Biochemical Engineering Journal (v.79, #C)

BEJ Keywords (IV).

Bioaugmentation is an effective treatment method to reduce recalcitrant pollutants from polluted sites. Dimethylformamide (DMF) is a very common toxic organic solvent among the effluents of textile and pharma industries. DMF was degraded by pre-adapted Paracoccus denitrificans SD1 with indigenous mixed cultures in both bioaugmentation and non-bioaugmentation conditions. In free cell condition, augmentation was not much significant due to competition among the bacterial cells and direct exposure of cells to toxic level of DMF. To enhance the degradation of DMF, cells were entrapped in PVA–alginate matrix individually and collectively for bioaugmentation experiments. Bioaugmentation is successful when immobilized P. denitrificans SD1 is introduced higher inoculum volume with indigenous cultures in continuous packed bed reactor system. This treatment has succeeded in removing 91.3% of 3% (v/v) DMF from the industrial effluent. This investigation advocates that bioaugmentation enhances the DMF removal efficiency by about 20% when compared to individual degradation by P. denitrificans SD1.
Keywords: N,N-Dimethylformamide; Bioaugmentation; Bioremediation; Packed bed reactor; Immobilization; Waste-water treatment;

There has been a broad spectrum of theoretical and experimental works on microorganism disruption methods undertaken in the past. However, there is a lack of understanding regarding the actual reasons for microorganism disruption using ultrasound and whether it is caused by shock or shear. In the case of shear stress, which is the focus of this paper, analysis of the intense turbulent flow region of an in-house built shear apparatus combined with the experimental results demonstrated that when the energy dissipation rate in the turbulence region is high, and the size of the eddy is smaller than the size of the cell, the likelihood of yeast disruption is high. The mechanical properties of yeast cells combined with the calculated energy dissipation rate were used to evaluate the yeast disruption efficiency (log reduction). The results show that the shear apparatus can efficiently and effectively disrupt S. cerevisiae at different treatment times, suspension temperatures and rotor speeds. The experimental work suggests that maximum yeast log reduction was achieved when the maximum power dissipation of 2.095 kW was recorded at 10,000 RPM, while suspension temperature was controlled below 35 °C. The corresponding shear stress at 10,000 RPM was 2586.2 Pa.
Keywords: Yeast; Shear treatment; Cell disruption; Energy; Water;

Modelling and simulation was done for a two-stage membrane-integrated hybrid reactor system for continuous production of L (+) lactic acid under non-neutralizing conditions. The model captures microbial conversion of sugar cane juice to lactic acid under substrate–product inhibitions with downstream purification by nanofiltration. All the major phenomena and the governing parameters like fluid flow, feed dilution, substrate–product inhibitions, Donnan and steric effects during micro and nanofiltration for cell recycle, product separation and purification have been reflected in the modelling. The model describes a green, integrated continuous process of direct lactic acid production starting with a cheap, renewable carbon source. The highest lactic acid concentration achieved after the final stage of nanofiltration was 66.97 g/L at 13 kg/cm2 operating pressure when the overall productivity reached 12.40 g/(L h). The developed model could successfully predict production, purification and transport of lactic acid through two stage membrane modules. Performance of the model was very good as indicated in the high overall correlation coefficient (R 2  > 0.980) and the low relative error (RE  < 0.1).
Keywords: Lactic acid; Membrane bioreactors; Scale-up; Modelling; Production kinetics;

A comparative study was made on immobilized Burkholderia cepacia lipase (PSL-C)-catalyzed acylation of lily polysaccharide (LP) with vinyl acetate in the ionic liquid (IL) 1-butyl-3-methylimidazolium tetrafluorobrate ([C4MIm][BF4]) under ultrasonic irradiation and magnetic stirring. The degree of substitution (DS) of the modified LP was used to evaluate the extent of acylation and thus enzymatic activity. The application of ultrasonic irradiation instead of magnetic stirring during acylation gave enhanced DS of the product in a shorter reaction time. The optimization studies also included variation in ultrasonic power, water activity and reaction time. PSL-C displayed higher operational stability and 56.0% of its original activity was maintained after being reused in 8 batches under ultrasonic irradiation. The acylation of LP in [C4MIm][BF4] was shown to be a regioselective one under both conditions, with C6―OH being acylated. The results demonstrated that the combined application of ultrasonic irradiation and ILs as a medium may be an efficient approach for enzymatic modification of LP.
Keywords: Lily polysaccharide; Ultrasonic irradiation; Enzyme biocatalysis; Lipase; Immobilized enzymes; Water activity;

This communication reports a new design of peptide disulfide, RKCGCFF, for facilitating oxidative protein refolding. The new design mimics the properties of protein disulfide isomerase (PDI) by introducing hydrophobic and positively charged patches into the two terminals of disulfide CGC. RKCGCFF was found more effective than the traditional oxidant oxidized glutathione (GSSG) as well as its counterpart, RKCGC, in facilitating the oxidative refolding of lysozyme. More importantly, RKCGCFF could improve lysozyme refolding yield at a high concentration (0.7 mg/mL). The research proved that incorporation of hydrophobic and charged patches into the CGC disulfide made the oxidant more similar to PDI in structure and properties.
Keywords: Protein; Refolding; Bioseparations; Protein recovery; Peptide disulfide; Foldase mimic;

Screening of peptide ligands that bind to the Fc region of IgG using peptide array and its application to affinity purification of antibody by Tomoya Sugita; Makoto Katayama; Mina Okochi; Ryuji Kato; Takamitsu Ichihara; Hiroyuki Honda (33-40).
Small peptides have attracted great interest for affinity purification since they are more stable, less immunogenic, and less expensive compared to protein ligands. In this study, screening of peptides that has affinity to Fc region of IgG was investigated from amino acid sequence of the IgG Fcγ receptor, which is well known as one of the IgG-Fc binding proteins, using a spot-synthesized peptide array. High affinity octamer peptides, NKFRGKYK and NARKFYKG, for mouse IgG were obtained by amino acid substitution assays. Both peptides also recognized human IgG-Fc. The association constants for human IgG-Fc were 8.9 × 106 (NKFRGKYK) and 6.5 × 106  M−1 (NARKFYKG). Purity of the human IgG-Fc from cell culture medium using the peptide immobilized resins was 83% for NKFRGKYK and 68% for NARKFYKG. These peptide ligands were used for the purification of monoclonal antibody (MAb) from cell culture supernatants. The yields obtained for the antibody were found to be 69% and 80% for NKFRGKYK and NARKFYKG, respectively. The residual DNA and host cell protein reduction obtained by these peptides resin were in the range of 4.2 and 1.3 log reduction value (LRV) (NKFRGKYK), 3.9 and 1.5LRV (NARKFYKG), respectively, comparable to those reported for Protein A. Therefore, it was indicated that the screened NKFRGKYK and NARKFYKG peptide would be useful as affinity purification ligands for IgG.
Keywords: Peptide ligand; IgG; Affinity purification; Peptide array;

