Biochemical Engineering Journal (v.69, #C)

BEJ Keywords (III).

► Improvement of the clavulanic acid production. ► Batch cultures with glycerol pulses at low temperature. ► Clavulanic acid production rate and clavulanic acid degradation rate as function of the temperature.Clavulanic acid (CA) is a potent beta-lactamase inhibitor produced by Streptomyces clavuligerus. Like other beta-lactam compounds, CA is chemically unstable at high temperatures. The decomposition of CA during bacterial fermentation reduces its concentration in the broth, resulting in low yields. This study investigated the use of temperature reduction to obtain high CA production during fermentation employing cultures of S. clavuligerus. Simple batch cultures, and batch cultures with glycerol pulses, were performed at different temperatures in shake flasks at 250 rpm, and pH 6.8. Firstly, three batch cultures using glycerol as carbon source were carried out at temperatures of 30 (control), 25, and 20 °C. Next, three batch cultures were carried out with temperature reductions from 30 to 25 °C, 30 to 20 °C, and 25 to 20 °C, after glycerol exhaustion. CA production generally increased in cultures with temperature reduction and glycerol feeding. Maximum CA concentration of 1534.3 mg L−1 was achieved in a culture maintained at a constant temperature of 20 °C, with one pulse of glycerol. Under these conditions, both glycerol uptake rate and CA degradation were low. The results obtained demonstrate the potential of temperature reduction in the optimization of CA production by S. clavuligerus.
Keywords: Clavulanic acid; Streptomyces clavuligerus; Temperature reduction; Glycerol uptake rate; Product degradation rate;

Enzyme kinetics and identification of the rate-limiting step of enzymatic arabinoxylan degradation by Louise E. Rasmussen; Cheng Xu; Jens F. Sørensen; Michael K. Nielsen; Anne S. Meyer (8-16).
► Wheat arabinoxylan was treated with designed combinations of pure enzymes. ► Endo-1,4β-xylanase was rate-limiting for enzymatic xylose release from arabinoxylan. ► A probability calculation is proposed for quantitation of β-xylosidase attack.This study investigated the kinetics of multi-enzymatic degradation of soluble wheat arabinoxylan by monitoring the release of xylose and arabinose during designed treatments with mono-component enzymes at different substrate concentrations. The results of different combinations of α-l-arabinofuranosidases (EC 3.2.1.55), one derived from Aspergillus niger (AFAn) and one from Bifidobacterium adolescentis (AFBa), respectively, a β-xylosidase (EC 3.2.1.37) from Trichoderma reesei, and an engineered D11F/R122D variant of Bacillus subtilis XynA endo-1,4-β-xylanase (EC 3.2.1.8) were examined. The two selected α-l-arabinofuranosidases catalyze liberation of arabinose residues linked 1 → 3 to singly (AFAn) or doubly (AFBa) substituted xyloses in arabinoxylan, respectively. When added to arabinoxylan at equimolar levels, the AFBa enzyme catalyzed the release of more arabinose, i.e. had a higher rate constant than AFAn, but with respect to the xylose release, AFAn – as expected – exhibited a better synergistic effect than AFBa with β-xylosidase. This synergistic effect with AFAn was estimated to increase the number of β-xylosidase catalyzed cuts from ∼3 (with β-xylosidase alone) to ∼7 in each arabinoxylan substrate molecule. However, the synergistic effects between β-xylosidase and the α-l-arabinofuranosidases on the xylose release were low as compared to the effect of xylanase addition with β-xylosidase, which increased the xylose release by ∼25 times in 30 min, to a yield equivalent to ∼104 β-xylosidase catalyzed cuts in each arabinoxylan substrate molecule. At equimolar addition levels of the four enzymes, the xylanase activity was thus rate-limiting for the β-xylosidase catalyzed depolymerization to release xylose from arabinoxylan. The work provides clues to design efficient enzymatic degradation of arabinoxylan into fermentable monosaccharides.
Keywords: Arabinoxylan hydrolysis; Enzyme kinetics; α-l-Arabinofuranosidase; β-Xylosidase; Endo-xylanase; Synergy;

► Fermenters scale-up based on volumetric mass transfer coefficient, k L a, is presented. ► Experimental k L a data for pilot-plant fermenter are presented. ► Process parameters are identified, the value of which depends on the fermenter size. ► The k L a correlations suitable for scaling-up are developed. ► The uncertainty is significantly reduced in the volume of the designed industrial fermenter.Mechanically agitated gas–liquid contactors are frequently used in the chemical, food and biochemical industries as fermenters and as hydrogenation or chlorination reactors. However wide the usage of such vessels is, their design is not based on chemical engineering data, but is still rather empirical. Thus, it is highly desirable to have a tool for the rational design of agitated gas–liquid contactors that is based on fundamental chemical engineering parameters that are transferable to other systems and operating conditions. Focusing on liquid film-controlled processes and using the data from fermenters of different scales, we develop k L a correlations that are suitable for scale-up.First, we discuss the way how to determine the proper experimental k L a values, which are not distorted by other equipment parameters as the gas residence time is. We demonstrate the possible k L a distortion on the pilot-plant experimental data by the comparison of the results obtained by two different experimental techniques. Further, we present physically correct k L a data for coalescent (pure water) and fully non-coalescent (sodium sulphate solution) batches. The data are presented both for laboratory and for pilot-plant fermenters. We identify the process parameters, the values of which are dependent on the vessel scale when operated under the same power input per unit volume, and, using these parameters, we develop common k L a correlations suitable to describe the data for various scales of the vessel.The correlations developed reduce the uncertainty in predicting the volume of industrial scale fermenters from almost 1/2 to 1/4 of their total volume, thereby enabling significant reductions in both the initial costs and operating costs.
Keywords: Fermenter; Mass transfer; Mass transfer correlations; Mixing; Multiple-impeller; Scale-up; Volumetric mass transfer coefficient;

