Biochemical Engineering Journal (v.53, #3)

BEJ Keywords (IV).

In situ phenol removal from fed-batch fermentations of solvent tolerant Pseudomonas putida S12 by pertraction by Louise Heerema; Nick Wierckx; Mark Roelands; Jan Henk Hanemaaijer; Earl Goetheer; Dirk Verdoes; Jos Keurentjes (245-252).
▶ Biomass growth and phenol production are inhibited at low phenol concentrations. ▶ A membrane unit in the reactor does not affect the growth and phenol production. ▶ Phenol removal by pertraction leads to an increase in total phenol production of 132%. ▶ Decrease overall mass transfer coefficient by fouling medium components. ▶ Decrease overall mass transfer coefficient by sterilization membrane. In situ phenol pertraction with 1-octanol has been experimentally studied to improve the production of the model component phenol by a recombinant strain of Pseudomonas putida S12. When the phenol concentration in the reactor reaches 2 mM, the cells in fermentations without phenol removal are inhibited in growth and phenol production. Growth and phenol production stop after approximately 80 h at a phenol concentration in the reactor of 3.8 mM. When phenol is removed from the fermentation broth by pertraction, a lower maximum aqueous phenol concentration of 2.6 mM is achieved, while the total phenol production increases to 132%, as compared to the fermentation without pertraction. There are indications that the volumetric productivity (mmol L−1  h−1) increases slightly in the fermentations with in situ pertraction compared to the reference experiments. As expected, the amount of phenol produced per gram biomass (the specific productivity, mmol g−1  L−1) remains constant in time for all fermentations. The use of pertraction for in situ phenol removal is compared to in situ second phase extraction, in situ solvent impregnated resins and in-stream pertraction. Although the system shows promising results, further modifications such as using a solvent with a higher partition coefficient can improve the overall performance.
Keywords: In situ phenol removal; Product inhibition; Fermentation; Pseudomonas putida S12; Membrane extraction; Mass transfer;

Cytoplasmic production of soluble and functional single-chain Fv-Fc fusion protein in Escherichia coli by Hiroyuki Sonoda; Yoichi Kumada; Tomohisa Katsuda; Hideki Yamaji (253-259).
▶ Soluble and functional scFv-Fc is expressed in E. coli trxB/gor double mutant. ▶ Solubility of scFv-Fc in the cytoplasm is improved by co-expression of GroELS. ▶ Productivity of more than 10 mg/L is achieved in shake-flask culture.We describe the soluble production of a mouse anti-bovine ribonuclease A single-chain variable fragment (scFv) fused with the Fc region of human IgG1 in Escherichia coli. Two production systems, secretory production using a pelB signal peptide and cytoplasmic production in a trxB/gor double mutant strain with an oxidizing cytoplasm, were investigated for efficient production of soluble and functional scFv-Fc fusion protein. Antigen-binding activity was observed in both systems but almost all of the scFv-Fc that was expressed formed insoluble aggregates. Hence, the co-expression of molecular chaperones was examined. Co-expression of GroEL/GroES showed a 4.6-fold increase in antigen-binding activity in the cytoplasmic production system but not in the secretory system. By contrast, the other two chaperones, DnaK/DnaJ/GrpE and trigger factor, had no effect in either production system. The protein solubility was also improved markedly by the co-expression of GroEL/GroES and approximately 70% of the 3A21 scFv-Fc protein was soluble. A practical productivity of more than 10 mg/L was achieved with a simple batch shake-flask culture. These results indicate that the E. coli cytoplasmic production system with oxidizing cytoplasm and molecular chaperones might be one of the choices for the soluble production of scFv-Fcs and other Fc fusion proteins.
Keywords: Single-chain variable fragment; Fc fusion protein; Molecular chaperone; GroELS; Soluble production; Escherichia coli;

