Biochemical Engineering Journal (v.83, #C)

IFC (CO2).

BEJ Keywords (II).

In this study, the abilities to produce enzymes by four Acremonium cellulolyticus strains were analyzed. Saccharification of potato pulp was performed to investigate the effects of the enzymes produced by A. cellulolyticus and to confirm the possibility of using A. cellulolyticus in the saccharification of potato pulp. Amylase, pectinase, galactosidase, and cellulase were produced by A. cellulolyticus from several carbon sources. Potato pulp was found to be a suitable substrate for A. cellulolyticus growth. The addition of cellulose not only improved the activity of cellulase but also improved the activity of α-galactosidase. Lactose and galactose induced the production of β-galactosidase and pectinase. Four strains of A. cellulolyticus were cultured in potato pulp to evaluate their abilities to produce cellulase, amylase, pectinase and galactosidase. Among them, A. cellulolyticus strain CF-2612 exhibited the highest production of all the enzymes. By using the crude enzymes from A. cellulolyticus strain CF-2612, 86% yield for glucose and 94% yield for galactose were achieved after 80 h of saccharification of potato pulp.
Keywords: Cellulase; Pectinase; Amylase; Galactosidase; Saccharification;

Highly efficient covalent immobilization of catalase on titanate nanotubes by Qinghong Ai; Dong Yang; Yuanbing Li; Jiafu Shi; Xiaoli Wang; Zhongyi Jiang (8-15).
In this study, titanate nanotubes (TNTs) with desirable biocompatibility and hydrophilicity have been synthesized by a facile and cost-effective alkaline hydrothermal method, and used to immobilize the enzyme. The characterization results reveal that the prepared TNTs have a regular tubular morphology with a length about 100–180 nm and an outer diameter about 10 nm, and a BET specific surface area of 305.4 m2  g−1. Catalase (CAT), as the model enzyme, was pre-modified by 3-(3,4-dihydroxyphenyl) propionic acid (3,4-diHPP) via 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) coupling chemistry, and then covalently immobilized on the TNTs surface by the chelation of catechol groups with Ti4+ ions. It is found that TNTs exhibits excellent performances as the immobilized supporter of enzyme: the enzyme loading is as high as 820 mg g of support−1; the relative activity of immobilized enzyme is about 60% of that of free enzyme; the immobilized CAT demonstrates enhanced storage and recycling stability.
Keywords: Enzymes; Immobilization; Titanate nanotubes; Chelation; Enzyme technology; Biocatalysis;

Bacillus coagulans JI12 was used to produce l-lactic acid from both cellulose and hemicellulose sugars of oil palm empty fruit bunch hydrolysate at 50 °C without sterilization prior to fermentation. In fermentation of mixed glucose and xylose (10 g/L:100 g/L or 50 g/L:53 g/L), both sugars were simultaneously converted to l-lactic acid. B. coagulans JI12 was tolerant against up to 4 g/L of furfural and 20 g/L of acetate and able to metabolize furfural to 2-furoic acid. After acid hydrolysis, both the hemicellulosic and cellulosic fractions of oil palm empty fruit bunch were fermented to lactic acid in a simultaneous detoxification, saccharification and co-fermentation process supplemented with 25 FPU Cellic® CTec2 cellulase per g cellulose, yielding 80.6 g/L of lactic acid with a productivity of 3.4 g/L/h. Neither pre-detoxification nor separation of fermentable sugars from lignin was required. These results indicate that B. coagulans JI12 is a promising strain for l-lactic acid production from lignocellulosic biomass.
Keywords: Simultaneous saccharification and co-fermentation; Detoxification; Bioprocess design; Cellulose; Lactic acid; Thermophiles;