Mixed culture of Saccharomyces cerevisiae and Acetobacter pasteurianus for acetic acid production by Zhi Wang; Mei Yan; Xiong Chen; Dongsheng Li; Li Qin; Zhijun Li; Juan Yao; Xinle Liang (41-45).
Mixed culture of Saccharomyces cerevisiae and Acetobacter pasteurianus was carried out for high yield of acetic acid. Acetic acid production process was divided into three stages. The first stage was the growth of S. cerevisiae and ethanol production, fermentation temperature and aeration rate were controlled at 32 °C and 0.2 vvm, respectively. The second stage was the co-culture of S. cerevisiae and A. pasteurianus, fermentation temperature and aeration rate were maintained at 34 °C and 0.4 vvm, respectively. The third stage was the growth of A. pasteurianus and production of acetic acid, fermentation temperature and aeration rate were controlled at 32 °C and 0.2 vvm, respectively. Inoculation volume of A. pasteurianus and S. cerevisiae was 16% and 0.06%, respectively. The average acetic acid concentration was 52.51 g/L under these optimum conditions. To enhance acetic acid production, a glucose feeding strategy was subsequently employed. When initial glucose concentration was 90 g/L and 120 g/L glucose was fed twice during fermentation, acetic acid concentration reached 66.0 g/L.
Keywords: Acetic acid; Acetobacter; Yeast; Mixed culture; Orthogonal experimental design; Fed-batch culture;

Inactivation of Lactococcus lactis ssp. cremoris cells in a droplet during convective drying by Nan Fu; Meng Wai Woo; Cordelia Selomulya; Xiao Dong Chen (46-56).
Spray drying is a less costly alternative to freeze drying in the mass-production of active dry microorganisms, if the drying conditions could be optimized to preserve cell viability. As spray drying is akin to a black-box process, in this study we used an alternative approach of a single droplet drying to study how drying conditions affect the inactivation of bacterial cells. The inactivation histories of Lactococcus lactis ssp. cremoris were investigated at air temperatures of 70, 90, and 110 °C. It was found that the viability of L. cremoris cells could be maintained at approximately the original level for extended drying durations (60–210 s), despite the high air temperatures. When plotted against droplet temperature T d , the inactivation rate k d at six drying conditions formed a general trend. An inactivation model was proposed to describe different inactivation histories under varied drying conditions. The description closely followed the experimental data, reported for the first time in literature. k d increased rapidly after T d passed a transition temperature range of 50–65 °C, coinciding with the onset temperature for denaturation of bacterial ribosomes. Other environmental parameters affecting inactivation are discussed to better understand the integrated effects of multiple stresses experienced by bacterial cells during convective drying.
Keywords: Viability; Kinetic parameters; Modelling; Food engineering; Inactivation kinetics; Culture preservation;

Five-step continuous production of PHB analyzed by elementary flux, modes, yield space analysis and high structured metabolic model by Markan Lopar; Ivna Vrana Špoljarić; Aid Atlić; Martin Koller; Gerhart Braunegg; Predrag Horvat (57-70).
A high structured metabolic model for PHB synthesis by Cupriavidus necator DSM 545 consisting of 43 mass balance equations related to the same number of intracellular compounds was established. The metabolic state of cells cultivated in a continuously operated five stage bioreactor cascade was analyzed by help of elementary flux modes and two-dimensional yield space. Two different C-source feeding strategies were performed. Concerning PHB and biomass yields, values of the more efficient strategy were used as the data source for elementary modes and metabolic flux calculations, respectively. Metabolic fluxes were calculated from experimental yield data using a combination of elementary modes by applying the quadratic programming approach, in which the sum of squared weighting factors was minimized. Two different metabolic situations concerning activity of glucose-6-phosphate isomerase were tested. The high structured metabolic model was validated by comparison of experimental data from 24 h batch cultivation and simulated results.
Keywords: Cupriavidus necator; Elementary modes; High-structured mathematical model; Metabolic fluxes; poly(3-Hydroxybutyrate); Yield space analysis;

Synthetic p-tetrasulphonatocalix[4]arene as novel excipient for lipase-complex by Irshad Ali Veesar; Shahabuddin Memon; Muhammad Noman Syed (71-76).
The present article describes formation of excipient-CRL complex from water soluble calix[4]arene derivative (3 as excipient) and Candida rugosa lipase (CRL), which is proposed as a reusable form of enzyme that is free from steric and diffusion limitations associated with those enzymes immobilized onto porous solid supports. The excipient-CRL could completely hydrolyze 50 mM p-nitrophenyl palmitate (p-NPP) in Tris–HCl buffer at a wide range of temperatures, i.e. 30–80 °C. It is stable under stirred conditions and could be reused multiple times without loss of enzyme activity. It was observed that excipient-CRL complex shows a significant effect on the enzyme activity with an enhancement in thermal stability, while pH and temperature affect the activity of excipient-CRL as well as free CRL. Consequently, the excipient-CRL was found more active than free CRL for the hydrolysis of p-NPP in respect of its reusability.
Keywords: Lipase; Enzyme; Excipient; Hydrolysis; Calixarene; p-Nitrophenyl palmitate;