Rheology and fluid dynamics properties of sugarcane juice by Zailer Astolfi-Filho; Vânia Regina Nicoletti Telis; Eduardo Basilio de Oliveira; Jane Sélia dos Reis Coimbra; Javier Telis-Romero (260-265).
▶ Values of viscosities were obtained for sugarcane juices. ▶ Data of friction factors were determined during the sugarcane juices flow. ▶ Data could be used in industrial practice to evaluate bioethanol process conditions.The sugarcane juice is a relatively low-cost agricultural resource, abundant in South Asia, Central America and Brazil, with vast applications in producing ethanol biofuel. In that way, a good knowledge of the rheological properties of this raw material is of crucial importance when designing and optimizing unit operations involved in its processing. In this work, the rheological behavior of untreated (USCJ, 17.9 °Brix), clarified (CSCJ, 18.2 °Brix) and mixed (MSCJ, 18.0 °Brix) sugarcane juices was studied at the temperature range from 277 K to 373 K, using a cone-and-plate viscometer. These fluids were found to present a Newtonian behavior and their flow curves were well-fitted by the viscosity Newtonian model. Viscosity values lied within the range 5.0 × 10−3  Pa s to 0.04 × 10−3  Pa s in the considered temperature interval. The dependence of the viscosity on the temperature was also successfully modeled through an Arrhenius-type equation. In addition to the dynamic viscosity, experimental values of pressure loss in tube flow were used to calculate friction factors. The good agreement between predicted and measured values confirmed the reliability of the proposed equations for describing the flow behavior of the clarified and untreated sugarcane juices.
Keywords: Bioprocess; Ethanol; Friction factor; Rheological properties; Temperature; Sugarcane;

Volumetric oxygen transfer coefficient and oxygen uptake rate in a dense activated sludge slurry bubble column were measured by varying activated sludge concentrations (from 2000 to 8000 mg/L) and/or aeration rates (from 0.3 to 1.5 L min−1). They were separately determined by the dynamic methods. The endogenous oxygen uptake rate of the activated sludge was estimated by monitoring the dissolved oxygen concentration change after turning off the air sparging in a stirred tank. The volumetric oxygen transfer coefficient in a dense activated sludge slurry bubble column was determined using the dissolved oxygen concentration profiles and the oxygen uptake rates predetermined from the separate measurements. While the oxygen uptake rate almost linearly increased with increasing the activated sludge concentration, the volumetric oxygen transfer coefficient decreased with an increase in the activated sludge concentration. Their empirical correlations were obtained as functions of activated sludge concentration by fitting the experimental data.For the aerobic biodegradation of p-nitrophenol (PNP) by acclimated waste activated sludge, the dynamic models for the dissolved oxygen mass balance and PNP degradation kinetics with the proposed correlations for volumetric oxygen transfer coefficient and oxygen uptake rate could successfully simulate the concentration profiles of dissolved oxygen and PNP during the time course of an aerobic biodegradation of PNP.
Keywords: Volumetric oxygen transfer coefficient; Oxygen uptake rate; Aerobic biodegradation; Acclimated waste activated sludge; Slurry bubble column;

Initial rates technique as a procedure to predict the anaerobic digester operation by Andrés Donoso-Bravo; Guzmán García; Sara Pérez-Elvira; Fernando Fdz-Polanco (275-280).
▶ Full-scale anaerobic plants operate in continuous mode. ▶ Batch tests are usually used to calculate kinetic parameters. ▶ In this case parameters were estimated through initial rate test. ▶ A simplified mathematical model of the process was used. ▶ A validation of the procedure shows good results in the sewage sludge treatment.In this study a novel and practical procedure was developed, that involves: initial methane production rate measurement in batch tests, kinetic parameters determination and modeling application in a continuous digester. The procedure was evaluated with three experimental conditions: raw sludge as substrate incubated at 35 °C and 55 °C and thermal pretreated sludge incubated at 35 °C. The initial specific methane production rate was fitted with the Monod type equation in order to calculate the kinetic parameters. The values obtained for the maximum specific methane production rate were 0.043, 0.143 and 0.052 gCH4  gVSI −1  d−1 for each experimental condition, aforementioned. The substantial increment of this parameter at thermophilic condition shows the differences in the specific maximum growth rate between thermophilic and mesophilic populations. The affinity constant values were 3.842, 4.790 and 4.623 g L−1 for each experimental condition; however, a significant uncertainty was obtained due to some identification problems. A preliminary validation of the procedure was applied for predicting the operation of a continuous digester treating raw sewage sludge. The overall behavior of the system was represented by the model, although it slightly underestimates the experimental values, by approximately 20%. The results achieved, indicate that the procedure may be used as a tool in a real scale operation; however, further research must be performed.
Keywords: Anaerobic digestion; BMP; Continuous operation; Initial rate; Mathematical model;

