Biochemical Engineering Journal (v.55, #2)

BEJ Keywords (II).

Influence of gellan gum concentration on the dynamic viscoelasticity and transient flow of fluid gels by M. Carmen García; M. Carmen Alfaro; Nuria Calero; José Muñoz (73-81).
Display Omitted► We prepare low-acyl gellan gum fluid gels by a low-energy mechanical treatment. ► CSLM shows fluid gels are dispersions of gel-like domains in an aqueous phase. ► The influence of gellan concentration on mechanical spectra of fluid gels is studied. ► Start-up tests avoiding pre-shear were used to model thixotropic viscosity decay. ► Flow interrupted tests illustrated the concentration dependence on viscosity recovery.Start-up at the inception of shear and flow interrupted experiments were carried out to illustrate the transient flow properties and thixotropic recovery of low-acyl gellan gum fluid gels prepared by a low-energy mechanical treatment. Small amplitude oscillatory shear tests were conducted to illustrate the rheological response under non-destructive conditions. Confocal laser scanning microscopy revealed the occurrence of a dispersion of gel-like domains in a continuous phase. Rheological methods were used to check that the samples characterised were free of thermo-mechanical history effects prior starting the transient flow tests. Start-up shear flow experiments at 10 s−1 resulted in a nearly instantaneous sharp rise of viscosity until a peak value was reached before it dropped following a sum of two first-order kinetic equations. The kinetic coefficients as well as the relative fall in viscosity were found to increase with gellan concentration. The equilibrium modulus as well as the peak and steady-state viscosities scaled with the gellan concentration following a power law equation with a characteristic exponent of about 2.0. Conversely, the degree of thixotropic recovery after a rest time of 10 s went down as gellan concentration increased, emphasising the need to balance both effects when formulating gellan fluid gels as stabilisers. The results obtained do a better job of highlighting the importance of peak viscosity than steady-state viscosity when data for pump selection and calculation are required.
Keywords: Rheology; Gellan gum; Fluid gels; Thixotropy; Biopolymers;

► An unstructured model is applied to predictive control of recombinant rhamnulose-1-phosphate aldolase production in E. coli fed-batch cultures. ► The model incorporates the effect of pulse induction on both growth rate and protein production at different induction loads. ► The model predicts glucose accumulation that is an indicator of activity reduction.A semi-empirical mathematical model for recombinant rhamnulose 1-phosphate aldolase (RhuA) production in high cell density cultures of Escherichia coli has been developed. An unstructured and non-segregated approach allowed to identify the inducer–biomass ratio (I/X) as the key parameter influencing initial specific production rate and specific growth rate reduction after induction. Biomass, substrate and RhuA concentration profiles have been properly fitted in fed-batch cultures induced by an IPTG pulse at different I/X ratio ranging from 0 to 3 μmol IPTG·g-1dry cell weight. The model has been validated firstly as able to predict recombinant aldolase and biomass production. In addition, since glucose accumulation in the induced period, which is an indicator of activity reduction by proteolysis, can be anticipated, the model has been used for control purposes with excellent results.
Keywords: Recombinant aldolase; Fed-batch; Escherichia coli; Mathematical modeling; Model-based control; Inducer–biomass ratio;