CO2 fixation for succinic acid production by engineered Escherichia coli co-expressing pyruvate carboxylase and nicotinic acid phosphoribosyltransferase by Rongming Liu; Liya Liang; Mingke Wu; Kequan Chen; Min Jiang; Jiangfeng Ma; Ping Wei; Pingkai Ouyang (77-83).
In wild-type Escherichia coli, 1 mol of CO2 was fixated in 1 mol of succinic acid generation anaerobically. The key reaction in this sequence, catalyzed by phosphoenolpyruvate carboxylase (PPC), is carboxylation of phosphoenolpyruvate to oxaloacetate. Although inactivation of pyruvate formate-lyase and lactate dehydrogenase is found to enhance the PPC pathway for succinic acid production, it results in excessive pyruvic acid accumulation and limits regeneration of NAD+ from NADH formed in glycolysis. In other organisms, oxaloacetate is synthesized by carboxylation of pyruvic acid by pyruvate carboxylase (PYC) during glucose metabolism, and in E. coli, nicotinic acid phosphoribosyltransferase (NAPRTase) is a rate-limiting enzyme of the NAD(H) synthesis system. To achieve the NADH/NAD+ ratio decrease as well as carbon flux redistribution, co-expression of NAPRTase and PYC in a pflB, ldhA, and ppc deletion strain resulted in a significant increase in cell mass and succinic acid production under anaerobic conditions. After 72 h, 14.5 g L−1 of glucose was consumed to generate 12.08 g L−1 of succinic acid. Furthermore, under optimized condition of CO2 supply, the succinic acid productivity and the CO2 fixation rate reached 223.88 mg L−1  h−1 and 83.48 mg L−1  h−1, respectively.
Keywords: Anaerobic processes; Fermentation; Enzymes; Glucose; CO2 fixation; Succinic acid;

The anaerobic co-digestion of cow manure and waste paper at ambient temperature condition was observed to be optimized at a mix proportion of 75:25 respectively. The development and testing of a set of simplified anaerobic digestion models (SADM's) for this mixture revealed that the Hill's based biogas yield rate model was most appropriate in describing the kinetics of biogas production. Parameter estimation using non-linear regression revealed that the half saturation constants expressed as acidified substrate and volatile solids equivalents were 0.228 g/L and 5.340 g VS/L respectively, and the maximum specific biogas yield rate and biodegradability were 2.2 mL/g VS/day and 0.313 respectively. The coefficients “n” and “m” indicative of acidogenic bacterial adaptation for degradation and acetogenic/methanogenic bacterial cooperativity were estimated to be 1.360 and 2.738 respectively, while hydrolysis/acidogenesis was considered the rate limiting step. The need of bacterial adaptation may be an important factor to consider during anaerobic modeling of complex biomass.
Keywords: Anaerobic process; Biodegradability; Biogas; Kinetic parameters; Growth kinetics; Rate limiting;

In our previous work, the ability of laccase enzymes to improve the fermentation performance of the thermotolerant yeast Kluyveromyces marxianus CECT 10875 on steam-exploded wheat straw slurry was demonstrated. As a continuation of this study, the present research evaluates different aspects, including pretreatment conditions, process configurations and substrate loadings, with the aim to proceed towards the use of K. marxianus and laccases for second generation ethanol production. For it, two wheat straw slurries resulting from different steam explosion pretreatment conditions (200 °C, 2.5 min and 220 °C, 2.5 min) were employed at various substrate loadings [5–14% (w/v)] under two process configurations: SSF (simultaneous saccharification and fermentation) and PSSF (presaccharification and simultaneous saccharification and fermentation). The better performance of K. marxianus was observed on the slurry produced at softer conditions. Its lower inhibitors content allowed to increase the total solids loading up to 10% (w/v) in both process configurations, reaching higher ethanol concentrations (12 g/L). Moreover, laccase detoxification improved these results, particularly in SSF processes, increasing the substrate loading up to 12% (w/v) and, consequently, obtaining the highest ethanol concentration (16.7 g/L).
Keywords: Lignocellulosic ethanol; Kluyveromyces marxianus; Laccase detoxification; Steam explosion; Process configurations; Substrate loadings;

Engineering of a bi-enzymatic reaction for efficient production of the ascorbic acid precursor 2-keto-l-gulonic acid by Vanja Kaswurm; Wouter van Hecke; Klaus D. Kulbe; Roland Ludwig (104-111).
The enzymatic production of the vitamin C precursor 2-keto-l-gulonate (2-KLG) from 2,5-diketo-d-gluconate (2,5-DKG) was catalyzed by coupling 2,5-diketo-d-gluconic acid reductase (2,5-DKG reductase) via its coenzyme to glucose dehydrogenase. This bi-enzymatic process shows complicated inhibition patterns caused by reaction products, NADP+ and NADPH. The bioconversion was optimized by modeling. Rate equations were derived for both enzymes and solved simultaneously by a numerical method to predict 2-KLG production. A series of batch conversions were used to verify the model and construct isocharts to determine optimal process parameters. The following key parameters for a fast and efficient conversion were found: (1) the NADP(H) concentration, (2) the volumetric activity of 2,5-DKG reductase, (3) the ratio of synthetic enzyme activity to regenerate enzyme activity and (4) the glucono-1,5-lactone concentration. By modeling the space-time yield of the process was nearly doubled and the coenzyme concentration reduced threefold.
Keywords: Coenzyme regeneration; 2-Keto-l-gulonic acid; NADP(H); Modeling; Process engineering;

Debaryomyces hansenii fermentation for arabitol production by Srujana Koganti; Lu-Kwang Ju (112-119).
The fermentation process for arabitol production from glycerol was developed using a Debaryomyces hansenii strain recently selected from a broad screening. The high-producing strain produced arabitol as the only detectable polyol from glycerol. In this work, the pH, dissolved oxygen concentration (DO), inoculum size and magnesium concentration, and the nitrogen-to-phosphorus (N/P) ratio were systematically evaluated for effects on cell growth rate and arabitol productivity. Among those evaluated, the medium with N/P = 9, DO of 5% air saturation and pH 3.5 supported the highest arabitol production. Under these optimal conditions, arabitol production of 40 g/L was achieved in 5 days compared to earlier studies with 15 g/L arabitol in 5 days. Volumetric productivity and specific productivity were successfully improved from 0.13 to 0.33 g/L-h and 0.007 to 0.02 g/g-h respectively with arabitol yield of 55% from glycerol.
Keywords: Arabitol; Glycerol; Debaryomyces hansenii; Osmotolerant yeast; Fermentation; Optimization;

