Biochemical Engineering Journal (v.56, #1-2)

BEJ Keywords (IV).

An efficient system for the production of useful metabolites by immobilized plant protoplasts (protoplasts with artificial cell walls) was developed. An advantage of the system is that the products are released readily into the culture medium with the double benefits of increasing overall productivity and facilitating downstream processing. This system solves many of the problems of conventional production systems using cells. This novel system has great potential in the production of useful metabolites; by using this system, it may become possible to efficiently produce many useful metabolites that are often difficult to produce with conventional methods.
Keywords: Alginate; Artificial cell wall; Elicitor; Immobilization; Plant protoplast; Useful metabolite production;

Simulation and optimization of a full-scale Carrousel oxidation ditch plant for municipal wastewater treatment by Wen-Ming Xie; Rui Zhang; Wen-Wei Li; Bing-Jie Ni; Fang Fang; Guo-Ping Sheng; Han-Qing Yu; Jing Song; De-Zhi Le; Xue-Jun Bi; Chang-Qing Liu; Min Yang (9-16).
► An integrated method with the activated sludge model 2d, support vector regression (SVR) and accelerating genetic algorithm (AGA) was developed in this study. ► The integrated model was used to simulate and optimize a full-scale Carrousel oxidation ditch wastewater treatment plant. ► The multiple-objective optimization with different weights indexes was used to achieve simultaneous nutrient removal.A full-scale Carrousel oxidation ditch wastewater treatment plant (WWTP) was simulated and optimized through integrating the activated sludge model 2d (ASM2d), support vector regression (SVR) and accelerating genetic algorithm (AGA). The ASM2d model after calibration and validation with the operating data was used to simulate the process. Operating parameters, including hydraulic retention times (HRTs) of anaerobic, anoxic and aerobic tanks, solids retention time (SRT) and internal recirculation ratio were subjected to optimization using SVR and AGA. The simulation results were normalized and SVR was employed to correlate the operating factors and the effluent quality. Then, the AGA approach was used to obtain the optimal operating conditions. The multiple-objective optimization with different weights indexes was adopted to achieve simultaneous nutrient removal. Compared with the present operating conditions, the HRT of the anoxic tank, the internal recirculation ratio and the SRT should be reduced, while the HRT of the aerobic tank should be prolonged to achieve better effluent quality. Such an integrated approach in this study offers an effective and useful tool to optimize the oxidation ditch process of WWTPs.
Keywords: Activated sludge; Bioreactor; Modeling; Optimization; Treatment plant; Wastewater;

A facile pretreatment method for efficient immobilization of penicillin G acylase by Pixiang Wang; Xiangyu Gong; Erzheng Su; Jingli Xie; Dongzhi Wei (17-22).
A facile pretreatment method was developed to obtain high specific activity of PGA from recombinant E. coli cells for preparing immobilized enzyme with high activity, which coupled selective extraction of PGA with butyl acetate and adsorption of butyl acetate with active carbon. Butyl acetate (5%, v/v) led to a 92.0% release of PGA, the specific activity of which in the extracting solution was twice that obtained by sonication. The negative effect on PGA immobilization due to residual butyl acetate in the extracting solution was removed by adding active carbon (8%, w/v). The final PGA solution meets requirement for industrial immobilization while eliminating the high cost of a traditional purification process. This pretreatment method developed in this work is simple, highly performing and cost-effective. It has been employed in a 10 tons/year immobilized PGA production line.
Keywords: Penicillin G acylase (PGA); Butyl acetate; Extraction; Active carbon; Aminated carrier; Immobilization;