Biosynthesis of CGTase by immobilized alkalophilic bacilli and crystallization of beta-cyclodextrin: Effective techniques to investigate cell immobilization and the production of cyclodextrins by Cristiane Moriwaki; Camila Sampaio Mangolim; Graziele Brescansin Ruiz; Gutierrez Rodrigues de Morais; Mauro Luciano Baesso; Graciette Matioli (22-32).
Cyclodextrin glycosyltransferase (CGTase) catalyses the degradation of starch, producing α-, β-, and γ-cyclodextrins (CDs). Immobilized cells offer several advantages, such as their prolonged and repeated use, ease separation from the fermentation medium, and reduced risk of contamination. The biosynthesis of CGTase and β-CD was optimized by immobilization of Bacillus firmus strain 37 and Bacillus sphaericus strain 41 cells on a loofa sponge. Both microorganisms produced significant levels of CGTase for three consecutive cycles (94.2 U/mL on average), increased the relative formation of the β-CD, and inhibited the formation of other CDs. The crystallization of the CD produced by both bacterial strains, when corn starch was used as the substrate, resulted in a 64% recovery of β-CD. The purity of the β-CD was 89.5% when maltodextrin was used as the substrate for B. firmus strain 37. The Tilden–Hudson technique was used as a simple, inexpensive, and efficient method for monitoring the continuous production of CDs using immobilized cells. The innovative use of the Fourier Transform Infrared Spectroscopy technique by means of the Attenuated Total Reflectance method suggested that the interaction between the B. sphaericus strain 41 cells and the loofa sponge occurred by natural adsorption.
Keywords: Cyclodextrins; CGTase; Biosynthesis; Enzyme production; Immobilized cells; Purification;

This study presents the ultrasound assisted ammonia pretreatment (UAAP) of sugarcane bagasse (SCB) and the influence of SCB particle size, liquid ammonia concentration, sonication time, temperature and liquid to solid ratio (LSR) on cellulose recovery and delignification. The maximum cellulose recovery and delignification observed at the optimum conditions (particle size 0.274 mm, sonication time 45 min, ammonia concentration 10%, LSR 10 mL/g and temperature 80 °C) were 95.78 and 58.14%, respectively. The dilute acid hydrolysis of pretreated SCB produced 16.58 g/L glucose, 8.21 g/L xylose, 2.78 g/L arabinose, 0.81 g/L furfural and 1.79 g/L acetic acid. The hydrolysate contained less inhibitors compared to the values reported in the literature during fermentable sugar production.
Keywords: Ethanol; Cellulose; Lignin; Glucose; Ultrasound; Ammonia pretreatment;

The reconstruction of the dynamically changing rates of O2 uptake and CO2 production during short term bioreactor perturbation experiments, from dissolved oxygen and offgas O2 and CO2 measurements, requires a model describing the mass transfer and dispersion of O2 and CO2 in the system as well as the sensor dynamics. Additionally, if perturbation experiments are carried out at near-neutral pH levels, also the inter conversion of dissolved carbon dioxide and bicarbonate needs to be taken into account. While developing such a model we found that for an accurate description of the systems dynamic response on a time-scale of seconds, it is required to incorporate not only the mass transfer between gas bubbles and broth, but also between headspace and broth. The delay and dispersion in the measurements of the gas analyser, due to the length and complexity of the offgas system, was accounted for by determining the impulse response of the offgas system. This model could be fitted excellently to an identification data set. Finally the model was successfully applied to reconstruct the dynamic rates of O2 uptake and CO2 production of a culture of Penicillium chrysogenum, which was perturbed by a glucose pulse.
Keywords: Oxygen uptake rate; Carbon dioxide evolution rate; Bicarbonate; Microbial fermentation; Dynamic conditions; Modelling; Impulse response; Parameter estimation; Glucose pulse;