Oriented immobilization of glucose oxidase on graphene oxide by Liya Zhou; Yanjun Jiang; Jing Gao; Xinqiang Zhao; Li Ma; Qianlin Zhou (28-31).
► GO was used as the immobilization support for GOD. ► GO was functionalized by Con A via diimide-activated amidation. ► GOD was oriented immobilized on a selective support composed of GO and Con A. ► Oriented immobilization of GOD had enhanced pH, thermal and storage stability. ► Oriented immobilized GOD had high affinity for glucose.The oriented immobilization of glucose oxidase (GOD) on a selective support composed of graphene oxide (GO) and concanavalin A (Con A) was prepared in this study. Specially, the amino groups of Con A were covalently attached to the carboxyl groups of pre-activated GO, and then GOD was oriented immobilized on the GO–Con A conjugate via strong biospecific affinity between the sugar residues of GOD and Con A. Compared with free GOD and randomly immobilized GOD (GO–GOD), the high-affinity of Con A and GOD endowed the oriented immobilized GOD (GO–Con A–GOD) with a wider pH stability range, a better thermal stability, a longer-term storage stability and a higher resistance ability toward the denaturing agents. The Michaelis constant (K m ) of GO–Con A–GOD was very close to that of free GOD. Hopefully, the GO–Con A–GOD may be further integrated into functional bioelectrodes for biosensor applications.
Keywords: Immobilized enzymes; Affinity; Glucose; Graphene oxide; Glucose oxidase; Kinetic parameters;

Producing 2,3-butanediol from agricultural waste using an indigenous Klebsiella sp. Zmd30 strain by Chiao-Ling Wong; Chien-Chang Huang; Wei-Bin Lu; Wen-Ming Chen; Jo-Shu Chang (32-40).
► Low cost and high efficiency 2,3-BDO production with Klebsiella sp. was developed. ► Agricultural wastes were used as feedstock for 2,3-BDO production. ► Medium composition was optimized using RSM to enhance 2,3-BDO production. ► Using rice straw gave a high 2,3-BDO productivity (2.08 g/l/h) and yield (62%).This study aimed to develop a low cost and high efficiency process for 2,3-butanediol (2,3-BDO) production by using Klebsiella sp. Zmd30 isolated from a hydrogen-producing anaerobic sludge. First, the effects of glucose concentration and nitrogen source on 2,3-BDO production with the Zmd30 strain were examined. The results show that the productivity and concentration of 2,3-BDO reached the highest values of 0.92 g/l/h and 58 g/l, respectively, when the glucose concentration was in the range of 160–180 g/l. Urea was a better nitrogen source when considering both the 2,3-BDO production performance and the cost. Next, medium composition for the growth of the Zmd30 strain was optimized using the response surface methodology (RSM) to enhance 2,3-BDO production. Based on the RSM analysis, the optimal composition of glucose, urea and MnCl2·4H2O, H3BO3 and CoCl2·6H2O was 160, 2.28, 7.01 × 10−4, 1.49 × 10−4 and 5.70 × 10−3  g/l, respectively. With this optimal medium, the 2,3-BDO productivity was improved to 1.15 g/l/h. To reduce the production cost, bagasse and rice straw were used as feedstock for 2,3-BDO production. It was found that using hydrolyzed rice straw resulted in better productivity than using hydrolyzed bagasse, as the highest productivity (2.08 g/l/h) and yield (62%) were obtained at an initial rice straw concentration of 150 g/l.
Keywords: Fermentation; Cellulose; Bioreactors; Microbial growth; 2,3-Butanediol; Klebsiella sp.;