Strain improvement and statistical media optimization for enhanced erythritol production with minimal by-products from Candida magnoliae mutant R23 by Laxman S. Savergave; Ramchandra V. Gadre; Bhalchandra K. Vaidya; Karthik Narayanan (92-100).
► Strain improvement of Candida magnoliae by random mutagenesis resulted in a mutant with improved erythritol production. ► Medium optimization for C. magnoliae mutant R23 by RSM. ► Glucose and yeast extract concentrations decide the production of erythritol as well as other by-products such as mannitol and glycerol. ► RSM was successfully used for minimizing by-products formation. ► Fermentative production of erythritol using RSM optimized media was studied in batch and fed-batch mode in 10 L laboratory fermentor.Mutants of Candida magnoliae NCIM 3470 were generated by ultra-violet and chemical mutagenesis to enhance erythritol production. The mutants were screened for higher reductase activity on agar plates containing high concentration of glucose and 2,3,5-triphenyl tetrazolium chloride (TTC). One of the mutants named as R23 gave maximum erythritol production, 60.3 g L−1, compared to 14 g L−1 of the parent strain. Glucose and yeast extract were identified as critical medium components which decide the ratio of polyols produced, mainly erythritol, mannitol and glycerol. In order to enhance the production of erythritol and to minimize the production of mannitol and glycerol, a four component-five level-three response central-composite-rotatable-design (CCRD) of response surface methodology (RSM) model was used. The optimum medium composition for erythritol production was found to contain (g L−1) glucose 238, yeast extract 9.2, KH2PO4, 5.16 and MgSO4 0.23. Moreover, erythritol production was studied in a 10 L fermentor in batch and fed-batch mode using RSM optimized medium. In fed-batch fermentation, 87.8 g L−1 erythritol was produced with 31.1% yield, without formation of any other polyols. Thus present study involving strain improvement followed by media and process optimization resulted in 6.2-fold increase in erythritol production and 3.4-fold increase in the yield.
Keywords: Candida magnoliae; Erythritol; Fermentation; Metabolite over-production; Modelling; Optimization;

Partial nitrification in a continuous pre-denitrification submerged membrane bioreactor and its nitrifying bacterial activity and community dynamics by Tadashi Nittami; Hiroaki Ootake; Yuko Imai; Yuya Hosokai; Ayako Takada; Kanji Matsumoto (101-107).
Display Omitted► Nitrogen removal via NO2 was achieved in a continuous pre-denitrification SMBR. ► The aeration saving was 29% compared to that required for N removal via NO3. ► N removal via NO2 can be achieved by inhibiting Nitrospira spp.In this study, nitrogen removal by partial nitrification via nitrite was completely achieved for >40 days in a continuous submerged membrane bioreactor configured with both a pre-denitrification (10 L) and a nitrification tank (20 L). The possible aeration saving achieved with this configuration was 29% compared to that required for nitrogen removal via nitrate. Partial nitrification in the submerged membrane bioreactor was achieved by controlling the influent ammonium concentration (161 mg N/L) and the dissolved oxygen concentration in the nitrification tank (0.6 mg/L). Measurement of oxygen uptake rates during batch experiments confirmed that increasing influent ammonium concentration (161 mg N/L) decreased nitrite oxidizing activity of the biomass to almost zero. Furthermore, real-time PCR assays revealed that increasing influent ammonium concentration led to a decrease not of Nitrobacter but Nitrospira in the submerged membrane bioreactor. These results suggest that partial nitrification can be achieved by inhibiting the activity and growth of Nitrospira spp. with high ammonium concentration.
Keywords: Activated sludge; Membrane bioreactor (MBR); Nitrifying bacteria; Nitrogen removal; Oxygen uptake rate (OUR); Wastewater treatment;