▶ We investigated elicitation of S. coelicolor by live E. coli in a bioreactor. ▶ Elicitation reversed production patterns of antibiotics. ▶ Elicitation increased undecylprodigiosin and decreased actinorhodin production. ▶ Increase in maximum undecylprodigiosin concentration was 6-fold.Elicitation mimics the inter-species interactions in nature resulting in complex metabolic responses in interacting microorganisms. In pure culture systems in industry and laboratory, most of these metabolic pathways are not active. We therefore, investigated for the first time in a bioreactor, the effect of introducing a live culture of another species to the Streptomyces coelicolor cultures that produce antibiotics such as undecylprodigiosin and actinorhodin. Recently, undecylprodigiosin has also been attributed with antitumor activities. Pure cultures of S. coelicolor produced higher concentrations of actinorhodin compared with undecylprodigiosin in a defined medium in a 2 L bioreactor. Elicitation by live cells of Escherichia coli altered this production pattern such that undecylprodigiosin production was enhanced and actinorhodin repressed. E. coli elicitor with a concentration of 1 × 107  cells mL−1 was added at 2.5% (v/v) so that it did not overtake the growth of S. coelicolor. Since the timing of elicitation is also important, we tested E. coli addition at the beginning, on the second and the fourth day of the start of S. coelicolor cultivation. Day zero elicitation gave the maximum enhancement of undecylprodigiosin production which was 3.5 mg L−1 compared with 0.58 mg L−1 in the pure culture.
Keywords: Streptomyces coelicolor; E. coli; Bioreactor; Elicitation; Undecylprodigiosin; Actinorhodin;

▶ Analysis of the interaction between protein surface structure and support surface facilitates the successful adsorption-depended enzyme immobilization. ▶ Substrate adsorption on the immobilization support changes the apparent activity of immobilized enzyme. ▶ Esterase BioH successfully immobilized on pro-hydrophilic polystyrene.After analyzing protein surface structure, the opposite side of the activity site of esterase BioH was observed to be hydrophilic. Hence subsequently, a hydrophilic-modified solid support was selected to immobilize enzyme through oriented adsorption. The immobilized enzyme remained 86% of its original activity under the acylation of 1-phenylethanol, much more than those immobilized on other solid supports with low hydrophilicity (20–35.6%), as the mode of the immobilization could facilitate enzyme to expose its active site to the substrate. This demonstrated that the strategy to select solid support based on the analysis of protein surface structure was successful. It was also noticed that the substrate of the reaction was obviously adsorbed onto the support, therefore the adsorption effect could not be ignored and it was necessary to obtain the adsorption isotherm of the substrate for a more authentic result. The parameter K m shifted from 884.56 to 296.09 mM after immobilization. This was possibly mainly caused by the increment of substrate concentration around the enzyme due to the substrate adsorbing on the support. It could be another explanation for why those parameters were changed after enzyme immobilization in addition to some existing proposed reasons such as stabilizing conformation, facilitating dispersion of enzyme, etc.
Keywords: Oriented immobilization; Substrate adsorption; Structure analysis; Polystyrene resin;

