Biochemical Engineering Journal (v.90, #C)

BEJ Keywords (II).

IFC (IFC).

Biodegradation of naphthalene using a functional biomaterial based on immobilized Bacillus fusiformis (BFN) by Chen Lin; Li Gan; Zuliang Chen; Mallavarapu Megharaj; Ravendra Naidu (1-7).
The degradation of naphthalene using immobilized Bacillus fusiformis (BFN) strain on alginate–polyvinyl alcohol (PVA)–clays bead was much higher than that of using a freely suspended BFN strain. This was due to the beads facilitating biodegradation by enhancing concentration of naphthalene using the beads as an adsorbent in the cells’ vicinity. This was confirmed by the kinetics of naphthalene on the beads, where its adsorption onto the beads was confirmed by pseudo-second-order kinetics. The biodegradation of naphthalene fitted well to the first-order rate model. More than 99.7% of naphthalene which was removed within 12 h, contained bentonite 2% (w/v), PVA 12% (w/v), alginate 0.3% (w/v) and 10% (v/v) initial biomass loading. Scanning electron microscopy (SEM) of the beads showed that B. fusiformis was evenly distributed within them. Storage stability and reusability results revealed that the ability to degrade naphthalene using beads with immobilized cells remained stable after storage at 4 °C for 35 days and being reused 8 times (12 days), respectively. Furthermore the naphthalene degradation rate of immobilized cells was maintained (94.3%) at the eighth cycle.
Keywords: Bacillus fusiformis; Naphthalene; Biodegradation; Immobilization; Biomaterial;

Embryonic stem cells (ESCs) possess great potential in many tissue engineering applications such as cardiac regeneration. Culture pH is important in stem cell cultures as they could impact vitality of the culture and cell fate decisions. In this study, we demonstrated how sensitive ESCs are to pH, utilizing murine ESCs as a cell model and experiments conducted at three different pH conditions (pH 6.8, 7.1 and 7.4). Maintenance of culture pH was achieved via a perfused rotary bioreactor while murine ESCs were encapsulated in alginate hydrogels, which served as a three-dimensional (3D) platform and matrix support for the ESC culture. Our results showed that at pH 6.8, ESC viability was inferior to those at pH 7.1 and 7.4. Lower cardiac gene expressions and percentage of cardiac troponin-I positive cells at pH 6.8 indicated that cardiac differentiation of ESCs was significantly compromised. Concurrently, residual pluripotency of ESCs was better conserved at pH 6.8 as compared to higher pH conditions. Finally increased levels of MAPK14 and HIF-2α suggest an impact of pH on kinase and HIF regulated pathways. This study highlights how a small change in pH could significantly affect the growth and differentiation of ESCs toward cardiomyocytes. Therefore, there is an important need for good control of culture parameters such as pH in ESC cultures, so as to obtain the optimal and desired cell output.
Keywords: Culture pH; Embryonic stem cells; Differentiation; Tissue engineering; Bioprocess;

Facile surface functionalization of multiwalled carbon nanotubes by soft dielectric barrier discharge plasma: Generate compatible interface for lipase immobilization by Zahra Rastian; Abbas Ali Khodadadi; Farzaneh Vahabzadeh; Christian Bortolini; Mingdong Dong; Yadollah Mortazavi; Azadeh Mogharei; Masoud Vesali Naseh; Zheng Guo (16-26).
To create compatible interface for enzyme immobilization, the surface of multi-walled carbon nanotubes (MWCNTs) was functionalized using soft technique dielectric barrier discharge plasma (DBDP) for carboxylation and amination; followed by further amidation of carboxyl group with alkylamine. Successful functionalization and enzyme immobilization were structurally confirmed using spectroscopic analysis Fourier-Transform Infrared Spectroscopy (FTIR) and X-ray Photoelectron Spectroscopy (XPS). The immobilization of Candida rugosa lipase (CRL) on functionalized MWCNTs was evidenced by clearly viewing with Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM) imaging. CRL showed more Freundlich equilibrium behavior upon immobilization on annealed and octadecylamidated MWCNTs, which suggested a multilayer adsorption; while upon physical adsorption on aminated and carboxylated MWCNTs, CRL, to more extent, demonstrated a Langmuir equilibrium property, producing an enzyme monolayer. It was proven that DBDP-mediated surface-functionalization could create compatible microenvironments for enzyme immobilization, resulted in improved specific activity and thermostability. The immobilized CRL on octadecylamidated MWCNTs displayed excellent reusability and operation stability, indicating its potential for industrial application.
Keywords: Dielectric barrier discharge plasma (DBDP); Multi-walled carbon nanotubes (MWCNTs); Biocatalysis; Immobilization; Lipase; Adsorption;