Improving the prediction of Pseudomonas putida mt-2 growth kinetics with the use of a gene expression regulation model of the TOL plasmid by Michalis Koutinas; Alexandros Kiparissides; Ming-Chi Lam; Rafael Silva-Rocha; Miguel Godinho; Victor de Lorenzo; Vitor A.P. Martins dos Santos; Efstratios N. Pistikopoulos; Athanasios Mantalaris (108-118).
► Combined genetic circuit–growth kinetic model of TOL plasmid of Pseudomonas putida. ► Model validation and predictive capability by independent experiments. ► Critical importance of gene expression in predicting unusual growth patterns.The molecular and genetic events responsible for the growth kinetics of a microorganism can be extensively influenced by the presence of mixtures of substrates leading to unusual growth patterns, which cannot be accurately predicted by mathematical models developed using analogies to enzyme kinetics. Towards this end, we have combined a dynamic mathematical model of the Ps/Pr promoters of the TOL (pWW0) plasmid of Pseudomonas putida mt-2, involved in the metabolism of m-xylene, with the growth kinetics of the microorganism to predict the biodegradation of m-xylene and succinate in batch cultures. The substrate interactions observed in mixed-substrate experiments could not be accurately described by models without directly specifying the type of interaction even when accounting for enzymatic interactions. The structure of the genetic circuit–growth kinetic model was validated with batch cultures of mt-2 fed with m-xylene and succinate and its predictive capability was confirmed by successfully predicting independent sets of experimental data. Our combined genetic circuit–growth kinetic modelling approach exemplifies the critical importance of the molecular interactions of key genetic circuits in predicting unusual growth patterns. Such strategy is more suitable in describing bioprocess performance, which current models fail to predict.
Keywords: Dynamic modelling; pWW0 (TOL) plasmid; Genetic circuit; Pseudomonas putida; m-Xylene;

Enhanced enzymatic transesterification of palm oil to biodiesel by Md. Mahabubur Rahman Talukder; Probir Das; Tan Shu Fang; Jin Chuan Wu (119-122).
► Comparison of methanol with methyl acetate (MA) as acyl acceptor for Novozym 435-catalyzed transesterification of palm oil to biodiesel. ► Methanol is more reactive than MA but deactivates Novozym 435. ► The presence of MA minimizes methanol deactivation of Novozym 435. ► Combined use of methanol and MA enhances the reaction 3-fold and reduces the enzyme load.Inefficient reaction is one of the drawbacks for the enzymatic production of biodiesel (BD). A method for enhanced Novozym 435-catalyzed transesterification of palm oil (PO) to BD using a mixture of methanol and methyl acetate (MA) as acyl acceptors has been developed. BD yield using methanol–MA mixture reached 95–96% after 8 h at a methanol to PO molar ratio of 1:1, a MA to palm oil molar ratio of 12:1, 2 g of PO, temperature of 50 °C and Novozym 435 loading of 0.6 g. In contrast with methanol–MA mixture, BD yield using MA alone after 24 h was 88–89% under the same reaction conditions. Novozym 435 was repeatedly used at 40 and 50 °C for 10 cycles. There was no considerable loss in the enzyme activity at 40 °C using either methanol–MA mixture or MA alone, while the remaining activity of the enzyme at 50 °C in both cases after 10 cycles was 82–85%. The developed method has a potential to be used for efficient enzymatic production of BD.
Keywords: Biodiesel; Novozym 435; Methanol–methyl acetate mixture; Palm oil; Transesterification;

Determination of apparent kinetic and stoichiometric parameters in a nitrifying fixed-bed reactor by in situ pulse respirometry by Alberto Ordaz; Catarina S. Oliveira; Joel Alba; Manuel Carrión; Frédéric Thalasso (123-130).
► The nitrification process was studied in a fixed-bed reactor. ► A combination of respirometry and tracer technique was used to characterize the process. ► Apparent kinetic and stoichiometric parameters were determined taking into consideration: mixing time in the reactor, nutrient and carbon limitation. ► We concluded that respirometry might be applied for the characterization of fixed-bed reactors.The determination of stoichiometric and kinetic parameters of biological processes occurring in fixed-bed reactors is usually achieved through mass balance methods or by the characterization of samples taken from the core of the reactor. Mass balance is time demanding and to obtain representative samples from the core of filter media is often difficult. A fast and non-invasive alternative method might be beneficial. In this paper we explored the potential for using in situ pulse respirometry in a submerged fixed-bed reactor for assessing biological processes. A nitrifying fixed-bed reactor was operated over 85 days while analyzing the dissolved oxygen response to pulses of ammonium. In situ pulse respirometry allowed us to determine the apparent maximum oxygen uptake rate and substrate affinity constant. Additionally, the apparent biomass growth yield was determined during the first 60 days. Unfortunately, after day 60, erratic biomass growth yield results were obtained, which we were unable to explain through hydrodynamic characterization of the reactor. However, it is concluded that in situ pulse respirometry is a potentially useful tool to characterize fixed-bed reactors as an alternative to other methods, such as mass balance.
Keywords: Nitrification; Respirometry; Biofilm; Kinetic parameters; Affinity constant; Hydrodynamic;