► A dissolved CO2 mass balance was developed for very-high-gravity ethanol fermentation. ► The equation considers biological and physiochemical properties of CO2. ► Henry's law becomes inapplicable for a small-scaled fermenter under very-high-gravity condition.The dissolved carbon dioxide (DCO2) evolved from Saccharomyces cerevisiae was measured for the duration of very-high-gravity (VHG) ethanol fermentation conducted at four glucose feeds (150, 200 ± 0.21, 250 ± 0.12, and 300 ± 0.28 g/L). The DCO2 concentration profiles depict evolution patterns that depend on the extent of glucose utilized. A mass balance for DCO2 was proposed by taking into account the physiochemical behavior of CO2 and biological processes occurring under VHG ethanol fermentation conditions. The proposed equations could interpret the underlying CO2 desorption, CO2 conversion, and CO2 evolution on the basis of yeast activity during VHG ethanol fermentation. Consequently for 150 and 200 ± 0.21 g glucose/L absence of yeast activity was interpreted in terms of zero CO2 evolution rate (CER(t)). In contrast, under 250 ± 0.12 and 300 ± 0.28 g glucose/L conditions the presence of non-zero residual glucose resulted in non-zero CER(t). The mass balance equation could also interpret that the physical desorption of CO2 from the fermentation broth did not follow the equilibrium relation between dissolved and off-gas CO2 concentrations as defined by Henry's law under current experimental conditions.
Keywords: Dissolved CO2 concentration; CO2 evolution rate; Mass balance; Very-high-gravity fermentation; Henry's law; Saccharomyces cerevisiae;

A low energy process for the recovery of bioproducts from cyanobacteria using a ball mill by Bangaru Balasundaram; Stephen C. Skill; Carole A. Llewellyn (48-56).
► The custom designed ball mill was found to be 34% more efficient than previous reports. ► Selective product recovery achieved by manipulating the mechanical forces involved. ► Critical tools needed for the design of algal biorefinery type process are identified. ► A unique study that develops a scalable differential recovery process for microalgae.Efficient and economical large scale bioprocessing of microalgae to produce a range of bio-products, working towards a biorefinery approach, is critical for the success of algal industrial biotechnology. The key process variables that affect the recovery of products from different sub-cellular locations were investigated using a high throughput cell disruption system and a cyanobacterium as model organism. This information was then used to design and test a differential recovery procedure at pilot scale using a custom designed ball mill that consumed 1.87 kWh/kg of dry biomass used approximately 34% lower than the best algal disruption system reported. The balance between the number of collisions and force of each collision between grinding media and the microorganism were manipulated to achieve differential recovery. Greater than 99 and 95% solids were recovered at the end of first and second ball milling step respectively through gravity sedimentation, an energy efficient solid-liquid separation technique. Based on these results and the release rates of intracellular and/or extracellular products tested, a theoretical framework is presented for the design of a differential recovery process using ball mills.
Keywords: Algal biorefinery; Ball mill; Cyanobacteria; Polysaccharides; Antioxidants;

► Agro-industrial residues and cow dung were used for the production of proteases. ► Cow dung supported more protease production than other tested substrates. ► Cow dung can be used as an effective substrate in an enzyme bioprocess ► This enzyme was detergent-, solvent-stable and had dehairing properties.The production of a halo-tolerant-alkaline protease by Halomonas sp. PV1 under solid state fermentation was optimized. Among the substrates evaluated, cow dung supported the maximum protease production (1351 U/g) when compared with wheat bran (1013 U/g). Process parameters such as, the fermentation period (72 h), pH (8.0), initial moisture (140%, v/w) and the inoculum level (15%, v/w) were optimized. The optimum enzyme production was achieved with 1.5% (w/w) xylose and a 1.25% (w/w) yeast extract. The partially purified protease enzyme was active over a temperature range of 30–50 °C and a pH range of 7–10. The protease was remarkably stable on sodium dodecyl sulfate and on various commercial detergents. The enzyme effectively dehaired goat hides, and makes it a potential source of alkaline protease. This substrate may have wide spread applications in enzyme bioprocesses like wheat bran.
Keywords: Solid-state fermentation; Alkaline protease; Optimization; Enzyme;

Production of ethanol from enzymatically hydrolyzed soybean molasses by Francielle Batista da Silva; Betânia Braz Romão; Vicelma Luiz Cardoso; Ubirajara Coutinho Filho; Eloízio Júlio Ribeiro (61-68).
► Soybean molasses is a by-product of soybean meal concentrate rich in carbohydrates. ► The aim the study was to evaluate the production of ethanol by fermentation of SM. ► Soybean molasses proved to be a potential substrate for ethanol production.Soybean molasses (SM) is a by-product of soybean meal concentrate. It is rich in sucrose, raffinose and stachyose. Stachyose and raffinose are not directly fermentable to ethanol but, when hydrolyzed, can be metabolized by Saccharomyces cerevisiae. In this work, we studied (1) the production of ethanol from natural SM by fermentation with S. cerevisiae, (2) the enzymatic hydrolysis of SM by α-galactosidase and the subsequent fermentation with S. cerevisiae (HF), and (3) ethanol production via the simultaneous hydrolysis and fermentation (SHF) of SM. The results showed that, although the fermentation of natural soybean molasses provided a fermentation yield of 72.9% under optimized conditions, hydrolyzed soybean molasses provided a fermentation yield 7.6% higher using the HF process and 8.2% higher using SHF. Both fermentation processes resulted in lower concentrations of residual sugar.
Keywords: Soybean molasses; Ethanol; Enzymatic hydrolysis;