Urea-induced protein denaturation can be effectively inhibited by trehalose, but the thermodynamic and kinetic behaviors are still unclear. Herein, the counteraction of trehalose on urea-induced unfolding of ferricytochrome c was studied. Thermodynamic parameters for the counteraction of trehalose were derived based on fluorescence spectroscopic data. Then the kinetics was emphatically investigated by stopped-flow fluorescence spectroscopy. Urea-induced unfolding of ferricytochrome c in 8.00 mol/L urea solution reveals two observable phases, including fast and slow phases following a burst phase. Trehalose has little influence on the burst phase amplitude. Nevertheless, the observable unfolding pathway is significantly affected by trehalose. At lower trehalose concentrations (<0.20 mol/L) in 8.00 mol/L urea, the unfolding pathways still keep to show two phases. However, the rate constant and amplitude for the fast phase diminish with increasing trehalose concentration. In contrast, the rate constant for the slow phase shows only a slight change with a significant increase of the amplitude. At higher trehalose concentrations (>0.30 mol/L), the unfolding pathway is transformed into a single slow phase. The rate constant and amplitude for the single phase also decrease with increasing trehalose concentration. The studies are expected to help our understanding of trehalose effects on protein stability.
Keywords: Protein; Biophysical chemistry; Protein denaturation; Kinetic parameters; Trehalose; Urea;

The effects of protein solubility on the RNA Integrity Number (RIN) for recombinant Escherichia coli by Mary Alice Salazar; Lawrence P. Fernando; Faraz Baig; Sarah W. Harcum (129-135).
High quality, intact messenger RNA (mRNA) is required for DNA microarray and reverse transcriptase polymerase chain reaction analysis and is generally obtained from total RNA isolations. The most widely recognized measure of RNA integrity is the RNA Integrity Number (RIN) obtained from the Agilent Bioanalyzer, as it provides sizing, quantification, and quality control measures. This work describes comparisons of the RIN values obtained for recombinant Escherichia coli. Uninduced recombinant E. coli cultures were examined, as well as induced cultures that produced either a soluble or insoluble recombinant protein. The uninduced cultures and the induced cultures producing soluble protein had higher RIN values than the induced cultures producing insoluble protein. These lower RIN values for E. coli producing the insoluble protein indicate that cellular degradation of the ribosomal RNA species is the likely cause of the lower RIN values. As the use of DNA microarrays and other gene expression tools increase in usage in the industrial recombinant protein production community, these results suggest the need for further studies to determine acceptable RIN ranges for gene expression analysis and effects of various culture conditions on RIN values for recombinant E. coli.
Keywords: Aggregation; Protease; Purification; Recombinant DNA; RNA; Inclusion bodies;

Product inhibition is a barrier for enzymatic conversion of cellulose into reducing sugar in single aqueous phase. In addition, the difficulty in the recovery of cellulase also leads to high cost for the enzymatic hydrolysis of cellulose. In this study, enzymatic degradation of cellulose was carried out in pH–pH recyclable aqueous two-phase systems (ATPS) composed by copolymers poly (AA-co-DMAEMA-co-BMA) (abbreviated P ADB3.8) and poly (MAA-co-DMAEMA-co-BMA) (abbreviated P MDB). In the systems, cellulase was immobilized on pH-response copolymer P MDB by using 1-Ethyl-3-(3-dimethyllaminopropyl)-carbodiimide hydrochloride (EDC) as cross-linker. Optimized partition coefficient of product in the systems was 2.45, in the presence of 40 mM (NH4)2SO4. Insoluble substrate and immobilized enzyme were biased to bottom phase, while the product was partitioned to top phase. Microcrystalline cellulose was hydrolyzed into reducing sugar, and the product entered into top phase. The yield of saccharification in ATPS could reach 70.57% at the initial substrate concentration of 0.5% (w/v), and the value was 9.3% higher than that in the single aqueous phase. Saccharification yield could reach 66.15% after immobilized cellulase was recycled five times in ATPS.
Keywords: Aqueous two phase; Cellulose; Cellulase; Immobilized enzymes; Biodegradation; pH-response copolymer;

Improvement of bilirubin adsorption capacity of cellulose acetate/polyethyleneimine membrane using sodium deoxycholate by Jia Ju; Gaohong He; Zhijun Duan; Wei Zhao; Yuanfa Liu; Lingling Zhang; Yinhua Li (144-152).
Bilirubin (BR) adsorbents have low removal efficiency because of the tight binding of BR with human albumin (HA) in a complicated blood system. Sodium deoxycholate (SDC) was selected as an adsorption promoter to improve the BR adsorption capacity of a cellulose acetate (CA)/polyethyleneimine (PEI) membrane. Static adsorption experiments show that the maximum BR adsorption capacity of the membrane with SDC in BR–HA mock solution is 100–200% at the molar ratio of SDC to HA ranging from 8 to 12, higher than that without SDC. It is also found that SDC is more efficiently adsorbed by the membrane than BR and HA. Absorption, circular dichroism, and zeta potential studies demonstrate that SDC can be bound with the BR–HA complex to form a ternary BR–HA-SDC m complex. On the basis, the facilitated adsorption mechanism of BR with SDC was proposed that SDC aggregates or micelles form a quasi-multilayer adsorption on the membrane, increase approachable binding sites, and prolong the distance between the BR–HA complex and the membrane. Thus, SDC as a spacer reduces the influence of the steric hindrance of HA, resulting in an enhanced BR adsorption capacity. Dynamic adsorption results further evidence the facilitated adsorption mechanism.
Keywords: Affinity; Microporous membranes; Chromatography; Bioseparations; Bilirubin; Bile salt;