Characterization of gas stripping and its integration with acetone–butanol–ethanol fermentation for high-efficient butanol production and recovery by Chuang Xue; Guang-Qing Du; Jian-Xin Sun; Li-Jie Chen; Shuai-Shi Gao; Ming-Liang Yu; Shang-Tian Yang; Feng-Wu Bai (55-61).
Two-stage gas stripping coupled with acetone–butanol–ethanol fermentation in a fibrous bed bioreactor was established for energy-efficient butanol recovery. The impacts of process parameters including butanol concentration, temperature and cell density with feed, gas flow rate, and cooling temperature on the efficiency of the gas stripping system were studied. High butanol concentration, low cell density and cooling temperature increased butanol titer in the condensate. The butanol titer in the condensate increased when stripping temperature increased from 25 to 55 °C, and decreased when temperature was above 55 °C. The optimal gas flow rate was 1.6 L/min, above which more water was stripped off and the condensate was diluted. After process optimization, 48.5 g/L butanol (73.3 g/L ABE) was produced in the fed-batch fermentation with in situ gas stripping due to the reduced butanol inhibition on cells. The condensate containing 147.2 g/L butanol (199.0 g/L ABE) was produced by the first-stage gas stripping, while a highly concentrated condensate containing 515.3 g/L butanol (671.1 g/L ABE) was obtained from the second-stage gas stripping. This process can significantly reduce energy consumption in the final product recovery.
Keywords: Fermentation; Fed-batch culture; Separation; Bioprocess design; Gas stripping; Butanol production;

Fed-batch production of l-phenylalanine from glycerol and ammonia with recombinant Escherichia coli by Michael Weiner; Christoph Albermann; Katrin Gottlieb; Georg A. Sprenger; Dirk Weuster-Botz (62-69).
Glycerol was used as carbon source for l-phenylalanine production with recombinant Escherichia coli. In contrast to glucose, no consumption of the precursor phosphoenolpyruvate (PEP) is necessary for glycerol uptake. Additional lactic acid feeding was necessary for growth because the genes encoding the PEP consuming pyruvate kinase isoenzymes have been deleted. Thus a fed-batch process was developed with feeding of lactic acid and glycerol for biomass formation followed by feeding of glycerol and ammonia for l-phenylalanine production. Unfortunately, plasmid instability was observed in the first process. Plasmid stability could be successfully assured by replacing an ampicillin resistance gene by a kanamycin resistance gene cassette. The resulting maximum l-phenylalanine concentration of 13.4 g L−1 was improved by 26% and biomass specific productivity ( 22  mg L-phe g CDW − 1  h − 1 ) was raised by 69%. The final l-phenylalanine concentration of 13.4 g L−1 was thus improved by a factor of 2.4 compared to earlier reports.
Keywords: l-Phenylalanine; Glycerol; Escherichia coli; Fed-batch; Plasmid stability;

A microrespirometric method for the determination of stoichiometric and kinetic parameters of heterotrophic and autotrophic cultures by Ivonne Esquivel-Rios; Rocio Ramirez-Vargas; Gabriel R. Hernandez-Martinez; Miguel Vital-Jacome; Alberto Ordaz; Frederic Thalasso (70-78).
Respirometry, which consists in the measurement of the oxygen uptake rate, is a method commonly used to determine kinetic and stoichiometric parameters of biological processes. In the present work, a novel microrespironetry method, which involves standard respirometry procedures but in a microreactor array, was developed and assessed, based on a standard commercial apparatus. First, the volumetric oxygen transfer coefficient (K L a), which is important for the interpretation of respirometric data, was determined under an exhaustive set of experimental conditions. K L a ranged from 2.2 to 48.0 h−1, which is similar to K L a typically found in standard respirometers. Then, the method was assessed through the determination of the maximum specific growth rate, the half-saturation constant and the growth yield of axenic and mixed cultures of autotrophic and heterotrophic bacteria. The selected cultures were Pseudomonas putida F1, Nitrobacter winogradskyi, an activated sludge and a nitrifying consortium. Additionally to reporting parameters for these cultures, this report shows that the microrespirometry method allowed for the determination of stoichiometric and kinetic parameters. Simultaneous tests done with a single culture sample, made possible by the microreactor array, allowed parameters determination with a lower standard error than observed with traditional respirometry and with much less experimental effort.
Keywords: Microtiter plate; Microwell plate; Bioreactors; Mass transfer; Kinetic parameters; Dissolved oxygen;

