurred mainly in the first 4 h. The Lactobacillus strains showed zones of clearing in the blood agar with a diameter <1 cm. The minimum surface tension value of the fermentation broth achieved was 39.5 mN/m for Lactobacillus pentosus CECT-4023 that represents a reduction in the surface tension of 10.5 mN/m comparing with the control.Time courses of glucose, biomass and biosurfactant were modeled according to reported models. Using MRS broth (Man, Rogosa & Sharpe medium for lactobacilli strains) as culture medium, the values estimated by the modeling of biosurfactant were P max  = 1.6 g of biosurfactant/L and r p/X  = 0.091 g/(L h), for Lactobacillus casei CECT-5275, P max  = 1.7 g/L and r p/X  = 0.217 g/(L h) for Lactobacillus rhamnosus CECT-288, P max  = 1.7 g/L and r p/X  = 0.069 g/(L h) for L. pentosus CECT-4023 and P max  = 1.8 g/L and r p/X  = 0.090 g/(L h) for Lactobacillus coryniformis subsp. torquens CECT-25600. P max is the maximum concentration of biosurfactant (g/L), and P r is the ratio between the initial volumetric rate of product formation (r p) and the initial product concentration P 0  (g/L). Using whey as production medium, the values estimated by the modeling of biosurfactant for L. pentosus CECT-4023 were P max  = 1.4 g of biosurfactant/L and r p/X  = 0.093 g/(L h).In conclusion, the results obtained for L. pentosus CECT-4023 showed that this is a strong biosurfactant producer strain and that cheese whey can be used as an alternative medium for biosurfactant production.
Keywords: Biosurfactant production; Surface tension; Fermentation; Lactobacillus;

In this study, sludge reduction in a conventional activated sludge (CAS) process where Aeolosoma hemprichi (one species of oligochaeta) was inoculated as a micro-fauna was tested. The factors affecting the growth of A. hemprichi and the influence of A. hemprichi on treatment performance were investigated. The observed specific growth rate of A. hemprichi decreased at lower SRT, and the SRT at which A. hemprichi would be totally exhausted was estimated about 3 days. Obvious appearance of A. hemprichi was observed when the F/M ratio was lower than 0.7 mg-COD/(mg-VSS day). A. hemprichi grew independent of sludge concentration when SS concentration was higher than 300 mg-VSS/L, and more easily predated on sludge floes with higher protein content or smaller particle size. The apparent sludge yield was found to have a negative correlation with the density of A. hemprichi in the CAS reactor. The relative sludge reduction by A. hemprichi was estimated to be about 39–65%, and the apparent sludge reduction rate per unite weight of A. hemprichi was from 0.53 to 6.32 mg-VSS/(mg-A. hemprichi day). The existence of A. hemprichi was beneficial to stabilize the sludge settleability and TP removal but did not affect COD and NH4 +-N removals in the process. The results indicated that using A. hemprichi to reduce sludge in wastewater treatment was feasible.
Keywords: Aeolosoma hemprichi; Predation; Sludge reduction; Wastewater treatment;

Feasibility of a membrane-aerated biofilm reactor to achieve controllable nitrification by A. Terada; T. Yamamoto; R. Igarashi; S. Tsuneda; A. Hirata (123-130).
A feasibility of a membrane-aerated biofilm reactor (MABR) for controllable nitrification was examined. The estimation of oxygen supply rate (OSR) with three polyacrylonitrile membrane modules revealed that specific OSR was equivalent in these membrane modules and OSR affected only air pressure, thus enabling control of aeration simply by adjustment of air pressure. A continuous nitrification experiment consisting of three reactors differing in membrane surface area investigated the reactor performance of the MABR at an air pressure of 23 kPa. The results indicated that the ammonia removal rate at steady state was dependent on membrane surface area, at rates nearly equivalent to that predicted by the above OSR experiment. The amount of bacteria adhering to the membrane surface was not completely proportional to membrane surface area due to clogging in a reactor with high membrane surface area, which accompanies a decrease in specific ammonia removal rate per biomass with membrane surface area. Stable ammonia removal rates at air pressures of 23, 45 and 100 kPa corresponded to the predicted values from the OSR experiment. Further, more than 80% oxygen utilization efficiency (OUE) was achieved under all operational conditions, indicating effective oxygen uptake by nitrifying bacteria under oxygen-depleted conditions. Based on these experiments, the MABR was shown to be a controllable nitrification system, and to be able to provide a reaction space for nitrification in a membrane-attached biofilm without altering the bulk conditions.
Keywords: Membrane-aerated biofilm reactor (MABR); Nitrification; Oxygen-depleted conditions; Oxygen supply rate (OSR); Oxygen utilization efficiency (OUE);