Effect of dissolved oxygen on biological phosphorus removal induced by aerobic/extended-idle regime by Hongbo Chen; Dongbo Wang; Xiaoming Li; Qi Yang; Kun Luo; Guangming Zeng; Maolin Tang; Weiping Xiong; Guojing Yang (27-35).
This paper showed that the aerobic/extended-idle (A/EI) regime drove superior biological phosphorus removal than the conventional anaerobic/oxic (A/O) process at low DO level.Previous researches have suggested that biological phosphorus removal (BPR) from wastewater could be achieved by the aerobic/extended-idle (A/EI) regime. This study further investigated the effect of dissolved oxygen (DO) concentration on BPR induced by the A/EI regime. The experimental results show that 1 mg/L of DO in mixed liquor benefited the BPR performance while a higher DO level of 5 mg/L deteriorated BPR. Fluorescent in situ hybridization analysis demonstrated that the improvement at 1 mg/L of DO was due to the shift in bacterial population from glycogen accumulating organisms (GAOs) to polyphosphate accumulating organisms (PAOs). The mechanism studies revealed that DO level affected the transformations of polyhydroxyalkanoates and glycogen and the activities of exopolyphosphatase and polyphosphate kinase. In addition, the BPR performances between the A/EI regime and conventional anaerobic/oxic (A/O) process were compared. The results showed that the A/EI regime drove better BPR performance than the A/O process at both low and high DO levels. More PAO and less GAO abundances in the biomass might be the principal reason for the higher BPR efficiency in the A/EI regime. Furthermore, controlling DO at a low level of 0.5 mg/L to promote BPR was demonstrated in a real municipal wastewater. The A/EI regime showed an excellent BPR performance at the low DO levels and had a better tolerance to oxygen-limited condition as compared to the A/O regime.
Keywords: Dissolved oxygen concentration; Biological phosphorus removal; Aerobic processes; Biosynthesis; Enzyme activity; Waste-water treatment;

Highly active CALB cross-linked enzyme aggregates (CLEAs) were synthesized using a layered methodology based on the synthesis of a cross-linked protein cofeeder core over which an external layer of lipase was later cross-linked. The layered CALB CLEAs were characterized in terms of their catalytic activity in three different test reactions: esterification of oleic acid and ethanol in absence of solvents, esterification of oleic acid and heptanol in organic medium, and hydrolysis of triolein in emulsioned medium. The impact of the cross-linker/protein mass ratio on CLEAs activity, and its evolution with storage time were evaluated in the solventless synthesis of ethyloleate. The amount of cross-linker used showed to be a key parameter for the evolution of the catalytic activity of CLEAs during storage. Under the best conditions found, hyperactivated CALB CLEAs with up to 188% of recovered activity in ethyl oleate synthesis were obtained. In terms of hydrolytic activity mature layered CALB CLEAs showed a retained activity of 68%. The assay of dried mature layered CALB CLEAs in heptyl oleate synthesis showed catalytic activities much higher than the one exhibited by free CALB, reaching 1 h-fatty acid conversions of 14% and 2%, respectively. The high catalytic activity shown by layered CALB CLEAs, suggests that they are an interesting alternative specially for the catalysis of fatty acid esterifications in both organic and solventless medium.
Keywords: Biocatalysis; Lipase; Immobilization; Cross-linked enzyme aggregates (CLEAs); Cofeeder core; Enzyme activity;

Recently, bioenergy recovery from sludge biomass has attracted increasing attention due to the high demand for renewable energy resources. In order to enhance methane production from sludge biomass, electrochemical treatment can be used as a novel and efficient pretreatment for the hydrolysis of sludge biomass. In this study, a combined electro-flotation and electro-oxidation pretreatment was employed to improve the anaerobic degradability of sludge biomass. Electro-flotation was efficient in separating flocs in the mixed liquor and led to a sludge volume reduction greater than 60% after 10 min of operation at a current density of 4.72 mA cm−2. Electro-oxidation using IrO2/Ti anode was performed to improve the anaerobic degradability of sludge and resulted in a 30% increase in COD solubilization after 30 min of operation at current density of 9.45 mA cm−2. The factors affecting electro-oxidation, i.e. the gap width between anode and cathode, current density and applied voltage, were investigated to optimize the operating conditions. A biochemical methane potential assay demonstrated that the anaerobic biodegradability of sludge was enhanced by combined electro-flotation and electro-oxidation pretreatment.
Keywords: Electro-flotation; Electro-oxidation; Biogas; Biodegradation; Cell disruption; Process integration;