Enzyme-immobilization in membranes accomplished by fostering membrane fouling was evaluated. Four different membrane configurations and five membranes were compared for immobilization of alcohol dehydrogenase (ADH) in terms of enzyme loading, permeate flux and final biocatalytic conversion. The membrane configuration impacted the efficiency of the enzyme-immobilization as well as the biocatalytic-membrane reaction, and the “sandwich mode”, with an extra polypropylene support above the membrane skin layer, worked best due to its high flux and stable conversion. Among the membranes, a GR51PP polysulphone membrane allowed for the highest flux during the reaction with the enzyme-immobilized membrane. At the same time, the lowest enzyme loading and low reaction stability were achieved for this membrane. Satisfactory enzyme loadings, stable conversions, but low flux rates were obtained for the PLTK and PLGC regenerated cellulose membranes. With these two highly hydrophilic membranes, the ADH enzyme activity was fully retained even after 24 h of storage of the membrane. Filtration blocking and resistance models were used to analyze the fouling/immobilization mechanisms and give explanations for the different results. The work confirms that fouling-induced enzyme immobilization is a promising option for enhancing biocatalytic productivity, and highlights the significance of the membrane type and configuration for optimal performance.
Keywords: Membrane fouling; Enzyme immobilization; Alcohol dehydrogenase; Filtration; Catalysis;

A milliliter-scale yeast-based fuel cell with high performance by Hiroyuki Kaneshiro; Kosuke Takano; Yogo Takada; Tomoyuki Wakisaka; Taro Tachibana; Masayuki Azuma (90-96).
Microbial fuel cells are attracting attention as one of the systems for producing electrical energy from organic compounds. We used commercial baker's yeast (Saccharomyces cerevisiae) for a glucose fuel cell because the yeast is a safe organism and relatively high power can be generated in the system. In the present study, a milliliter (mL)-scale dual-chamber fuel cell was constructed for evaluating the power generated by a variety of yeasts and their mutants, and the optimum conditions for high performance were investigated. When carbon fiber bundles were used as an electrode in the fuel cell, high volumetric power density was obtained. The maximum power produced per volume of anode solution was 850 W/m3 under optimum conditions. Furthermore, the power was examined using seven kinds of yeast. In Kluyveromyces marxianus, not only the power but also the power per consumed glucose was high. Moreover, it was suggested that xylose is available as fuel for the fuel cell. The fuel cell powered by K. marxianus may prove to be helpful for the effective utilization of woody biomass.
Keywords: Glucose; Xylose; Microbial fuel cell; Yeast; Saccharomyces cerevisiae; Kluyveromyces marxianus;

A novel biocatalytic route for (S)-3-cyano-5-methylhexanoic acid, the key chiral intermediate of pregabalin, was successfully developed using whole cells of newly isolated Arthrobacter sp. ZJB-09277. Kinetic resolution of a series of rac-3-cyano-5-methylhexanoic acid esters bearing a β-stereocenter indicated that steric effect of the leaving alcohol moiety played an important role in determining activity and enantioselectivity of Arthrobacter sp. ZJB-09277 esterase. Enantiomeric ratio of the esterase toward rac-3-cyano-5-methylhexanoic acid ethyl ester was significantly increased from 33 to 80 by addition of 50% (v/v) DMSO and key reaction parameters optimization. The whole-cell catalysts exhibited strong tolerance against high DMSO concentration up to 80% (v/v). Enzymatic resolution of the substrate at a concentration of 100 mM gave (S)-3-cyano-5-methylhexanoic acid in 44.6 mM and 95.1% ee, which greatly increased the feasibility of this bioprocess to become an industrial approach.
Keywords: Biocatalysis; Chiral systems; Enantioseparation; (S)-3-Cyano-5-methyl hexanoic acid; High DMSO concentration tolerance; Optimization;