Modelling and dynamic simulation of hybrid moving bed biofilm reactors: Model concepts and application to a pilot plant by Giorgio Mannina; Daniele Di Trapani; Gaspare Viviani; Hallvard Ødegaard (23-36).
► Reliability of a mathematical model of hybrid bed biofilm reactors was presented. ► Effectiveness of hybrid bed biofilm reactors for biological removal was shown. ► Good development of nitrifying bacteria to the detriment of heterotrophic one was recorded.In the recent years, there has been an increasing interest in the development of hybrid reactors, especially in the up-grading of existing activated sludge plants that are no longer able to comply with concentration limits established by regulatory agencies. In such systems the biomass grows both as suspended flocs and as biofilm. In this way, it is possible to obtain a higher biomass concentration in the reactor, but without any significant increase of the load to the final clarifier. The paper presents the setting-up of a dynamic mathematical model aimed at quantitatively describing the biokinetic processes occurring in a hybrid moving bed biofilm reactor (HMBBR), and, more in general, in integrated fixed-film activated sludge (IFAS) processes, as well as to compare the simulation results with measured data from a HMBBR pilot plant built at the Norwegian University of Science and Technology in Trondheim (Norway). Particularly, the pilot plant consisted of three aerobic tanks in series; the first and third aerobic reactors were pure suspended biomass systems, while the second aerobic reactor was filled with the AnoxKaldnes™ K1 carriers for biofilm development. The mathematical model consists of two connected models for the simulation of both suspended biomass and biofilm. Biochemical conversions are evaluated according to the well known matrix notation used in the Activated Sludge Model No. 1 (ASM1) for both attached and suspended biomass and, in addition to biochemical conversion, the model contains the simulation of particulate detachment from the biofilm into the bulk liquid. The results showed an overall good agreement between measured and simulated data, for both biofilm and suspended biomass, with a good reproduction of dynamic processes in the hybrid moving bed pilot plant, and they are encouraging for further developments.
Keywords: Biofilm modelling; Dynamic simulation; Hybrid moving bed biofilm reactors; Kinetic parameters; Wastewater treatment;

► Optimized the conditions for thicker biofilm formation by P. aeruginosa CPCL on PP. ► Increase in the concentration of glucose favours thicker biofilm formation. ► Temperature has negative effect on the formation of thicker biofilm. ► Shear stress has no effect on the formation of biofilm by P. aeruginosa on PP. ► Rhamnolipid regulates the spatial arrangement of the biofilm.The effect of rhamnolipid during the formation of initial biofilm (48 h) by Pseudomonas aeruginosa CPCL on polypropylene surface was studied. This strain was isolated from a petroleum contaminated soil and it produces a mixture of rhamnolipids. The nutritional and environmental conditions necessary for optimal production of rhamnolipid, attachment of rhamnolipid and formation of thicker biofilm on the polypropylene surface were found to be different from one another. Comparatively lower pH and temperature, higher concentration of ammonium chloride and rotation velocity of the shaker were required for the biofilm formation and the attachment of rhamnolipid on the polypropylene surface. Screening design showed that the extent of viable cells and rhamnolipid attached to the polypropylene surface was positively correlated (r  = 0.474; p  < 0.01). However, central composite design did not show any such relationship, possibly because of the different experimental ranges used in central composite design. There were considerable variations in the thickness and spatial arrangement of biofilms grown under these three different optimized conditions. Higher substratum coverage and thicker biofilm were observed under the condition optimized for biofilm formation when compared to the other two conditions. This study is essential since P. aeruginosa biofilms are found to have implication in bioremediation of recalcitrant pollutants.
Keywords: Fractional factorial design; Rhamnolipid; Biofilm; Polypropylene; Central composite design;

