Biochemical Engineering Journal (v.66, #C)

BEJ Keywords (IV).

► Immobilized catechol 2,3-dioxygenase (C23O) exhibits improved storage stability. ► Immobilization of C23O enhances its resistance to inactivation by autooxidation. ► Immobilization of C23O from KB2 strain protects the enzyme from the inhibition.In this paper we report the immobilization of catechol 2,3-dioxygenase from Stenotrophomonas maltophilia KB2 in alginate hydrogel with the aim of improving its functional stability by increasing structural rigidity of the enzyme. Immobilization yield and expressed activity were 49.4% and 49.4%, respectively. The storage stability of entrapped catechol 2,3-dioxygenase at 4 °C was found up to 35 days (266.3 mU/mg protein), while at 4 °C the free enzyme lost its activity within 24 h. Immobilization of dioxygenase increased the optimum temperature for activity by 10 °C, while both soluble and immobilized enzyme showed maximum activity at the same pH. The K m, V max, and Hill constant values for immobilized enzyme were 0.2 μM, 604.6 mU/mg protein, and 1.00, respectively, whereas those for the free enzyme were 46.3 μM, 1602.0 mU/mg protein, and 4.1, respectively.The immobilized catechol 2,3-dioxygenase from KB2 strain showed relatively higher activity against 3-methylcatechol, 4-methylcatechol, 4,5-dichlorocatechol, 3,5-dichlorocatechol, hydroquinone and tetrachlorohydroquinone than soluble enzyme. Immobilization of catechol 2,3-dioxygenase from KB2 strain protected the enzyme from the inhibition and enhanced its resistance to inactivation during catalysis. That makes the enzyme suitable for the bioremediation and detoxification of xenobiotic-contaminated environments.
Keywords: Immobilization; Biodegradation; Kinetic parameters; Substrate inhibition; Stenotrophomonas; Catechol 2,3-dioxygenase;

► We examine the phospholipids profiles in biofilms exposed or unexposed to inhibitor. ► We examine the O2 respiration profiles in biofilms exposed or unexposed to inhibitor. ► Reduction in total phospholipid and O2 respiration were used to evaluate inhibition. ► We found the discrepancy in results from two methods. ► We attribute it to the delay of loss of phospholipids to decrease in O2 respiration.To elucidate the toxic effect of pentachlorophenol (PCP) on the microbial activities of biofilms, we used two methods to evaluate the inhibition caused by PCP. The spatial distribution of phospholipids in biofilms was determined by phospholipid analysis combined with the micro-slicing technique. The inhibition was derived from the reduction in total phospholipid concentrations on the basis of the phospholipid profile in biofilms before and after exposure to PCP. Moreover, using O2 microprofiles as bases, we determined the spatial distributions of the net specific O2 respiration in biofilms with and without exposure to PCP. Furthermore, the inhibition was derived from the reduction in respiratory rates in biofilms. We attribute the discrepancy in results from the two methods to the delay of the loss of phospholipids compared with the decrease in the oxygen uptake rate in response to PCP.
Keywords: PCP; Biofilms; Phospholipid analysis; Micro-slicing; Microelectrode; Oxygen respiration rate;

► We study the production of l-DOPA using a biocatalytic zeolite membrane. ► We study the role of the zeolite membrane as free radical scavenger. ► Results: Biocatalytic zeolite membrane enhances the performance of the tyrosinase. ► Results: Possibility to regenerate the zeolite membrane by thermal treatment. ► Results: The specific activity of the immobilised enzyme was higher than free form.The production of l-3,4-dihydroxyphenylalanine (l-DOPA) from l-tyrosine catalysed from mushroom tyrosinase immobilised in a continuous membrane reactor was studied and compared with free enzyme used in a stirred tank reactor. The enzyme was immobilised by cross-flow filtration in asymmetric tubular membranes made of polyamide having nominal molecular weight cut off of 20 kDa.Results showed that the immobilization procedure did not alter the catalytic properties of the enzyme. The immobilised tyrosinase showed high stability over 30 h. In a continuous bioreactor the specific activity of tyrosinase is higher than that obtained with the free enzyme and those reported in the literature. The continuous operation minimizes the oxidation of the l-DOPA and for this reason the productivity (22.54 mg L−1  h−1) is 95% higher than that obtained with the total recycle reactor.Results obtained in the present study are promising for the development of the bioreactor at larger scale.
Keywords: Immobilised enzyme; Tyrosinase; Polyamide membranes; Continuous membrane bioreactor; l-DOPA production; Specific activity and productivity;