Physiological characteristics of predominant ammonia-oxidizing bacteria enriched from bioreactors with different influent supply regimes by Akihiko Terada; Sho Sugawara; Tomoko Yamamoto; Sheng Zhou; Keisuke Koba; Masaaki Hosomi (153-161).
Two acclimatized biomasses exposed to ammonium (NH4 +) concentration of 600 mg N L−1, one from a completely stirred tank reactor (CSTR), the other from a sequencing batch reactor (SBR), were assayed for nitritation performance, predominant nitrifying bacterial population and nitrous oxide (N2O) production. By virtue of fluctuating and constant NH4 + concentrations respectively, the SBR and CSTR wastewater supply regimes were hypothesized to support different predominant ammonia-oxidizing bacteria (AOB) exhibiting distinct biokinetic properties. Nitritation efficiency (NO2 -N/NO2+3 -N) was higher in the SBR (89%) than the CSTR (30%) likely due to free ammonia and dissolved oxygen concentration. Quantitative fluorescence in situ hybridization (FISH) analyses revealed that fast-growing (r-strategist) AOB of halophilic and halotolerant Nitrosomonas lineage were more highly enriched in the SBR (76 ± 4.2%) than the CSTR (38 ± 6.0%). The CSTR predominantly enriched slow-growing (K-strategist) AOB Nitrosospira spp. (42 ± 1.9% versus 1.4 ± 0.8% in the SBR). Biokinetic parameter estimation consolidated the FISH result: the maximum growth rate and half-saturation coefficients for NH4 + were higher in the SBR (μ max  = 0.92 day, K NH4+  = 28.9 mg N L−1) relative to the CSTR (μ max  = 0.42 day, K NH4+  = 3.47 mg N L−1), suggesting that the extent of nitritation may be controlled by choice of wastewater influent operational regime, which itself determines predominant AOB. N2O production was a maximum of 25 times higher (10.2 mg N-N2O h−1 at 0.5 mg O2  L−1) in CSTR-enriched biomass than in SBR-enriched biomass (0.41 mg N-N2O h−1 at 0.5 mg O2  L−1).
Keywords: Ammonia-oxidizing bacteria; Biokinetic parameters; Halophilic and halotolerant Nitrosomonas spp.; Nitritation; Nitrosospira spp.;

The impact of the timing of induction on the metabolism and productivity of CHO cells in culture by Zahra Sheikholeslami; Mario Jolicoeur; Olivier Henry (162-171).
Efficient inducible mammalian expression systems are becoming increasingly available and are particularly attractive from a process optimization point of view in that they allow decoupling the growth and production phases. In biphasic processes, the timing of induction is one of the most critical factors to consider for maximizing the productivity, since it will affect both the cumulative biomass concentration and the cell specific productivity. In an effort to assess how these two factors must be balanced for optimal productivity, we have performed a metabolic characterization of CHO cells expressing a recombinant antibody and harboring an efficient inducible expression system. Cells taken at different stages of growth were transferred and induced in fresh medium at their corresponding cell densities, and the kinetics of growth, nutrient consumption and product formation were compared during the production phase. Low cell density inductions achieved lower maximum cell concentrations, but exhibited higher cell specific productivity and greater culture longevity, and ultimately led to increased final product titers. To gain more physiological insights into the observed differences, 13C metabolic flux analysis was performed to characterize and compare the metabolism of cells induced at respectively low and high cell concentrations. A number of key intracellular fluxes were found to be affected by the cell density and the corresponding availability of nutrients during the induction phase. While glucose utilization efficiency is increased in high cell density induction, thereby reducing the specific lactate production rate, it appears to be compensating for the decreased catabolic rates of amino acids. The comprehensive metabolic characterization performed in this work can help guide the development of an efficient feeding strategy pre- and post-induction.
Keywords: Inducible expression; 13C-metabolic flux analysis; CHO cell cultures;

Fed-batch operational strategies for recombinant Fab production with Pichia pastoris using the constitutive GAP promoter by Xavier Garcia-Ortega; Pau Ferrer; José Luis Montesinos; Francisco Valero (172-181).
Carbon source and growth rate are two major parameters affecting recombinant protein production in Pichia pastoris. The effect of the most commonly used carbon sources (glycerol or glucose) and the specific growth rate (μ) has been studied on the production of a human antigen-binding fragment (Fab) in this cell factory under the constitutive GAP promoter in fed-batch cultures.Glycerol for batch phase and glucose for fed-batch phase was the most successful carbon source combination. During batch phase, by-products were detected when glucose was used, despite maintaining DO at values higher than 35%. Also, the presence of cell aggregates was detected affecting the reproducibility and operability of the bioprocess.Conversely, glucose was the best substrate for fed-batch phase. When working at C-limiting conditions, neither by-products nor aggregates were detected and Fab production levels were comparable to those obtained with glycerol. In addition, the lower heat yield (Y Q/X ) and oxygen to biomass yield ( Y O 2 / X ) for glucose-supported cultures made this substrate the best alternative from an industrial operational point of view.In addition, the effect of specific growth rate on fed-batch Fab production was studied. Medium and high μ (0.10 and 0.15 h−1) set-points showed similar Fab production yield. However, in terms of total and volumetric productivity, higher μ was the best process condition.
Keywords: Pichia pastoris; Recombinant protein production; Antibody fragment; P GAP ; Fed-batch; Specific growth rate; Substrates;

Esterase BioH is a critical enzyme for Biotin synthesis in Escherichia coli, which has been previously found to be active in the acylation of secondary alcohols and amines. Directed evolution towards improved acylation activity requires a high-throughput screening method. The aim of this study is to explore whether the acylation activity of BioH can be improved by directed evolution of its hydrolysis activity. A colorimetric method based on p-nitrophenyl butyrate hydrolysis was adopted in the high-throughput determination of hydrolysis activity. The wild-type BioH showed a hydrolysis activity of 18 U/mg, and the specific activities for α-phenylethanol and α-phenylethylamine acylation were 12.8 U/mg and 3.5 U/mg, respectively. After two rounds of directed evolution, seven mutants with improved hydrolysis activity were obtained, among which, K213E, Q70L/M170T and M197L/K213E also showed improvement in acylation activity. To further improve the acylation activity, site mutations were generated in different combinations at the four hot spots Q70L, M170T, M197L and K213E. Among the resulting mutants, Q70L/M197L/K213E showed the highest activity in α-phenylethylamine acylation with a 120% improvement, while Q70L/K213E had the highest α-phenylethanol acylation activity, which was improved by 70%. The results demonstrated that directed evolution of the hydrolysis activity might be a possible approach to improve the acylation activity of the esterase BioH.
Keywords: Enzyme technology; Biocatalysis; Enantioseparation; Enzyme activity; Escherichia coli BioH; Directed evolution;