Most of the controlled-release systems developed for drug delivery applications depend on membrane technology. The dense structure of some membranes used in controlled-release systems can excessively prolong the release of drug due to the low permeability of the coating to drug. To increase the drug release rate, asymmetric-membrane tablet coatings were prepared by a phase-inversion technique using cellulose acetate/acetone/water solution. The roles of the composition of the membrane solution and the evaporation condition on the release rate of drug were determined using in vitro dissolution and morphological studies and predicted phase diagrams. Results show that drug release from asymmetric-membrane based tablet coatings is primarily governed by the dynamics of the phase-inversion process with zero-order or near-zero-order release easily achievable. In an attempt to derive an empirical expression for the release rate of drug as a function of composition of the coating solution, a statistical experimental design was used. Good fit of the experimental data by the empirical expression was obtained. In addition, the predictive capability of the model equation was also found to be satisfactory. Analysis of the significance of each term in the expression indicates that the cellulose acetate:acetone ratio has the most significant influence on the release rate of theophylline.
Keywords: Asymmetric-membrane; Phase-inversion; Statistical design; Cellulose acetate; Theophylline;

Polyethyleneimine (PEI) was grafted onto the surface of silica gel particles via the coupling effect of γ-chloropropyl trimethoxysilane (CP), and the novel composite support PEI/SiO2 was prepared. The chemical structure and composition were characterized with infrared spectrum and conductometric titration, and its surface electrical property was also measured. The character of the immobilized enzyme on the novel composite support was studied by using penicillin G acylase as a model enzyme. By using the double functional group reagent glutaraldehyde, penicillin G acylase was immobilized on the composite support PEI/SiO2. The effects of various immobilization conditions on the apparent activity of the immobilized enzyme were examined, such as time, the used amount of glutaraldehyde, pH value and so on. The mechanism of immobilizing enzyme was probed into. The suitable temperature and pH value for using the immobilized enzyme to catalyze the hydrolysis reaction of penicillin G were researched, and the continuous operating stability of the immobilized enzyme was examined. The experiment results show that the combining of less quantity of covalent linking with a great deal of physical adsorption caused by a great number of amino groups in PEI macromolecules not only can lead to faster immobilizing of penicillin G acylase, but also can result in retaining well the conformation of enzyme, and the immobilized enzyme has higher activity. Compared to the free enzyme, the ranges of suitable temperature and pH value are all widened as the immobilized enzyme is used, the thermal stability of the immobilized enzyme is also enhanced distinctly, and the immobilized enzyme has better continuous operating stability and its activity can keep at the level of 87.5% of fresh one all along after using 15 times repeatedly.
Keywords: Penicillin G acylase; Polyethyleneimine; Silica gel; Composite support; Immobilization;

This study explored the effect of temperature and carbon source on the denitrification process. Batch experiments were conducted within the 10–30 °C range, using either acetic acid, propionic acid or an 1:1 mixture (by weight) of the same acids as an external carbon source. A 5 M NaNO3 solution was added to yield nitrate-nitrogen concentrations of 50, 100 and 200 mg/L, and carbon to nitrogen (C:N) ratios of 2:1 and 4:1, respectively. It was observed that denitrification was feasible under all conditions investigated, provided that the initial reactor pH was adjusted to approximately 6.5. Overall, a temperature change from 10 to 20 °C exerted a greater effect on both the specific denitrification and carbon consumption rates than a further temperature increase from 20 to 30 °C, which was also evident in the corresponding temperature coefficient values. At a given temperature, the specific denitrification rate appeared to depend on the initial nitrogen concentration, while the specific carbon consumption rate was a function of the initial carbon content. Furthermore, a C:N ratio of 2:1 was sufficient for complete denitrification at all temperatures and types of carbon investigated. During the mixed VFA experiments, acetic acid was utilized faster than propionic acid, indicating a preferential utilization pattern. Finally, substantial alkalinity recovery was also observed in all runs, in accordance with the theoretically calculated values.
Keywords: Denitrification; Volatile fatty acids; Temperature; Nitrate; Specific rate; Carbon consumption;

Solubilization of horse heart cytochrome c into reverse micelles, a self-assembled nanostructure composed of sodium di-2-ethylhexyl sulfosuccinate (AOT), enhances the peroxidase activity in the presence of hydrogen peroxide. The catalytic activity of cytochrome c hosted in reverse micellar solution is 10-fold higher than that in water, and which depends on Wo, a molar ratio of water to AOT, and pH in aqueous droplets. In addition, the fluorescence intensity based on the tryptophan residue and the visible absorption identifying the axial Met 80-Fe bond imply that the reverse micellar solubilization induces the cleavage of the heme crevice and thus enables the formation of peroxidase-like peroxide-heme complexes. These results suggest that cytochrome c solubilized in reverse micelles provides a homogeneous catalyst as peroxidase available in organic media.
Keywords: Microemulsion; Peroxidase; Surfactant; Oxidation;