▶ Elicitation and in situ adsorption can act synergistically. ▶ Established the addition time and addition concentration of [1-13C]-l-Phe. ▶ Hyper-production of 13C-labeled trans-resveratrol. ▶ The 13C labeling position of 13C-labeled trans-resveratrol was verified.Bioproduction of 13C-labeled trans-resveratrol in plant cell culture has attracted considerable attention with regard to potential applications for human benefit and to better understanding their absorption and in vivo metabolism in humans and animals. In the present work, two elicitors (SA and JA) and adsorbents (HP2MGL) were introduced into the Vitis vinifera cell suspension culture, and the results indicated that they could work synergistically to improve the production of trans-resveratrol (2666.7 mg L−1). Afterward, 1 mM [1-13C]-l-phenylalanine (Phe) was added to bioproduce 13C-labeled trans-resveratrol with the 13C enrichment from 35.7% at day 5 to 20.8% at day 10. Purification of the products by several chromatographic steps was reported, and the 13C labeling position was verified using 13C NMR. Our results indicated that, in this case, the rate limiting step of production was the post-biosynthetic events rather than the biosynthesis in elicited culture. Furthermore, our results suggested the importance of simultaneous optimization of biosynthetic pathways of metabolites and their post-biosynthetic steps toward achieving commercial plant cell culture. With the help of this technique, we could expect to do industrial scaling-up in a bioreactor to produce high amounts of the non-labeled and 13C-labeled trans-resveratrol in the near future.
Keywords: Vitis vinifera; 13C-labeled trans-resveratrol; Secondary metabolites; Plant cell tissue; Culture engineering; Metabolic control; Adsorption;

Jatropha seed protein functional properties for technical applications by Dianika Lestari; Wim J. Mulder; Johan P.M. Sanders (297-304).
▶ We investigated functional properties of Jatropha seed press cake protein. ▶ Oil removal from press cake decreased protein emulsifying properties. ▶ Oil removal from press cake improved protein foaming and adhesive properties. ▶Jatropha press cake protein film forming properties is still need to be improved. ▶Jatropha press cake protein has the most potential as emulsifier or paper adhesive. Jatropha press cake, by-product after oil expression from Jatropha seeds, contains 24–28% protein on dry basis. Objectives of this research were to investigate functional properties, such as solubility, emulsifying, foaming, film forming, and adhesive properties, of Jatropha press cake proteins and compared those with relevant industrial proteins. From our study, we found that protein extracted from press cake proteins had a solubility of about 90% above pH 9. Emulsifying properties of press cake protein were comparable to sodium caseinates and reached the highest value at pH 9–10. Jatropha proteins formed films with tensile strength of 0.4–1.8 MPa with 10–75% elongation, which were below soy protein or wheat-gluten. Further oil removal from press cake decreased emulsifying properties, while increased foaming and adhesive properties of the extracted proteins. Protein extracted from de-oiled press cake showed better foaming properties than sodium caseinate at pH 10, but lower than egg white protein at all pH. Furthermore, press cake protein showed better adhesive properties than casein adhesives at the same dry matter content. Based on these results, Jatropha press cake protein showed most promising results on adhesive and emulsifying properties, which indicate the potential of Jatropha press cake protein as emulsifier or paper adhesive.
Keywords: Jatropha seed protein; Protein extraction; Biorefinery; Protein functional properties; Solubility; Technical applications;

Bi-objective optimisation of the enzymatic hydrolysis of porcine blood protein by Raúl Pérez-Gálvez; M. Carmen Almécija; F. Javier Espejo; Emilia M. Guadix; Antonio Guadix (305-310).
▶ Porcine blood protein was hidrolysed with Alcalase. ▶ Response surface methodology was employed for empirical modelling. ▶ Bi-objective optimisation was performed on degree of hydrolysis and suspended solids.Protein from porcine blood meal was hydrolysed with Alcalase to obtain a final revalorised product suitable, for example, to take part in the composition of an organic fertiliser. Three experimental factors of the reaction (pH, temperature and enzyme–substrate ratio) were optimised by means of a statistically designed experiment and response surface methodology. The goal of the optimisation problem was to maximise both the degree of hydrolysis and solubilisation of the substrate, obtaining a maximum degree of hydrolysis (28.89%) with pH 6.24, 54.2 °C and enzyme–substrate ratio of 10%. Regarding the content of suspended solids, its minimum value (30.29% related to the initial weight of blood meal) was attained at pH 7.5, 59.8 °C and enzyme–substrate ratio of 10%. The controversial effects of pH and temperature on the substrate solubilisation and the final degree of hydrolysis, suggested employing a multiobjective optimisation technique. A Pareto Front was generated in order to find a set of intermediate solutions which satisfied both objectives in an adequate degree.
Keywords: Blood meal; Enzyme bioreactors; Modelling; Bi-objective optimisation; Proteolysis; Response surface methodology;