Hydroquinone glycosides were produced by transglycosylation reactions catalyzed by cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. (Toruzyme® 3.0L). The reactions were carried out in an aqueous system containing hydroquinone (HQ) and maltodextrin as acceptor and donor substrate molecules respectively. The conditions for the synthesis of hydroquinone glucoside (α-arbutin) were 9 mM hydroquinone, maltodextrin (5%, w/v) in 20 mM citrate phosphate buffer, pH 5.5 and 0.025 mg/ml toruzyme at 40 °C for 24 h. The transfer efficiency of hydroquinone glycosylation was 31.8% and 29.2% respectively, when α-cyclodextrin and maltodextrin were employed as donor substrates. The major glycoside product was identified as hydroquinone-1-O-α-d-glucopyranoside (α-arbutin) on the basis of mass spectrometric, nuclear magnetic resonance analysis and component analysis of its enzymatic hydrolysates. The highest molar yield of α-arbutin (21.2%) was obtained when α-cyclodextrin was used as the donor substrate. A two step enzymatic reaction system comprising of CGTase and amyloglucosidase helped to attain a molar yield of 30% for α-arbutin. At room temperature the solubility of α-arbutin in water was determined to be 12.8 g/100 ml which is approximately 1.8 fold higher than that of hydroquinone.
Keywords: Enzymes; Biocatalysis; Purification; Bioconversion; Arbutin; Transglycosylation;

In this study, a biphasic fermentation system coupling use of surfactant and in situ extractant was established to enhance Antrodin C production in submerged fermentation of Antrodia camphorata. Effects of several crucial factors such as the selection, concentration and addition time of surfactants and in situ extractants on the mycelial growth and production of Antrodin C were systematically studied. Results showed that the optimal surfactant and extractant were Tween-80 and soybean oil, respectively. The maximum production of Antrodin C (247.09 ± 0.97 mg/L) was obtained when 0.1% (w/v) Tween-80 (as surfactant) and 10% (v/v) soybean oil (as in situ extractant) both supplied at the logarithmic growth phase of fermentation (the 72th h and the 96th h, respectively), corresponding to 3.6 times significant increase compared with that of the control (53.23 ± 0.86 mg/L).
Keywords: Antrodia camphorata; Antrodin C; Biosynthesis; Bioprocess design; Submerged culture; Integrated processing;

Enhanced d-hydantoinase activity performance via immobilized cobalt ion affinity membrane and its kinetic study by Yi-Miao Ko; Chih-I Chen; Chia-Chi Lin; Shu-Chen Kan; Chi-Zong Zang; Chiung-Wen Yeh; Wei-Feng Chang; Chwen-Jen Shieh; Yung-Chuan Liu (200-205).
Various immobilized metal ions affinity membranes (IMAMs) were prepared from the regenerated cellulose membrane (RC membrane) and chelated with various metal ions such as Co2+, Ni2+, Cu2+ and Zn2+. The D-hydantoin-hydrolyzing enzyme (DHTase) harboring a poly-His tagged residue was used as a model protein to be immobilized on the prepared IMAMs through the direct metal–protein interaction forces. The adsorption isotherm and the kinetic parameters V max, K m,app of DHTase on IMAMs were studied. The cobalt ions chelated IMAM (Co-IMAM) was found to yield the highest specific activity of DHTase. Under the immobilization condition, the cobalt ion chelated amount was 161.4 ± 4.7 μmol/disk with a DHTase activity of 4.1 ± 0.1 U/disk. As compared to the free DHTase, the immobilized DHTase membrane could achieve a broader pH tolerance and higher thermal stability. In addition, 98% of the residual activity could be retained for 7-times repeated use. Only little activity loss was observed within 36-day storage at 4 °C. This is the first report concerning about using cobalt ion as the effective chelated metal ion for simultaneous purification and immobilization operation.
Keywords: Enzyme biocatalysis; Immobilization; Membrane bioreactors; Affinity; poly-His tag; Hydantoinase;

Magnetic molecularly imprinted polymers for improved extraction of tanshinones from herbs via integrated extraction and cleanup system by Wensong Li; Liangrong Yang; Fuchun Wang; Huacong Zhou; Xingfu Yang; Yinbin Huang; Huizhou Liu (206-213).
A novel separation technology at room temperature for traditional Chinese medicines was proposed in this work by adding magnetic molecularly imprinted polymers (M-MIPs) into extraction solution and sample matrix. The M-MIPs show a more adsorption capacities and higher selectivity for the template than magnetic non-molecularly imprinted polymers (M-NMIPs) without the specific binding sites. Addition of the M-MIPs to the extraction solution provides one-step extraction and cleanup, the improvement of extraction rate and extraction yields of three tanshinones (from 0.40, 0.23 and 0.12 mg g−1 in 240 min by solvent extraction to 0.52, 0.27 and 0.19 mg g−1 in 5 min by 200 mg sorbent), and reusability of extraction solvent. The extraction yields of three tanshinones by this technology at room temperature in 5 min were higher than those by ultrasonic extraction in 30 min, by heat reflux extraction in 45 min and by solvent extraction at room temperature in 4 h. The integrated technology has the advantages of one-step extraction and cleanup, high extraction efficiency, low solvent consumption and room temperature.
Keywords: Magnetic molecularly imprinted polymers; Enhanced extraction; Process integration; Adsorption; Separation; Purification;

This study was on the kinetics and process parameters for ultrasound-assisted extraction (UAE) of water-soluble components and polysaccharides (PS) from the dry mycelium of a medicinal fungus, Cordyceps sinensis Cs-HK1. Four process variables (factors) were evaluated at different levels, ultrasound intensity (2.44–44.1 W/cm2), temperature (40–70 °C), solid particle size (156.5–750 μm), and solid-to-liquid ratio (1/30–1/70 g/mL). The experimental data of yields versus time in most cases were fitted closely to two empirical kinetic models for solid–liquid extraction, parabolic diffusion equation (y  =  y o  +  y 1 t 1/2) and power law (y  =  βt n ) with high correlation coefficients (R 2) of 0.95–0.99 for total extract yield, and 0.90–0.96 for PS yield. The PS yield was increased more significantly than the total extract yield with the ultrasound intensity. Reducing the particle size and increasing the extraction temperature led to a higher yield and extraction rate; increasing the solid-to-liquid ratio (or decreasing the liquid volume) increased the PS yield and extraction rate but had little influence on the total extract. Significant correlations were found between extraction rate (dy/dt) and ultrasound power density (P/V), and between extract yield (y) and energy density (Pt/V). The kinetic and process parameters are useful for rational design and efficient operation of UAE processes.
Keywords: Medicinal fungus; Polysaccharide; Ultrasonic extraction; Kinetic model;