Characterization of the response of GFP microbial biosensors sensitive to substrate limitation in scale-down bioreactors by Frank Delvigne; Alison brognaux; Nathalie Gorret; Peter Neubauer; Angélique Delafosse; Marie-Laure Collignon; Dominique Toye; Michel Crine; Mathieu Boxus; Philippe Thonart (131-139).
► GFP microbial biosensors have been applied to the detection of bioreactor mixing imperfections. ► Substrate limitation can be easily detected at the single cell level. ► GFP leakage can be correlated to bioreactor mixing efficiency.The dynamics of microbial stress response in intensive cultivation conditions remains not completely understood. In this work, two green fluorescent protein (GFP) transcriptional reporters have been used as biosensors of the heterogeneities generated in a two-compartment scale-down reactor. The stress promoters have been chosen for their responsiveness to carbon limitation corresponding to the global substrate profiles encountered in intensive fed-batch cultures. From our results, it can be concluded that the exposure of microbial cells to substrates heterogeneities tends to decrease the GFP expression level in fed-batch mode. Fluorescence intensities have been monitored at the single cell level by using flow cytometry. During the course of the fed-batch culture, a drop at the level of the intracellular GFP content has been observed for the two scale-down operating conditions and for the two promoters sensitive to substrate limitation (rpoS and csiE). The fluorescence drop can be attributed to the repression of these promoters but also to the release of GFP to the extracellular medium according to the increase of the fluorescence level of the supernatant. This leakage has been observed for all the operating conditions, i.e. the scale-down reactors and the culture operating in the normal mode, i.e. in a well-mixed bioreactor. Interestingly, GFP leakage is more pronounced in the case of the cultures operated in the normal mode. Indeed, staining by propidium iodide tends to be more elevated for the microbial cells cultured under the normal mode by comparison with those cultured in scale-down conditions, indicating a higher permeability of the membrane. These results suggest that GFP microbial biosensors could be used to detect simultaneously mixing imperfections and their impact on the viability of microorganisms.
Keywords: Microbial stress; Bioreactor mixing; Protein leakage;

Preparation of protein nanoparticles using AOT reverse micelles by K. Naoe; S. Yoshimoto; N. Naito; M. Kawagoe; M. Imai (140-143).
► Protein nanoparticles were prepared via cross-linking reaction using sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse micelle system as a nanoreactor. ► Water-soluble ribonuclease A nanoparticles were successfully prepared in the size range of 6–100 nm by controlling with water content. Solvent species formulating the reverse micellar organic phase also can control the nanoparticle size. ► Cytochrome c nanoparticles prepared in this system were slightly hydrophobic and stably dispersed in ethanol, not in aqueous solutions.Biopolymeric nanoparticles made of protein via cross-linking reaction was prepared using sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse micelle system as a nanoreactor. The prepared ribonuclease A nanoparticles were stably dispersed in aqueous solutions and the size of the nanoparticles was controlled in the range of 6–100 nm with water content in the system. Solvent species formulating the reverse micellar organic phase also can control the nanoparticle size. These effects on the nanoparticle size would be attributed to the dynamic behavior of reverse micelles. In contrast, cytochrome c nanoparticles were stably dispersed in ethanol while completely precipitated in both aqueous solutions and non-polar organic solvents. These findings indicate that a specific interaction of protein with AOT, which depends on the solubilization site of each protein in reverse micelles, is a significant factor to affect the surface properties of protein nanoparticles.
Keywords: Reverse micelle; Protein; Nanoparticle;