Parameter estimation in kinetic models for large scale biotechnological systems with advanced mathematical programming techniques by Jimena Di Maggio; Cecilia Paulo; Vanina Estrada; Nora Perotti; Juan C. Diaz Ricci; M. Soledad Diaz (104-115).
In the present work, we formulate parameter estimation problems for kinetic models of large-scale dynamic biotechnological systems. We propose dynamic models of increasing complexity for metabolic networks and continuous bioreactors. The differential algebraic equations (DAE) system for the metabolic network represent the glycolysis, the phosphotransferase system and the pentose-phosphate pathway of Escherichia coli, with modifications proposed for several enzyme kinetics. The most sensitive parameters have been ranked by performing global sensitivity analysis on the dynamic metabolic network. Since the kinetic parameters for the enzymes have been obtained from in vitro experiments, the formulation of a detailed kinetic model for the metabolic network allows parameter adjustment for in vivo conditions. We formulate an unstructured non-segregated model for a chemostat to study the dynamic response to a glucose pulse in a continuous culture of E. coli. Moreover, we perform parameter estimation by formulating a maximum likelihood problem, subject to the DAE systems, within a control vector parameterization approach. Nine kinetic parameters in the metabolic network model have been estimated with good agreement with published experimental data. For the bioreactor model, seven parameters have been tuned based on experimental data obtained in this work. Numerical results show a good agreement between the observed data and the predicted profiles.
Keywords: Dynamic metabolic network; Dynamic optimization; Control vector parameterization;

Efficient preparation of enantiopure l-tert-leucine through immobilized penicillin G acylase catalyzed kinetic resolution in aqueous medium by Weiming Liu; Jixing Luo; Xiaojian Zhuang; Wenhe Shen; Yang Zhang; Shuang Li; Yi Hu; He Huang (116-120).
Racemic DL-tert-leucine (DL-Tle) was resolved to obtain enantiopure L-Tle through enantioselective hydrolysis of its N-phenylacetyl derivative with immobilized penicillin G acylase (PGA). The effects of pH, reaction temperature, substrate concentration and reaction time on the reaction were investigated. The reaction was conveniently carried out at 0.4 M substrate concentration in water at pH 8.0 and 30 °C. Under the optimized reaction conditions, L-Tle was obtained in an enantiopure form (>99% ee) with 45.8% substrate conversion after 4 h. The thermal stability and operational stability of immobilized PGA were examined. Furthermore, the preparation of L-Tle was successfully performed in a recirculating packed bed reactor (RPBR) system and immobilized PGA exhibited a long-term stability for 51 days with a slight decrease of activity. The isolated D-enantiomer was racemized at 160 °C for 15 min and reused as substrate. The results obtained clearly demonstrated a potential for industrial application of immobilized PGA in the preparation of L-Tle through enantioselective hydrolysis of its N-phenylacetyl derivative.
Keywords: L-tert-leucine; Immobilized penicillin G acylase; Biocatalysis; Bioprocess design; Optimization; Packed bed bioreactors;

Characterization of a thermophilic l-arabinose isomerase from Thermoanaerobacterium saccharolyticum NTOU1 by Xing-Guang Hung; Wen-Chi Tseng; Shiu-Mei Liu; Wen-Shyong Tzou; Tsuei-Yun Fang (121-128).
l-Arabinose isomerase (EC 5.3.1.4, l-AI) mainly catalyzes the reversible aldose–ketose isomerization between l-arabinose and l-ribulose. l-AIs can also catalyze other reactions, such as the conversion of d-galactose to d-tagatose. In this study, the araA gene encoding l-AI was PCR-cloned from Thermoanaerobacterium saccharolyticum NTOU1 and then expressed in Escherichia coli. The recombinant l-AI was purified from the cell-free extract using nickel nitrilotriacetic acid metal-affinity chromatography. The purified enzyme showed an optimal activity at 70 °C and pH 7–7.5. The enzyme was stable at pHs ranging from 6.5 to 9.5 and the activity was fully retained after 2 h incubation at 55–65 °C. The low concentrations of divalent metal ions, either 0.1 mM Mn2+ or 0.05 mM Co2+, could improve both catalytic activity and thermostability at higher temperatures. The recombinant T. saccharolyticum NTOU1 l-AI has the lowest demand for metal ions among all characterized thermophilic l-AIs. This thermophilic l-AI shows a potential to be used in industry to produce d-tagatose from d-galactose.
Keywords: Arabinose isomerase; Bioconversion; Recombinant DNA; Enzyme activity; Thermoanaerobacterium saccharolyticum; Thermophiles;