Display Omitted► The kinetic of the enzymatic synthesis of prunin 6″-O-laurate was studied. ► Mechanism ordered Bi Bi without inhibition was consistent with experimental data. ► Fastest initial rate was obtained working with prunin in colloidal state. ► Optimised kinetic expression was fitted to experimental data even in solvent-free system.Alkyl prunin esters are new compounds, which are soluble in lipophilic media. They possess antioxidant and antibacterial properties, so they may have useful applications. The current work studied the kinetic of prunin 6″-O-laurate synthesis catalysed by Novozym 435 from vinyl laurate and prunin in acetone completely solubilised, forming colloids or scattered in solid state, according to its concentration in the reaction media.The kinetic study was determined at 50 °C using initial concentrations between 20 and 220 mM for prunin and between 20 and 4000 mM (solvent free system) for vinyl laurate. When prunin completely solubilised or forming colloids was used, a model based on mechanism ordered Bi Bi without inhibition, neither by alcohol nor acyl group, which was the best fit in the initial rate data. The determined model was used to simulate initial reaction rate and these values were plotted against the experimental data. The model was consistent with the experimental data (slope 0.97 ± 0.01, R 2 0.993, n  = 72), even on solvent-free systems.
Keywords: Alkyl prunin ester; Lipase; Kinetic study; Transesterification;

Bioaugmentation: Possible solution in the treatment of Bio-Refractory Organic Compounds (Bio-ROCs) by Samer Semrany; Lidia Favier; Hayet Djelal; Samir Taha; Abdeltif Amrane (75-86).
► Some emerging pollutants are refractory to conventional biological treatment. ► Acclimation, biostimulation and bioaugmentation processes were defined. ► Experimental approach to evaluate bioaugmentation's performance is exposed. ► Recently published papers dealing with bioaugmentation are reviewed. ► Some engineered new approaches of bioaugmentation are also noticed.Water quality is questionable when looking at the emerging pollutants that accumulate in hydrosphere day after day. In terms of water treatments to eliminate organic compounds, biological treatments are the most used technique, especially the conventional activated sludge (CAS) process. Unfortunately, several of these compounds such as petroleum and industrial wastes, some pesticides, pharmaceuticals, and hormonal substances appear to be refractory to such treatments and can join the different water surfaces from other natural environments especially contaminated soils. Physical and chemical treatments, in particularly the advanced oxidation processes (AOPs) are able to degrade some of these pollutants, but the nature and the possible effects of oxidation by-products remain controversial when complete mineralization does not occur. The aim of this article is to shed light on new and renewed biotechnological techniques recently introduced to resolve recalcitrance problem. Amending activated sludge with additional microorganisms able to produce versatile enzymes to enhance biodegradability or “bioaugmentation”, is gradually considered as a possible solution for the recalcitrance of Bio-Refractory Organic Compounds (Bio-ROCs). The use of this technique, traditionally in soil remediation field, and recently in water and sediments treatment is reviewed in this paper.
Keywords: Water pollution; Bioremediation; Activated sludge; Biostimulation; Bioaugmentation;

Effect of biotin and a similar compound on succinic acid fermentation by Actinobacillus succinogenes in a chemically defined medium by Yong-lan Xi; Ke-quan Chen; Rong Xu; Jiu-hua Zhang; Xue-fei Bai; Min Jiang; Ping Wei; Jie-yu Chen (87-92).
► An efficient chemically defined medium (CDM) was created. ► Biotin concentration in CDM was optimised. ► Biotin can be replaced by 5-aminolevulinate in CDM. ► The mechanism underlying the metabolism of biotin was studied.A minimal chemically defined medium (CDM) was created and used to study the effect of biotin on the metabolism of Actinobacillus succinogenes NJ 113. The medium contained four amino acids and vitamins as key growth factors. To gain insight into the effect of biotin, different concentrations of this compound were added, and glucose utilisation, cell growth, and the succinic acid concentration were investigated. Anaerobic batch fermentation was carried out at optimised conditions in a 3 L fermentor with a succinic acid concentration of 45.2 g/L and a yield of 90.4%. A metabolic pathway was proposed after further study of the mechanism underlying the metabolism of biotin. The same results were obtained when 5-aminolevulinate (5-ALA) was used instead of biotin. We suggest that 5-ALA is a great alternative to biotin for the preparation of succinic acid by biological fermentation.
Keywords: Anaerobic processes; Biotin; Optimisation; Succinic acid; Fermentation; Batch processing;