► A new wastewater treatment process combining an AO-MBR with thermochemical sludge disintegration is developed and tested. ► The influence of FeSO4 on the sludge disintegration process is first examined. ► The observations showing that the thermochemical sludge disintegration is fully compatible with conducted AO-MBR system. ► The thermochemically sludge disintegration process is only slightly affected by FeSO4 addition. ► The effluent water qualities and membrane operation are not significant affected during over 9 months of operation.A new wastewater treatment process combining an anoxic–oxic membrane bioreactor (AO-MBR) with thermochemical sludge disintegration step was developed and tested in laboratory scale experiments. The study was performed on two developed AO-MBR systems, namely a P-MBR (practical system) and a C-MBR (control system), run in parallel and continuously fed with real domestic wastewater. In Run 1, in the P-MBR, a part of the mixed liquid (1.5% of the influent flow rate) was disintegrated thermochemically (at 80 °C, pH 11 and 3 h) and was sent back to the bioreactor. During this period, sludge solubilization efficiency was about 20–22%. Sludge production rate of the P-MBR was less than that of the C-MBR about 33%. In Run 2, ferrous sulfate (FeSO4·7H2O) was added into the P-MBR to control the total phosphorus in the effluent less than 1.0 mg/L. It is interesting that the sludge solubilization efficiency in this period was reduced down to 17–19% showing that the thermochemically sludge disintegration process was only slightly affected by the FeSO4·7H2O addition. The reduction of sludge production rate of the P-MBR compared with that of the C-MBR was only 24%. The MLVSS/MLSS ratio was reduced from 75% to 68%. Regardless of the FeSO4·7H2O addition, the TN removal efficiencies were maintained around 62% in both systems. In conclusion, the sludge disintegration plays an important role in reduction of sludge production rate. Although the adding FeSO4·7H2O caused a reduction in sludge production rate, the effluent water qualities and membrane operation were not significant affected during the operation of over 9 months.
Keywords: Ferrous sulfate; Membrane bioreactor; Nutrient removal; Thermochemical disintegration; Sludge reduction;

Design of a microfluidic respirometer for semi-continuous amperometric short time biochemical oxygen demand (BODst) analysis by Albert Torrents; Jordi Mas; Francesc Xavier Muñoz; Francisco Javier del Campo (27-37).
► A highly compact, novel electrochemical dual flow cell has been designed. ► The design is optimized for manufacturing using mass production techniques. ► The system targets process control in wastewater treatment and food technology. ► Theory demonstrates feasibility of the semi-continuous determination of the BODst. ► The determination of a wide range of organic loadings may be accessible.This paper presents the design of a miniaturized electrochemical respirometer to monitor organic content in water samples semi-continuously, in contrast to current biochemical oxygen demand, BOD, methods. We demonstrate the use of finite element method simulations as design tool for a novel concept microfluidic respirometer. The device is based on a flow cell separated by a thin membrane from a bioreactor. Simulations show that once membrane material and thickness are chosen, oxygen supply rate still provides sufficient flexibility to allow the measurement of samples spanning a very wide range of organic matter concentrations. The design presented here uses an electrochemical oxygen sensor, and the whole system is amenable to fabrication using standard microfabrication and rapid prototyping techniques.
Keywords: Modeling; BOD biosensor; Respirometry; Electrochemistry; Microfluidics; Finite element method;