► Two techniques were used to immobilize P. putida in PVA gel particles. ► Technique T2 avoids subjecting the bacteria to sub-freezing temperature. ► The immobilized bacteria were used to degrade phenol in two reactor configurations. ► Both immobilization techniques and both reactors proved to be effective. ► SBBR was more effective than bubble column reactor due to the better mixing. Pseudomonas putida, immobilized in polyvinyl alcohol (PVA) particles, has been successfully utilized for the bioremoval of phenol from simulated wastewater, using two immobilization techniques and two types of bioreactors. The biodegradation efficiency of P. putida immobilized within the PVA gel before the cross-linking stage of the polymer (T1) was compared to that of the same bacteria immobilized by soaking blank PVA particles in bacterial suspension (T2), a procedure that avoids subjecting the bacteria to sub-freezing temperature during the cross-linking stage. The effects of nutrient deprivation and exposure to high phenol concentrations on the activity of P. putida were also evaluated. The experimental results indicated that the immobilized bacteria remained active for a period of 72 h, even without the addition of nutrients. Subsequently the activity gradually decreased, but the bacteria easily regained their original activity with the addition of nutrients. Sudden exposure to high phenol concentrations resulted in immediate decline in the biodegradation activity, but the bacteria adapted to the new concentrations and regained their activity within 24 h. The biodegradation experiments were carried out in two types of bioreactors namely, bubble column and spouted bed bioreactor (SBBR). Both reactor configurations and both immobilization techniques proved to be effective in the biodegradation of phenol.
Keywords: Phenol degradation; Spouted bed bioreactor; Bubble column; Immobilized bacteria; Substrate inhibition;

► Chitin and chitosan were prepared from shrimp waste within a short processing time. ► Optimum decalcification depends upon protein and the moisture content of the waste. ► Chitosan viscosity was not dependent on acetic acid release during deacetylation. ► Conditions for chitin preparation have a high influence on chitosan viscosity. ► The response surface analysis confirmed the experimental results.Deproteination of decalcified chitin from Crangon crangon shells and deacetylation to chitosan was investigated to find out optimum conditions for a high viscosity chitosan. Deproteination was carried out with an optimum shrimp shell:alkali (s:a) ratio of 1:4 from 30 °C to 65 °C and at each temperature, incubation times were varied from 2 to 5 h for maximal efficiency. Viscosity of the chemically extracted chitin samples at different temperature–time conditions ranged from 195 to 391 mPa s. A two-way ANOVA with Bonferroni post test was performed on experimental data. Chitin extracted at various temperatures and incubation times was deacetylated with 50% (w/w) NaOH at increasing reaction times. The highest chitosan viscosity of 1976 mPa s was observed in a sample deproteinated for 5 h at 55 °C and deacetylated for 1 h at 105 °C and 2 bar N2 pressure. One-way ANOVA test for chitosan viscosity measurements indicated a significant P value of <0.05. Viscosity was not dependent on acetic acid release during deacetylation. A central composite design (CCD)–response surface analysis (RSA) was carried out and the resulting optimised model solutions confirmed the results. Highly viscous chitin and chitosan compared to commercially available products could be prepared within a short time by selecting suitable process conditions.
Keywords: Waste treatment; Separation; Optimisation; Shrimp shell:alkali ratio; Viscosity; Response surface analysis (RSA);

► We report the development of the first available growth kinetic model specific to redox potential-controlled very-high-gravity (VHG) ethanol fermentation process. ► We propose and implement a two-step parameter-estimating strategy to estimate kinetic parameters pertinent to yeast growth, ethanol production, and glucose consumption. ► We validate that the parameters obtained by using the proposed strategy could result in a high accuracy of data prediction (R 2  > 0.95). ► We construct operating diagrams to select the best fermentation conditions towards high fermentation efficiency, short fermentation time, and low residual glucose after fermentation.A growth kinetic model for redox potential-controlled very-high-gravity (VHG) fermentation was developed. The model is semi-empirical and two important VHG fermentation operating conditions including glucose feed and redox potential level were incorporated in the model. The model consists of a substrate inhibition term and a product toxification term. A two-step parameter-estimating strategy was proposed and implemented. In the first step, the first few experimentally collected data points were used to estimate parameters relating to the substrate inhibition term; in the second step, the complete data set was used to estimate parameters relating to the product toxification term. The developed growth model could fit the original data with an R 2 value >0.95. Three operating diagrams were constructed by using the results generated from the developed model. Each respective diagram could be used to select the best operating condition for the shortest fermentation time, the highest final ethanol concentration, or the highest fermentation efficiency.
Keywords: Saccharomyces cerevisiae; Ethanol; Kinetic models; Very-high-gravity fermentation; Redox potential; Operating diagram;