► The partitioning of amino acids is investigated in PEG/nonionic surfactant ATPS. ► The hydrophobicity of PEG/nonionic surfactant ATPS are estimated. ► The partitioning of lysozyme in ATPS is further predicted. ► The hydrophobic interaction play an important role in the biomolecules partitioning.The partitioning of various hydrophobic or hydrophilic amino acids was investigated in aqueous two-phase systems (ATPS) composed of polyethylene glycol (PEG) and nonionic surfactant (Triton X-100 and Tween 80), respectively. The influences of amino acids side chain group, tie line length (TLL) of ATPS, nonionic surfactant type and PEG molecular weight on the partitioning coefficients of amino acids were extensively studied. The results displayed that the hydrophilic amino acid preferred to partition in the top PEG-rich phase, and the hydrophobic amino acid preferred to the bottom surfactant-rich phase. These distribution trends of amino acids in PEG/nonionic surfactant ATPS were more obvious with increasing TLL, while the PEG molecular weight had no significant effect on the amino acids partitioning in ATPS. Moreover, the estimated hydrophobic factor (HF) values of PEG/nonionic surfactant ATPS should be about −10−2  mol/kJ according to the amino acids partitioning, which demonstrated that the bottom surfactant-rich phase was generally more hydrophobic than the top PEG-rich phase. PEG20000/Triton X-100 ATPS had more extensive HF values scope (−0.0038 to −0.0496 mol/kJ) than PEG20000/Tween 80 ATPS (HF −0.0162 to −0.0306 mol/kJ) in the investigated TLL due to the different micelles size and number. The partitioning of model protein lysozyme was predicted in PEG/nonionic surfactant ATPS based on ATPS HF and lysozyme hydrophobicity. The experimental and predicted partitioning coefficients of lysozyme displayed a good consistency in the PEG/nonionic surfactant ATPS. It was shown that the hydrophobic interaction played an important role in both the partitioning of amino acids and the partitioning of the small model protein in PEG/nonionic surfactant ATPS.
Keywords: Aqueous two-phase system; Nonionic surfactant; Amino acids; Hydrophobic; Lysozyme; Partitioning;

Deciphering butanol inhibition to Clostridial species in acclimatized sludge for improving biobutanol production by Bor-Yann Chen; Fu-Yu Chuang; Chung-Liang Lin; Jo-Shu Chang (100-105).
► Effect of butanol toxicity on butanol production by a mixed culture was studied quantitatively. ► Butanol inhibition to cell growth and butanol production was analyzed with dose–response curves. ► Feasibility of butanol formation by a bacterial consortium was evaluated in toxicological terms.This study aimed to quantitatively evaluate the correlations between butanol (BtOH) tolerance of solvent-producing bacteria (SPB) and the performance of fermentative butanol production. The toxicity potency of BtOH was revealed to suggest the feasibility of butanol formation with a Clostridial species-dominated bacterial consortium. When the mixed culture was grown on the optimal medium comprising 60 g/L glucose, 0.5 g/L FeSO4·7H2O and 5.13 g/L yeast extract, the maximal tolerant butanol concentration, butanol production, hydrogen production and glucose consumption were ca. 16 g/L, 10.64 ± 0.60 g/L, 4153 ± 815 mL/L and 54.99 ± 1.92 g/L, respectively. Moreover, almost all the dose–response curves representing toxicity potency of butanol on microbial characteristics was nearly identical in order of magnitude. Thus, although generations of by-products during butanol fermentation are interactive, the maintenance of microbial growth capability still plays a crucial role to control the performance of butanol production. The quantitative findings in toxicological terms directly suggest that the BtOH toxicity seemed to be inevitable during BtOH production. In addition, butanol inhibition could be reversibly attenuated by removal of butanol to make it below the critical level (ca. 7.83–9.52 g/L or EC50).
Keywords: Fermentation; Microbial growth; Product inhibition; Butanol tolerance; Dose response assessment;

Facilitated oxidative refolding of ribonuclease A from inclusion bodies with a new redox system by Guang-Jie Han; Xiao-Yan Dong; Lin Zhang; Li-Tang Fu; Guo-Zhen Wang; Yan Sun (106-112).
► 4-Mercaptobenzeneacetate and hexanoyl cystamine was proposed as a new redox system. ► The system was more efficient than GSH/GSSG in accelerating protein refolding. ► The high efficiency was proven in the refolding of RNase A from inclusion bodies.Oxidative refolding is a crucial step in the bioseparation process of bioactive RNase A from inclusion bodies (IBs) overexpressed in Escherichia coli, and it has been well recognized that RNase A refolding suffers from the slow formation of correct disulfide bonds with traditional redox agents such as reduced glutathione (GSH) and oxidized glutathione (GSSG). Hence, there is demand of developing new redox systems that can accelerate the oxidative refolding of this protein. In this work, a new redox system composed of 4-mercaptobenzeneacetate (ArSH) and hexanoyl cystamine (HCA) was proposed. It was found that the oxidative refolding of RNase A reached a refolding yield of 94% in 2 h with ArSH/HCA at optimized concentrations, which was two times faster than that with GSH/GSSG. Moreover, the refolding yield of RNase A from IBs could also reach 89% in 3 h with ArSH/HCA, much better than the performance with GSH/GSSG, which was only 69% in 8 h refolding. Therefore, the use of the new redox system led to a significant increase of refolding yield and over 60% reduction in the refolding time in the inclusion body protein refolding. The results indicated that the new redox system was much more efficient in the oxidative refolding of RNase A than the traditional redox system.
Keywords: Protein; Refolding; Bioseparations; Kinetic parameters; Ribonuclease A; Redox system;