The objective of this study was to enhance the solubility of Simvastatin by nanosizing approaches for improving its bioavailability. Nanosuspension (Sim-NS) formulated by media milling was optimized by 32 factorial design keeping the volume of milling media (X 1) and surfactant concentration (X 2) as independent and the particle size (Y) as dependent variable. A minimum particle size of 250.8 ± 12.6 nm was achieved. Simvastatin was also size reduced by supercritical antisolvent (SAS) method using CO2 as antisolvent, wherein particle size was reduced from 71.3 ± 0.87 μm to 16.54 ± 0.38 μm. Sim-NS showed significant increase in saturation solubility and decrease in crystallinity of the drug as compared to Simvastatin supercritical antisolvent product (Sim-SAS) and plain drug suspension. In vitro release showed increased dissolution rate of the drug for Sim-NS followed by Sim-SAS and plain drug. Pharmacodynamic evaluation in rats showed significant decrease in the total cholesterol and triglyceride levels for Sim-NS followed by Sim-SAS and plain drug. In vivo pharmacokinetics in rats showed an increase in bioavailability of Sim-NS (2.6 times) and Sim-SAS (1.8 times) compared to plain drug. Hence, particle size engineering has tremendous potential to enhance the bioavailability and hypolipidemic activity by altering physicochemical properties of the poorly water soluble drug.
Keywords: Optimization; Absorption; Biomedical; Nanosuspension; Supercritical antisolvent process; Kinetic parameters;

Effect of heating rate on pDNA production by E. coli by Karim E. Jaén; Alvaro R. Lara; Octavio T. Ramírez (230-238).
The effects of heating rate (HR) on the performance of two-phase (batch followed by fed-batch) high cell-density cultivations (HCDC) of E. coli DH5α for the production of plasmid DNA (pDNA) were investigated. Optimal temperatures for the HCDC, as selected from shake flask experiments at constant temperatures between 30 and 45 °C, were 35 °C for biomass accumulation in the batch phase and 42 °C for inducing pDNA replication during the fed-batch. In HCDC the temperature was increased at HR of 0.025, 0.05, 0.10 and 0.25 °C/min and the performance of the cultivations were compared to a HCDC run at constant temperature (35 °C). Compared to constant 35 °C, heat-induced HCDC accumulated up to 50% less biomass within the same cultivation time and acetate and glucose accumulated to high concentrations. The overall specific productivity (Q P ) and average pDNA yield (Y p/x ) in HCDC at 35 °C were 0.22 ± 0.02 mg/g h and 5.3 ± 0.00 mg/g, respectively. Such parameters were maximum at a HR of 0.05 °C/min, reaching 0.56 ± 0.06 mg/g h and 9.3 ± 0.6 mg/g, respectively. At HR above 0.5 °C/min, Y p/x remained relatively constant, whereas Q P tended to decrease. The supercoiled pDNA fraction remained around 80% at all HR. Bioreactors were equipped with a capacitance/conductivity probe. In all cases biomass concentration correlated closely with the capacitance signal and acetate and glucose accumulation was accompanied by an increase in the conductivity signal. Thus, it was possible to calculate acetate and biomass concentrations, as well as μ, from online capacitance and conductivity signals using estimators. Altogether, in this study it was shown that it is possible to maximize pDNA productivity by choosing an appropriate HR and that relevant parameters can be estimated by capacitance/conductivity signals, which are useful for better process control and development.
Keywords: pDNA vaccine; Temperature induction; Acetate; E. coli; High cell-density; Capacitance sensor;

Bisulfite as scavenger for enhanced biotechnological production of 3-hydroxypropionaldehyde by Lactobacillus reuteri by Marc J.A. Stevens; Sabine Vollenweider; Peter Mertes; Christophe Lacroix (239-245).
Lactobacillus reuteri converts glycerol into 3-hydroxypropionaldehyde (3-HPA), a promising preservative and chemical precursor. Production of 3-HPA by L. reuteri is limited due to the toxicity of 3-HPA to the producing cells. To circumvent this toxicity, the suitability of bisulfite as 3-HPA scavenger during production was assessed. Concentrations of 400 mM 3-HPA or bisulfite caused a viable cell number decrease from 1010 to 107 and 108  cells/ml, respectively, in 1 h. In contrast, the 3-HPA-bisulfite adduct was not toxic for the cells.Cells exhibiting high specific 3-HPA yield in a standardized glycerol conversion test were used to test bisulfite as scavenger during 3-HPA production. Addition of bisulfite (pH 5.8) after 2, 25, and 50 min resulted in conversion of 591 ± 15 mM glycerol within 2 h, similar to the rates under control conditions without bisulfite (501 ± 22 mM). Furthermore, cells from the production with bisulfite were still biological active after 3 consecutive production cycles, converting in total 974 ± 8 mM glycerol in 4 cycles, whereas cells from the control virtually lost their capability to utilize glycerol after two cycles and converted 695.5 ± 3.5 mM.Our results demonstrate the suitability of bisulfite to scavenge 3-HPA to obtain high yields during biotechnological production by the food grade microorganism L. reuteri.
Keywords: In situ product removal; Reuterin; Glycerol;