An evaluation of the utility of the hepatic differentiation method using hollow fiber/organoid culture for the development of a hybrid artificial liver device by Naoki Amimoto; Hiroshi Mizumoto; Kohji Nakazawa; Hiroyuki Ijima; Kazumori Funatsu; Toshihisa Kajiwara (69-74).
► We developed a HF/organoid culture method to induce the differentiation of ES cells into hepatocytes. ► We compared the results of the hepatic differentiation using the HF/organoid culture with those using monolayer culture. ► The up-regulation of hepatocyte markers and the liver-specific functions were observed in the HF/organoid culture. ► However, they were not observed in the monolayer cultures. ► The HF/organoid culture method is an effective tool for inducing the differentiation of ES cells into hepatocytes.To put the hybrid artificial liver (HAL) using cultured hepatocytes into practical use, it is necessary to develop a high-performance artificial liver device. We developed a novel hollow fiber (HF)/organoid culture method to induce the differentiation of pluripotent stem cells into hepatocytes. In this study, we compared the results of the hepatic differentiation using the HF/organoid culture with those using monolayer culture to evaluate its utility as a hepatic differentiation method. In both cell cultures, ES cells showed high proliferative activity immediately after cell seeding. The up-regulation of hepatocyte-specific markers such as albumin (ALB), carbamoyl phosphate synthetase 1 (CPS-1) and tryptophan 2,3-dioxygenase (TDO) were observed as the culture progressed, and the expression of liver-specific functions such as the removal of ammonia and albumin secretion were detected after about 2 weeks of the hepatic differentiation induction in the HF/organoid culture. However, the results were not observed in the monolayer culture. In conclusion, the HF/organoid culture method has promise as an effective tool for the differentiation of ES cells into hepatocytes.
Keywords: Embryonic stem cells; Hepatic differentiation; Three dimensional culture; Hollow fiber; Hybrid artificial liver;

Liquid–liquid extraction by mixed micellar systems: A new approach for clavulanic acid recovery from fermented broth by Valéria Carvalho Santos; Francislene Andréia Hasmann; Attilio Converti; Adalberto Pessoa (75-83).
► Clavulanic acid (CA) is a potent inhibitor of β-lactamases. ► Aqueous two-phase micellar systems were used with success to extract CA from broth fermented. ► Under all conditions studied CA partitioned to the dilute, micelle-poor phase. ► The system used has potential as a new first-step operation of CA purification.This work is the first attempt to apply aqueous two-phase mixed micellar systems (ATPMS) of the nonionic surfactant Triton X-114 and the anionic one AOT to extract clavulanic acid (CA) from broth fermented by Streptomyces clavuligerus. Cloud points were determined in McIlvane buffer pH 6.5 with or without NaCl, and diagram phases/coexistence curves were constructed. CA partition was investigated following a 24-full factorial design in which AOT (0.022, 0.033 and 0.044% w/w), Triton X-114 (1.0, 3.0 and 5.0% w/w) and NaCl (0, 2.85 and 5.70% w/w) concentrations and temperature (24, 26 and 28 °C) were selected as independent variables, and CA partition coefficient (K CA ) and yield in the top phase (η CA ) as responses. CA partitioned always to the top, micelle-poor phase. The regression analysis pointed out that NaCl concentration and interaction between temperature and Triton X-114 concentration had statistically significant effects on K CA , while η CA was mainly influenced by temperature, Triton X-114 concentration and their interaction. Different ATPMS compositions were then needed to maximize these responses, specifically 0.022% (w/w) AOT, 5% (w/w) Triton X-114 for K CA (2.08), and 0.044% (w/w) AOT, 1% (w/w) Triton X-114 for η CA (98.7%), both at 24 °C without NaCl. Since at 0.022% (w/w) AOT, 1% (w/w) Triton X-114 and 28 °C without NaCl the system was able to ensure satisfactory intermediate results (K CA  = 1.48; η CA  = 86.3%), these conditions were selected as the best ones. These preliminary results are of concern for possible industrial application, because CA partition to the dilute phase can simplify the subsequent purification protocol.
Keywords: Clavulanic acid; Aqueous two-phase mixed micellar systems; Extraction purification;