► First study on Penicillin G hydrolysis in a basket bioreactor with immobilized penicillin amidase. ► Modeling of Penicillin G concentration in the outer and inner regions of the immobilized particles. ► Modeling of Penicillin G mass transfer and hydrolysis under substrate and products inhibitions. ► Quantitative comparison between the magnitudes of diffusivity and enzymatic hydrolysis rate.The external and internal mass transfers of Penicillin G in the process of its enzymatic hydrolysis to 6-Aminopenicillanic acid under competitive and non-competitive inhibitions using a bioreactor with stationary basket bed of immobilized penicillin amidase have been analyzed. By means of the Penicillin G mass balance for a single particle of biocatalysts, considering the specific kinetic model proposed by Warburton et al., mathematical expressions have been developed for describing the profiles of Penicillin G concentrations and mass flows in the outer and inner regions of biocatalyst particles, as well as for estimating the influence of internal diffusion on its hydrolysis rate. The results indicated that very low values of internal mass flow could be reached in the particles centre. The corresponding region was considered an “enzymatic inactive region”, its extent varying from 0 to 51% from the overall volume of each biocatalyst. By enzyme immobilization and using the basket bed, the rate of enzymatic reaction is reduced over 160 times compared to the process with free enzyme
Keywords: Penicillin; Bioreactors; Immobilized enzymes; Penicillin amidase; Mass transfer; Diffusion;

► Modeling comparison of co- and counter-diffusion biofilms for nitritation efficiency (NE). ► Higher NE in counter-diffusion biofilm (27.5% vs. 7.9% in co-diffusion biofilm). ► Oxygen affinity constants determine NE to a higher extent in the co-diffusion biofilms. ► Maximum permissible oxygen concentration at the interfaces higher in counter-diffusion biofilms.The effect of biokinetics on nitritation was investigated in two biofilm geometries, the Membrane Aerated Biofilm Reactor (MABR) and a conventional biofilm system. A 1D biofilm model was used and evaluated by global sensitivity analysis using the variance based Sobol method. The main focus was on the influence of key biokinetic parameters (maximum specific growth rates, oxygen and nitrogen affinity constants of AOB (ammonium oxidizing bacteria) and NOB (nitrite oxidizing bacteria)) and their ratios on nitritation efficiency in these geometries. This exhaustive simulation study revealed that nitritation strongly depends on the chosen kinetic parameters of AOB and NOB. The maximum specific growth rates (μ max,AOB and μ max,NOB) had the strongest impact on nitritation efficiency (NE). In comparison, the counter-diffusion geometry yielded more parameter combinations (27.5%) that resulted in high NE than the co-diffusion geometry (7.9%). The oxygen concentrations at the relevant biofilm interfaces (membrane/biofilm for counter-diffusion or bulk/biofilm for co-diffusion) were not predictive of NE. However, the maximum allowable oxygen concentration to maintain higher NE was higher for the counter-diffusion geometry.
Keywords: Nitritation; Biofilm modeling; Biofilm geometry; MABR; Oxygen affinity; Specific growth rate;

Purification of lactic acid from microfiltrate fermentation broth by cross-flow nanofiltration by J. Sikder; S. Chakraborty; P. Pal; E. Drioli; C. Bhattacharjee (130-137).
► Cross flow nanofiltration. ► Study of nanofiltration membrane with a cell-recycle fermentation unit for high purity l (+) lactic acid. ► Lactic acid fermentation broth produced by fermentation of sugar cane juice with the help of Lactobacillus plantarum. ► Recovery and recycle of unconverted sugars making the process economically attractive.Experimental investigations were carried out on cross-flow nanofiltration of microfiltrate fermentation broth of lactic acid during membrane-integrated fermentative production of monomer grade lactic acid. Three types of composite polyamide nanofiltration membranes (NF2, NF3 and NF20) manufactured by Sepro Co. (USA) were used in nanofiltration. Lactic acid fermentation broth produced by fermentation of sugar cane juice by Lactobacillus plantarum in a membrane-integrated hybrid reactor system was filtered by NF membranes after preliminary filtration through microfiltration membranes for cell separation and recycle. Effects of cross flow rate, transmembrane pressure and pH on flux and rejection of both acid and unconverted sugars for recycle were analyzed. NF3 membrane was successful in retaining 94% unconverted sugars while allowing 32% lactic acid to permeate at a flux of 113 L m−2  h−1 at pH 5.5, temperature 37 °C, transmembrane pressure of 13 bar and cross-flow velocity 2.48 m s−1. The study shows that integration of a properly selected nanofiltration membrane with a cell-recycle fermentation unit and lactate conversion unit can yield l (+) lactic acid with an optical purity of 85.6% through an economically attractive route by recovering and recycling of unconverted sugars. The nanofiltration combine with bipolar electrodialysis in downstream purification can replace the multiple purification steps by two steps while yielding a monomer grade lactic acid from a mixture of unconverted sugars and lactic acid.
Keywords: Cross-flow; Membrane separation; Fermentation broth; Lactic acid; Nanofiltration; Sugar recovery;