► Efficient removal of anti-inflammatory compounds in the STRs and FBRs. ► Significant removal of CBZ and DZP were found in both bioreactors. ► The operation of STRs and FBRs was stable for 50 and 100 days, respectively. ► Gas supply in FBRs diversely affected the degradation efficiency of DZP and CBZ. ► The major degradation products of DCF, IBP and NPX were identified.Stirred tank reactors (STRs) and fixed-bed reactors (FBRs) were applied for the removal of diclofenac, ibuprofen, naproxen, carbamazepine and diazepam by Phanerochaete chrysosporium. The operation of STRs with free pellets and immobilized fungus attained stable operation for 50 days. Both bioreactors achieved high removal efficiencies for diclofenac, ibuprofen and naproxen while partial removal of carbamazepine and diazepam. The configuration of the FBR maintained a steady and feasible operation for 100 days with complete removal of diclofenac, ibuprofen and naproxen regardless of the aeration system (air or oxygen) and remarkably high removal percentages of carbamazepine and diazepam: 60–90%. The extraction of the target compounds from the biomass and the support showed low residual concentrations of all the compounds both on fungal pellets and on the support of the FBR. Only significant values were detected in the STR with immobilized mycelium, demonstrating partial adsorption. A tentative identification of the degradation products of the three anti-inflammatories was performed, indicating the presence of 4-hydroxy-diclofenac, 1-hydroxy-ibuprofen-, 6-O-desmethyl-naproxen, as the major degradation products of the three parent compounds.
Keywords: White-rot fungus (WRF); Stirred tank reactor (STR); Fixed-bed reactor (FBR); Degradation; Pharmaceutical compounds;

Biological treatment of leachate from solid wastes: Kinetic study and simulation by Muliye Tamrat; Carlos Costa; M. Carmen Márquez (46-51).
► Leachate from solid wastes has been stabilized and degraded by aerobic treatment. ► High content in organic matter and nutrients makes it suitable for agricultural use. ► Dynamic modelling predicts effluent COD value after biological treatment. ► Leachate is a half-biodegradable liquid (K s  = 18,950 mg/L, μH,max  = 0.21 d−1).Leachate from municipal solid wastes processed in the treatment plant in Salamanca, Spain has been stabilized by aerobic biological treatment. High organic load (42,310 mg O2/L COD) and high concentration of K (4376 mg/L) and N (3320 mg/L) make this leachate suitable for agricultural applications, but before agricultural use, biological treatment has to be applied for stabilization and odour reduction. Biodegradability has been studied in aerobic and anaerobic media, showing aerobic (64%) to be more effective than anaerobic (40%) in COD removal. Kinetic study of aerobic–anaerobic biodegradation is presented and a dynamic model for effluent COD prediction in aerobic biological treatment is developed.
Keywords: Waste treatment; Biodegradation; Aerobic processes; Dynamic simulation; Leachate; Solid wastes;

Extended kinetic model for DBT desulfurization using Pseudomonas Putida CECT5279 in resting cells by J. Calzada; A. Alcon; V.E. Santos; F. Garcia-Ochoa (52-60).
► We carry out the kinetic modeling of the 4S route for P. putida CECT 5279 resting cells. ► We consider the influence of deactivation of enzymes involved in the 4S route. ► We consider the influence of biomass concentration in the process. ► The model proposed explains closely the behavior of the P. putida 4S route.Dibenzothiophene desulfurization by Pseudomonas putida CECT5279, genetically modified microorganism, in resting cells is studied. In previous works, operational conditions were established and a kinetic model describing the four serial reactions was proposed. Later studies showed the existence of two characteristic growth times of this bacterium, 5 and 23 h, offering maximum activities in the desulfinase and the monooxygenase enzymes of this route. The combination of cells collected at 5 and 23 h of growth time was proved to be a very effective biocatalyst for desulfurization in resting cells. In this work, the previously proposed kinetic model is extended and applied to these cells with different ages. Moreover, other extension is considered, taking into account the activity loss of the enzymes involved in 4S route, and the influence of biomass concentration employed. These extensions are of considerable importance in order to scale-up the process. The kinetic model developed is able to fit the experimental results for resting cell operation with cells of different ages, in different concentration taking into account the enzyme deactivation.
Keywords: Biodesulfurization; Pseudomonas putida CECT5279; Kinetic modeling; Dibenzothiophene desulfurization; 4S route; Enzyme activity decay;