Preparation and antioxidant activity of Radix Astragali residues extracts rich in calycosin and formononetin by Cai-Yun Chen; Yuan-Gang Zu; Yu-Jie Fu; Meng Luo; Chun-Jian Zhao; Wei Wang; Bao-Shan Zhao; Ji Li; Thomas Efferth (84-93).
► This is the first report on simultaneous enrichment and separation of calycosin and formononetin with excellent free radical-scavenging activity from extracts of Radix Astragali residues by macroporous resin. ► Macroporous resins possess ideal pore structure and various functional surface groups, which are advantageous in high adsorption properties, simple procedure, high efficiency, low cost, little environmental pollution and easy regeneration. ► Nowadays, the wild natural resources of Astragalus membranaceus are diminishing and the bulk of the commercial supply is mainly from farming sources in China. Therefore, recycling of Radix Astragali residues is reasonable for a more efficient utilization of Radix Astragali resources.Radix Astragali is considered an excellent source of Traditional Chinese Medicine and a kind of famous restorative food. To make full usage of Radix Astragali, calycosin and formononetin were enriched through various macroporous resins from its residues, HPD500 resin, compared with other resins, offered the best effectiveness for the enrichment and separation of compounds. 35 °C was selected for its highest adoption capacity during static adsorption process, which can be obtained from adsorption isotherms. And then dynamic adsorption and desorption tests were carried out to optimize the operating parameters. After one run treatment with HPD500 resin, the contents of calycosin and formononetin in the product were 4.41-fold and 4.60-fold with recovery yields of 75.16% and 77.06%, respectively. Investigations on the antioxidant activity showed that eluate with 60% ethanol exhibited better free radical-scavenging activity (IC50 0.03 mg/mL) than crude extracts (IC50 0.05 mg/mL). Thus, this fast and effective method was established and could be a promising for the large-scale preparation of calycosin and formononetin from Radix Astragali residues.
Keywords: Enrichment; Calycosin; Formononetin; Radix Astragali residues; Antioxidant activity; Macroporous resins;

Enhancement of enterocin A production by Enterococcus faecium MMRA and determination of its stability to temperature and pH by Amel Rehaiem; Nelson Pérez Guerra; Zouhaier Ben Belgacem; Paula Fajardo Bernárdez; Lorenzo Pastrana Castro; Mohamed Manai (94-106).
► Enterocin A synthesis was assayed in MRS broth in batch and fed-batch fermentations. ► The realkalized fed-batch fermentation technique enhanced enterocin A synthesis. ► The enterocin A production system in this culture was satisfactorily modelled. ► This bacteriocin was classified as a primary metabolite dependent on the pH drop. ► Bacteriocin stability was high around 84 °C, at low pH, and short incubation times.The growth, nutrient (total sugars, phosphorous, nitrogen and proteins) consumption and product formation by Enterococcus faecium MMRA in de Man, Rogosa and Sharpe (MRS) broth was followed in non-realkalized batch, realkalized batch and realkalized fed-batch cultures. In the latter fermentation, the growing culture was fed with a mixture of MRS medium (20 g of glucose/L) and a 400 g/L concentrated glucose. In the three cultures, a typically homolactic fermentation (only lactic acid was produced) was observed. The realkalized fed-batch culture was mainly characterized with higher biomass (2.4 g/L), lactic acid (32.5 g/L) and enterocin A (35.9 AU/mL) concentrations compared with the two batch processes.Mathematical models were developed to describe the productions of biomass, enterocin A and lactic acid in the two realkalized cultures. The growth and lactic acid production were successfully modelled with the Monod and the Luedeking and Piret models, respectively. Enterocin A was modelled by using a modified form of the Luedeking and Piret model, which includes a term for the effect of the pH drop rate on bacteriocin synthesis. Furthermore, the maximum enterocin A stability was obtained at temperatures below 100 °C, at acidic pH values and short incubation times.
Keywords: Enterococcus faecium MMRA; Enterocin A; Realkalized batch; Fed-batch; Fermentation; Modelling; Temperature; pH;