Modulation of yeast hexokinase on bio-inspired membranes by Hiroshi Umakoshi; Atsushi Nishida (138-143).
Display Omitted► The positively charged liposome enhanced the enzymatic activity of hexokinase (HK). ► HK activity was enhanced 1.8-fold on liposome with positively-charged microdomain. ► HK activity was enhanced because of its slight conformational change on the liposome.Hexokinase (HK) is the first enzyme of the glycolytic pathway and is known to modulate its own activity by binding to the mitochondrial membrane. In this study, the enzymatic activity of HK was measured in the presence of various liposomes. The positively charged liposome with an appropriate charge density was found to increase the HK activity. The HK activity was enhanced 1.5-fold in the presence of 5 mol% didodecyldimethylammonium bromide (DDAB) and 1.8-fold with 5–10 mol% 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol hydrochloride (DC-Cholesterol) on the POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline) liposome. Analysis of (i) HK binding onto liposome, (ii) intrinsic Trp fluorescence, and (iii) circular dichroism of HK suggested that the HK activity was enhanced on positively charged microdomain because of its slight conformational change through the electrostatic and hydrophobic interactions.
Keywords: Membranome; Membrane stress biotechnology; Liposome; Hexokinase; Positive-charge;

A multi-level meta-heuristic algorithm for the optimisation of antibody purification processes by Ana S. Simaria; Richard Turner; Suzanne S. Farid (144-154).
► A decisional tool to optimise biopharmaceutical purification was created. ► An industrial case study on the production of therapeutic antibodies was addressed. ► Chromatography sequences were optimised to meet cost and impurity removal targets. ► Genetic algorithms optimised column sizing strategies to meet time constraints. ► Graphical tools visualised user preferences for selecting alternative strategies.Multi-product biopharmaceutical facilities need flexible process configurations that can adapt to products with diverse characteristics and impurity loads so as to avoid bottlenecks and delays, whilst meeting final product specifications and cost targets. In order to aid the design of such facilities, this work presents a meta-heuristic optimisation approach using genetic algorithms where different levels of decision are addressed (facility, product sequence and unit operation) and multiple process and business criteria are used to evaluate each alternative generated. This is applied to a case study on the production of therapeutic monoclonal antibodies (mAbs), with a focus on the optimal purification sequences and chromatography column sizing strategies to cope with different facility configurations of upstream and downstream trains and different product impurity loads. The industrial case study provides novel insights that allow the identification of the most cost-effective purification sequences and column sizing strategies that meet demand and purity targets for each product in the facility. Emphasis is placed also on providing methods to visualise the trade-offs in the set of optimal solutions with similar cost values so as to enhance the decision making process.
Keywords: Biopharmaceutical manufacturing processes; Antibody purification; Process economics; Genetic algorithms; Multi-product facility design;

The schematic representation of l-NIBC-Au (a) and d-NIBC-Au (b) interact with HSA. The molecular configuration of l-NIBC had matched better with HSA than the case of d-NIBC.Display Omitted► Electrochemical methods were used to investigate the enantioselective interaction. ► HSA showed different behaviors on N-isobutyryl-cysteine enantiomers chiral surfaces. ► Larger electrochemical response signals were observed after HSA adsorbed on N-isobutyryl-l-cysteine-Au than N-isobutyryl-d-cysteine-Au. ► This work provides a reference for studying chiral drugs.The stereoselective interaction between human serum albumin (HSA) and N -isobutyryl-cysteine (NIBC) enantiomers was investigated by cyclic voltammetry and electrochemical impedance spectroscopy. The electrochemical responses of the NIBC enantiomers modified surfaces were varied along with the inserted time and the concentration of HSA. The results showed that HSA molecules displayed different adsorption behaviors on NIBC enantiomers modified surfaces and preferred to be adsorbed on the l-NIBC modified surface. That is to say, l-NIBC modified surface had stronger interaction with HSA molecules than d-NIBC modified surface, suggesting that the molecular configuration of l-NIBC had matched better with HSA than the case of d-NIBC. The investigation of the interaction between protein and chiral molecules may not only help to understand the high selectivity of chirality in biosystems, but also provide a reference for studying chiral drugs.
Keywords: N-isobutyryl-cysteine; Human serum albumin; Adsorption; Amino acids; Protein; Sensors;

► We validate instrumented flow followers under real flow conditions in a stirred vessel of a highly viscous bio-substrate. ► The sensor particles resist the harsh environments of the mixing process. ► The acquired data reflect the internal conditions of the vessel correctly. ► Parameter profiles such as residence profiles and velocity profiles are extracted from the measured data. ► Flow dynamics and macro-mixing effects are evaluated from the measured data.A group of flow following sensor particles was validated under real flow conditions in a highly viscous substrate in a 1000 L model fermenter vessel, equipped with a pitched blade impeller, which was operated at two different axial positions in an intermittent mixing regime. The neutrally buoyant sensor particles track basic hydrodynamic and process parameters, namely hydrostatic pressure (giving the axial position), temperature and acceleration. The sensors are connected to a measurement electronics, which is enclosed in a robust capsule that can resist the harsh conditions in an industrial mixing process. The results show that the sensor particles still reflect the flow conditions in the vessel qualitatively. Moreover, the sensor particle data allow estimation of macro-mixing parameters, such as circulation time distributions and average circulation times.
Keywords: Flow follower; Sensor particle; Autonomous sensor; Macro-mixing; Circulation time; Parameter tracking; Bio-substrate; Biogas; Bioreactor;