Purification of potato polyphenol oxidase (PPO) by partitioning in aqueous two-phase system by Bhalchandra K. Vaidya; Hitesh K. Suthar; Sangita Kasture; Sanjay Nene (161-166).
The aim of this work is to extract and purify potato polyphenol oxidase (PPO) from the potato tuber (Solanum tuberosum) using aqueous two-phase system (ATPS). The PPO was extracted by partitioning in ATPS composed of polyethylene glycol (PEG) and phosphate. Effect of phase composition, molecular weight of PEG and pH of the system on enzyme partitioning and selective enrichment was studied. The optimum system was found at pH 7.0 containing 5% PEG-8000 and 28.5% phosphate, with K a of 32.3, purification factor of 15.7 and a 97.0% yield of enzyme activity in the top phase.
Keywords: Aqueous two-phase systems; Polyphenol oxidase; Downstream processing; Protein recovery; Enzyme purification;

An improved differential evolution method for efficient parameter estimation in biofilter modeling by T.R. Bhat; Divya Venkataramani; V. Ravi; C.V.S. Murty (167-176).
The task of mathematical modeling of biofilters is weighed down by a lack of reliable information about some parameters, which decisively influence the substrate biodegradation process. Biofilm thickness is one such parameter. Biofilters handle diverse substrate loading rates and gas phase velocities, which have a significant effect on the biofilm thickness. The problem of evaluation of this parameter is further compounded by the fact that there are other related issues, which need to be addressed. Secondly, information about biokinetics is rather hard to get because of problems with experimentation. Thirdly, air/water distribution coefficients are used as a rule for predicting the amount of substrate going into the biolayer from the gas phase, when, in fact, air/biofilm distribution coefficients should be used. Henry's law distribution coefficients for the substrate in actual conditions are equally difficult to determine. Inverse modeling is a viable strategy for estimation of these critical parameters. An important component of the inverse modeling is an efficient optimization technique. In this study, an improved differential evolution method (IDE) has been proposed and applied for the evaluation of the parameters mentioned above, in the framework of the solution of an inverse problem. It has been demonstrated that, the IDE, which is a variant of the original DE method, is twice as efficient as the DE method in terms of computational speed for the same level of accuracy. A case study involving the biodegradation of phenol in air streams has been investigated to validate the models proposed.
Keywords: Biodegradation; Biofilms; Biofilters; Improved differential evolution; Optimization; Parameter estimation;

This manuscript presents the UV–vis study of the aniline elimination by polymerization using peroxidases from different sources and hematin, free and supported. The use of magnetite-supported hematin has potential in the implementation of aniline elimination by polymerization in acidic media at pH 4. The temperature must be selected between 45 and 65 °C. Crude preparations of Pleurotus sajor caju (PSC) are an alternative to purified Horseradish peroxidase (HRP) as the catalyst of the reaction. Pernigraniline is the main species found in the reaction media after aniline elimination procedure. Supported oxidoreductases are active biocatalysts and conversion of aniline in the 30–90% range can be obtained, depending on the catalyst. The UV–vis spectra show bands corresponding to base and protoned (blue and purple-deep red) pernigraniline, a contribution of quinoid species at 480 nm and a minor contribution of base blue emeraldine and another oxidation intermediate states between pernigraniline and emeraldine. At selected conditions, the recovered solid showed greenish/reddish coloration. The goal is the precipitation of the aniline as polyaniline. Supported biocatalysts were less active than free enzymes.
Keywords: Magnetite; Peroxidase; Hematin; Enzymatic;

The kinetics and thermodynamics of copper(II) and lead(II) biosorption onto Aspergillus niger pretreated with NaOH were studied with respect to pH, temperature and initial metal ion concentration. The optimum pH values were determined as 5.0 and 4.0 for copper(II) and lead(II) at 25 °C, respectively. Biosorption capacity values of the biomass increased with increasing initial metal ion concentration and temperature. The maximum biosorption capacities were obtained as 28.7 and 32.6 mg g−1 at 250 mg dm−3 initial copper(II) and lead(II) concentration at 35 °C, respectively. The equilibrium data were analyzed using Freundlich, Langmuir and Redlich–Peterson adsorption models. The model parameters were estimated by the non-linear regression analysis. It was seen that equilibrium data fitted very well to the Langmuir adsorption model at all temperatures studied. The applicability of the saturation type kinetic model for metal–A. niger system was tested at different temperature in the range of 20–35 °C. The activation energies of the biosorption were determined as 27.48 and 36.76 kJ mol−1 for Cu(II) and Pb(II)–A. niger systems, respectively. Using the thermodynamic equilibrium coefficients obtained at different temperature, the Gibbs free energy changes (−18.97 kJ mol−1 for Cu(II) and −22.97 kJ mol−1 for Pb(II) (at 35 °C)), enthalpy changes (28.9 kJ mol−1 for Cu(II) and 38.3 kJ mol−1 for Pb(II)) and entropy changes (0.168 kJ mol−1  K−1 for Cu(II) and 0.186 kJ mol−1  K−1 for Pb(II)) of biosorption were also determined. The results showed that biosorption of copper(II) and lead(II) on A. niger was endothermic and spontaneous.
Keywords: Biosorption; Copper(II); Lead(II); Aspergillus niger; Adsorption isotherm; Saturation type kinetic model; Activation energy; Gibbs free energy; Enthalpy; Entropy;