To maintain long-term lignin-degrading enzyme production under non-sterile conditions was a key to the technical application of white rot fungi in wastewater treatment. In this work, a novel open fungal reactor system with ozone as the bactericide, and using immobilized Phanerochaete chrysosporium, was built and operated continuously to produce the manganese peroxidase and decolorize the Acid Blue 45. The results showed that an average of 84% Acid Blue 45 decolorization, the manganese peroxidase production with its activity ranging from 63 U L−1 to 5 U L−1, was achieved during about 25 days system continuous operation. The contaminating bacteria in the reactor can be controlled at a level of 4.65 × 104  CFU ml−1 that did not adversely affect the fungal activity. The result of this study provides a new practical way for future design and operation of white-rot fungi reactor under non-sterile conditions.
Keywords: White-rot fungi; Bioreactors; Ozone; Immobilization; Enzyme production; Biodegradation;

Effect of thermochemical pretreatment on the solubilization and anaerobic biodegradability of the red macroalga Palmaria palmata by G. Jard; C. Dumas; J.P. Delgenes; H. Marfaing; B. Sialve; J.P. Steyer; H. Carrère (253-258).
The rapid development of anaerobic digestion brought with it the problem of biomass resources and supply. Marine biomass is emerging as an advantageous substrate. Such macroalgae as Palmaria palmata are promising substrates for anaerobic digestion as they possess a high methane potential (308 ± 9 mL gVS −1). The aim of this paper was to study the efficiency of the anaerobic digestion of P. palmata after a range of thermal and chemical pretreatment. The anaerobic digestion of raw and pretreated macroalgae was carried out in batch mesophilic biomethane potential tests (BMP). Thermal (between 20 and 200 °C) and thermo-chemical (addition of NaOH and HCl) pretreatment were performed on P. palmata. Thermal pretreatments at 20, 70, 85 and 120 °C and acid or soda pretreatments at 160 °C had no significant effect on P. palmata's methane potential. After high temperature pretreatment (180–200 °C), the BMP decreased with the temperature which can be explained by the formation of refractory compounds in the liquid fraction. In contrast, the addition of 0.04 gNaOH gTS−1 at 20 °C led to a release of proteins and induced an increase in the BMP from 308 ± 9 mL gVS −1 (untreated) to 365 ± 9 mL gVS −1. Thus, P. palmata can be used advantageously as a substrate for anaerobic digestion and its methane production enhanced by the addition of NaOH.
Keywords: Anaerobic digestion; Biogas; Biodegradation; Methane potential; Pretreatment; Seaweed;

Production of stable quinine nanodispersions using esterified γ-polyglutamic acid biopolymer by Christoph Hoennscheidt; Dirk Kreyenschulte; Argyrios Margaritis; Rainer Krull (259-266).
Novel methods are needed for the development of nanodispersed drug formulations to enhance bioavailability of many hydrophobic pharmaceuticals. The poorly water-soluble quinine is a well-known anti-malaria drug which can be used as a promising model compound for the development of novel nanodispersed formulations. In addition to hydrophobic drug's own affecting properties, surfactants play an important role for the enhancement of their low bioavailability by preparing stable dispersions. Amphiphilic compounds can efficiently be used to stabilize colloidal fragments by preventing the precipitation or crystallization of poorly water-soluble active ingredients during fabrication. A novel biopolymer derivative based on the biotechnologically produced γ-polyglutamic acid (γ-PGA) from Bacillus licheniformis cultivation was developed for encapsulation of the active ingredient. High-molecular γ-PGA is an anionic polyelectrolyte that was optimized and modified with hydrophobic l-phenylalanine ethyl ester (l-PAE) to form an amphiphilic comb polymer P(γ-GA-r-l-PAE) with surfactive properties. The approach of the nanodispersion polymer concentration, molecular weight and grafting degree enables the efficient stabilization of the poorly water-soluble model drug. The research presented in this report indicates the potential benefits of hydrophobically modified γ-PGA and suggests its potential role in forming stable dispersions for future pharmaceutical applications.
Keywords: γ-Polyglutamic acid; Biopolymer; Surfactant, Dispersion; Quinine; Solvent evaporation;

Nonporous and mesoporous silica-coated magnetite cluster nanocomposites particles were fabricated with various silica structures in order to develop a desired carrier for the lipase immobilization and subsequent biodiesel production. Lipase from Pseudomonas cepacia was covalently bound to the amino-functionalized particles using glutaraldehyde as a coupling agent. The hybrid systems that were obtained exhibited high stability and easy recovery regardless of the silica structure, following the application of an external magnetic field. The immobilized lipases were then used as the recoverable biocatalyst in a transesterification reaction to convert the soybean oil to biodiesel with methanol. Enzyme immobilization led to higher stabilities and conversion values as compared to what was obtained by the free enzyme. Furthermore, the silica structure had a significant effect on stability and catalytic performance of immobilized enzymes. In examining the reusability of the biocatalysts, the immobilized lipases still retained approximately 55% of their initial conversion capability following 5 times of reuse.
Keywords: Silica structure; Lipase; Immobilized enzymes; Enzyme activity; Biodiesel; Biocatalysis;

A hollow fiber membrane bioreactor using trioctylphosphine oxide (TOPO) impregnated in polypropylene hollow fiber membranes was developed for two-phase biodegradation of phenol using Pseudomonas putida ATCC 11172. Scanning electron microscopy revealed white deposits of TOPO impregnated non-uniformly within the cross sections and surfaces of the membranes. The extractant impregnated membranes exhibited high adsorption capacity and rates, whereas biodegradation of 800–2500 mg/L phenol at 200 mL volume in the extractant impregnated hollow fiber membrane bioreactor (EIHFMB) was characterized by high cell growth and biodegradation rates. For example, 1000 mg/L phenol was completely degraded within 12 h at a specific growth rate of 0.73 h−1 while the biomass yield and average biodegradation rate were 0.31 g/g and 86 mg/L h, respectively. The biodegradation capacity and rate in the EIHFMB were improved by increasing the effective length of the fibers by 50%, as demonstrated during the biodegradation of 3000 mg/L phenol. The adsorption/desorption rates were also enhanced with increasing aqueous phase flow rate. EIHFMB performance remained unchanged over 400 h of operation under various operating conditions suggesting the stability of TOPO impregnation within the membrane. These results indicate the use of EIHFMB as a promising technology in solvent-free two-phase biodegradation of phenolic compounds.
Keywords: Biodegradation; Liquid–liquid extraction; Membrane bioreactors; Phenol; Substrate inhibition; Two phase partitioning bioreactor;