Development of growth factor-immobilizable material for hepatocyte transplantation by Yung-Te Hou; Hiroyuki Ijima; Nana Shirakigawa; Takayuki Takei; Koei Kawakami (172-181).
► Growth factor-immobilizable material was developed for hepatocyte transplantation. ► Partial hepatectomy were used to promote growth factor secretion. ► This material is effective for the in vitro hepatocyte culture. ► Angiogenesis and viability of transplanted cells were improved in vivo.Growth factor (GF)-immobilizable materials were developed as a practical hepatocyte transplantation method for reconstructing a tissue-like structure in liver tissue engineering. Two GF-immobilizable scaffolds, namely single hepatocyte-embedded, heparin-immobilized, collagen-gel-filled polyurethane foam, and hepatocyte spheroid-embedded, heparin-immobilized, collagen-gel-filled polyurethane foam were developed by covalently incorporating heparin into collagen gel, using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide/N-hydroxysuccinimide for hepatocyte transplantation. Seventy percent partial hepatectomy (PH) was performed at the same time after hepatocyte transplantation. Angiogenesis efficiency and viability of transplanted cells are discussed in terms of normalized hemoglobin content, nuclear density and histological observations after transplantation. In summary, the normalized hemoglobin content and viability of transplanted cells were higher in GF-immobilized scaffolds with PH pretreatment than in the other scaffolds with/without PH pretreatment. These materials have the potential for in vivo hepatocyte transplantation, as GFs released from remnant liver were easily incorporated into the heparin-immobilized collagen gel system. These GF–heparin complexes may promote the survival of embedded cells. Furthermore, the transplantation of spheroids promoted increased angiogenesis compared with hepatocytes, and resulted in sufficient vascularization for cell survival.
Keywords: Growth factor-immobilizable scaffold; Partial hepatectomy; Heparin; Hepatocyte transplantation;

Dialysis shake flask for effective screening in fed-batch mode by Cornelia Bähr; Bernd Leuchtle; Christian Lehmann; Julia Becker; Markus Jeude; Frank Peinemann; René Arbter; Jochen Büchs (182-195).
► Parallel small-scale fed-batch cultivations by continuous diffusion driven nutrient release. ► Screening in fed-batch mode similar to conditions in production process possible. ► Feed rate can be adjusted in a broad range and any desired nutrient can be fed. ► Up to 1000fold increase of catabolite repressed production in fed-batch mode. ► In combination with Respiration Activity Monitoring System powerful screening tool.In the traditional bioprocess development, the operation mode changes from batch in screening to fed-batch in lab and production scale. The presented dialysis shake flask allows small-scale fed-batch cultivations by a diffusion driven nutrient release. It consists of a feed reservoir system which is filled with a highly concentrated nutrient solution. The nutrient diffuses through a dialysis membrane into the medium. Cultivations with Escherichia coli BL21 (DE3) pRhotHi-2-EcFbFP and Hansenula polymorpha RB11 pC10-FMD (PFMD-GFP) proved the feasibility of the concept. As positive effects of these small-scale fed-batch cultivations, enhanced (up to 1000fold) yields for catabolite repressed product formation, reduced overflow metabolism and diminished medium acidification were observed. The new dialysis fed-batch shake flask is characterised by a high flexibility regarding the attainable feed rates and the spectrum of feedable compounds. The feed rate can be adjusted in a broad range by simply adapting the diffusion parameters. Based on the working principle, the release of any desired compound is possible. The dialysis fed-batch shake flask allows the detailed investigation of strains and process conditions under operation mode similar to industrial production process. Combined with the Respiration Activity Monitoring System (RAMOS), it represents an effective screening tool for the first steps of bioprocess development.
Keywords: Bioreactors; Diffusion; Fed-batch culture; Shake flask; Dialysis; Screening;

► A multi-promoter construct was built to co-express a whole cellulase system. ► A multi-cistronic construct was built to co-express a whole cellulase system. ► Synergistic effect of cellulases expressed in the work was investigated. ► Significantly higher glucose was produced with co-expression strategy.Usually, glucose production from cellulose hydrolysis involves synergistic actions by exoglucanase, endoglucanase and β-glucosidase. To co-produce a whole cellulase system in Escherichia coli, a multi-promoter construct and a multi-cistronic construct harboring an exoglucanase (CbhA), an endoglucanase (CenA) and a β-glucosidase (BGL) were built herein. Synergistic effect of three enzymes was also investigated. The results showed synergism of CenA and BGL played a critical role in degrading filter paper to glucose. Compared with monocistronic constructs, multi-promoter and multi-cistronic constructs harboring three enzymes showed significantly higher glucose yield. The specific activity of multi-promoter construct was 22.5-fold and 6.9-fold higher than CbhA and CenA, respectively and that of multi-cistronic construct was 33.6-fold and 10.3-fold higher than CbhA and CenA, respectively. In conclusion, a whole cellulase system could be co-produced in E. coli for converting cellulose to glucose. Moreover, the strategy could be easily applied to other cellulolytic enzymes, as well as for other multi-enzymatic systems.
Keywords: Cellulase; Co-expression; Escherichia coli; Enzyme production; Enzymes; Cellulose;