The interaction between digestion conditions and the sludge physical characteristics and behaviour was investigated for anaerobically digested primary sludge in completely stirred tank reactors (CSTRs). The CSTRs were operated to maintain sludge retention times (SRTs) of 10, 15, 20 and 30 days and temperatures of 25 and 35 °C. The change of the floc size as a result of digestion was examined using wet sieve analysis (0.100, 0.125, 0.200, 0.500 and 1.000 mm). The results reveal a substantial reduction in all floc sizes with improving digestion conditions. Digestion leads to the transfer of bigger flocs into smaller ones, which has a remarkable effect on the sludge physical behaviour. The majority of the raw and digested flocs are smaller than 0.100 mm. The dewatering results showed the existence of an optimal SRT for dewaterability at 20 and 15 days for the reactors operated at 25 and 35 °C, respectively. The dewaterability of sludge digested at less favourable conditions, viz. 10 days at 25 °C deteriorates due to increase of small flocs generated from destruction of larger flocs. The digested sludge settling results showed a slight worsening but insignificant trend with increasing the SRT.
Keywords: Anaerobic processes; Dewaterability; Floc size distribution; Mixing; Optimisation; Waste treatment;

The biooxidation of ferrous ion into ferric ion by Acidithiobacillus ferrooxidans can be potentially used for the removal of H2S from industrial gases. In this work, Fe3+ ions were obtained through the oxidation of Fe2+ using the LR strain of At. ferrooxidans immobilized in PVC stands in a pilot-scale bioreactor, while H2S was removed in an absorption tower equipped with Rasching rings. At. ferrooxidans LR strain cells were immobilized by inoculating the bacterium in a Fe2+-mineral medium and percolating it through the support. After complete Fe2+ oxidation, which took around 90 h, the reactor was washed several times with sulfuric acid (pH 1.7) before a new cycle was started. Four additional cycles using fresh Fe2+ mineral medium were then run. During these colonization cycles, the time required for complete iron oxidation decreased, dropping to about 60 h in the last cycle. The batch experiments in the H2S gas removal trials resulted in a gas removal rate of about 98–99% under the operational conditions employed. In the continuous experiments with the bioreactor coupled to the gas absorption column, a gas removal efficiency of almost 100% was reached after 500 min. Precipitate containing mainly sulfur formed during the experimental trial was identified by EDX.
Keywords: Biological oxidation; Bioreactors; Acidithiobacillus ferrooxidans; Ferrous sulfate; Immobilization; Hydrogen sulfide;

Performance and mechanism in binding of Reactive Orange 16 to various types of sludge by Sung Wook Won; Sun Beom Choi; Yeoung-Sang Yun (208-214).
Four types of waste sludge were collected from a drinking water treatment plant (waterworks sludge; WS), sewage treatment plant (sewage sludge; SS), anaerobic digestion (digested sludge; DS) and landfill site (landfill sludge; LS). These sludges were evaluated as biosorbents for the removal of Reactive Orange 16 (RO 16). The maximum sorption capacities of the four waste sludges estimated using the Langmuir equation at pH 2 were in order LS (159.0 ± 6.0 mg g−1) > SS (114.7 ± 4.7 mg g−1) > DS (86.8 ± 4.5 mg g−1) > WS (47.0 ± 5.8 mg g−1). However, the desorption efficiency of LS was insufficient (43.2%) compared with that of SS (90.0%), indicating that the SS has the potential to be regenerated as a biosorbent. From the potentiometric titration and FTIR study, the binding sites present in the SS were considered to be protonated amine groups (–NH3 +), which play a role in the electrostatic interaction with the negatively charged sulfonate groups of RO 16.
Keywords: Biosorption; Batch processing; Separation; Affinity; Waterworks sludge; Sewage sludge; Digested sludge; Leachate sludge; Reactive Orange 16;