Real-time PCR quantification of the population dynamics of ammonia-oxidizing bacteria in a pilot-scale wastewater treatment plant by Xiaohui Wang; Ya Zhang; Xianghua Wen; Yu Xia; Man Hu; Zhaofang; Kun Ding (61-65).
► We monitored AOB number in a pilot-scale WWTP. ► Stable function of nitrification was correlated with a stable AOB number. ► Ammonia removal efficiency was positively correlated with AOB number.To determine whether the stable nitrification was correlated with a stable AOB population size in a pilot-scale wastewater treatment plant (WWTP) and to relate the AOB population to the ammonia removal efficiency, the population dynamics of AOB in a pilot-scale WWTP was monitored over a half-year period using real-time PCR assay. During the study period, the effluent ammonia concentrations were below 2.5 mg/L almost all the time, indicting a stable function of nitrification. The change of AOB population was relatively stable, ranging from 0.7 × 1010 to 7.9 × 1010 cells per liter mixed liquor suspended solids (MLSS) mixture, indicating that the stable function of nitrification was correlated with a stable AOB number in the pilot-scale WWTP. Correlation analysis demonstrates a significantly positive correlation between ammonia removal efficiency and total AOB population number (r  = 0.711, P  < 0.01). The findings enrich the theory involving the relation between AOB population dynamics and function of nitrification in WWTPs.
Keywords: Waste water treatment; Biodegradation; Bioreactors; DNA; AOB population dynamics; Real-time PCR;

Kinetic model and study of the influence of pH, temperature and undissociated acids on acidogenic fermentation by D. Infantes; A. González del Campo; J. Villaseñor; F.J. Fernández (66-72).
► Acidogenic fermentation of glucose was studied. ► A model describing the kinetics of the fermentation was developed. ► The model developed predicted accurately the fermentation process taking into account the pH, temperature and undissociated acids concentration.Acidogenic fermentation is a technology able to produce hydrogen and volatile fatty acids in friendly environmental way. Because of that, it is important to improve the knowledge in this area. In this work, glucose batch fermentations were carried out by a mixed culture at different pH (4, 5, and 6 units) and temperatures (26, 33 and 40 °C). These operating conditions caused an important effect on the fermentation process, such as inhibition of cell growth and substrate consumption. From these results, the critical undissociated acid concentration that caused important shifts in biomass growth and substrate consumption was found. A mathematical model was fitted to the experimental results, and it was observed that the undissociated acid concentrations affected the fermentation in an exponential way, being the process more sensitive when temperature was high. The energy required for cell maintenance increased when pH was low and temperature was high. The specific growth rate increased as pH and temperature increased. Moreover interesting relationships were found between kinetic parameters.
Keywords: Fermentation; Glucose; Modelling; Kinetic parameters;

Screening for silver nanoparticle-binding peptides by using a peptide array by Masashi Kuboyama; Ryuji Kato; Mina Okochi; Hiroyuki Honda (73-77).
Display Omitted► Core fragment, LFRYL, from AgNP-binding peptide AG4 was identified. ► Peptide array was utilized for identification of the short peptide. ► The analogue peptide, LFRKL, was shown to bind AgNPs. ► Two peptides bind AgNPs specifically among nanoparticles of other metal oxides. ► Their specificity is comparable with that of AG4.The functional fragment of the silver nanoparticle (AgNP)-binding peptide AG4 (NPSSLFRYLPSD) was identified by screening using a peptide array. The short pentameric functional peptide LFRYL was identified as the core peptide that is responsible for binding of AgNPs by AG4. The analogue LFRKL was also shown to bind AgNPs. Tests with nanoparticles of other metal oxides showed that these two peptides bind AgNPs specifically and that their specificity is comparable with that of AG4.
Keywords: Amino acids; Adsorption; Affinity; Polypeptides; Silver nanoparticles; Peptide arrays;