The present work aims to investigate the surface activity of the biosurfactant produced by Acinetobacter baylyi ZJ2 isolated from crude oil-contaminated soil sample in China and evaluate its potential application in microbial enhanced oil recovery. The biosurfactant produced by A. baylyi ZJ2 was identified as lipopeptide based on thin-layer chromatography, Fourier transform infrared spectroscopy and nuclear magnetic resonance techniques. This biosurfactant could reduce the surface tension of water from 65 mN/m to 35 mN/m, and interfacial tension against oil from 45 mN/m to 15 mN/m. Moreover, surface activity stability results showed that this biosurfactant was effective when the salinity was lower than 8% and the pH value was 4–9, and it was especially stable when the salinity was lower than 4% and pH was 6–7. Based on the result of gas chromatography, there was a decrease in heavy components and an increase in light components, which indicated that A. baylyi ZJ2 exhibited the biodegradability on the heavy components of crude oil. Furthermore, the ability of recovering oil from oil-saturated core showed that nearly 28% additional residual oil was displaced after water flooding. The lipopeptide biosurfactant produced by A. baylyi ZJ2 presented a great potential application in microbial enhanced oil recovery process, owing its good surface activity and satisfying degradation ability to crude oil.
Keywords: Optimization; Microbial growth; Purification; Biodegradation; Biosurfactant; Acinetobacter baylyi;

Process engineering studies of free and micro-encapsulated β-galactosidase in batch and packed bed bioreactors for production of galactooligosaccharides by Pramita Sen; Arijit Nath; Chiranjib Bhattacharjee; Ranjana Chowdhury; Pinaki Bhattacharya (59-72).
Keywords: Galactooligosaccharides; Immobilized enzyme; Enzyme activity; Packed bed bioreactor; GOS yield; Batch processing;

The P170 expression system enhances hyaluronan molecular weight and production in metabolically-engineered Lactococcus lactis by Ashish Singh Chauhan; Sneh Sanjay Badle; K.B. Ramachandran; Guhan Jayaraman (73-78).
Recombinant Lactococcus lactis strains based on the P170 expression system were developed for hyaluronan (HA) production, by incorporating genes from the has operon of Streptococcus zooepidemicus and compared with nisin-inducible recombinant L. lactis strains containing the hasABC and hasABD constructs. It was found across all batch and fed-batch experimental studies that HA concentration and molecular weight (MW) were higher for the P170 expression systems than the corresponding NICE-based strains. The highest hyaluronan MW was obtained for all constructs in batch studies at 60 g/L initial glucose concentration, the highest being 2.94 MDa for the P170 strains with hasABC construct (L. lactis APJ3). In fed-batch studies with constant feed rate, the L. lactis APJ3 gave better HA yield (0.03 g/g) than the NICE-based strain. A higher hyaluronan MW was obtained for all strains in pulse fed-batch compared to constant feed experiments, the highest being 2.52 MDa for L. lactis APJ3.
Keywords: Hyaluronan; Biosynthesis; Recombinant DNA; Lactococcus lactis; Batch Processing; Fed-Batch Cultures;

A novel pH-sensitive and water-soluble polyvinyl alcohol (PVA)-cellulose acetate phthalate (CAP) composite-based biomaterial was prepared, in which the multi-scale web of copper (Cu)-grown carbon micro-nanofibers (Cu-ACF/CNFs) was in situ dispersed during a synthesis stage. PVA-CAP and Cu-nanoparticles (NPs) were used as an encapsulating agent and nano-antibiotics, respectively. The web of Cu-ACF/CNF was prepared by growing CNFs on the activated carbon microfiber (ACF) substrate by chemical vapor deposition using Cu NPs as the catalyst. The novel step of the synthesis included esterification of polyvinyl acetate (PVAc) to produce a PVA gel to which the ball-milled Cu-ACF/CNF was blended at the incipience of the gel formation to produce the PVA-CAP-Cu-ACF/CNF metal-carbon-polymeric composite film. The in vitro dissolution tests revealed that the encapsulating polymeric composite was dispersible in water and its rate of dissolution was high at pH > 6.5. The antibacterial tests performed on the material demonstrated its effectiveness against both gram negative Escherichia coli and gram positive Staphylococcus aureus bacterial strains. The Hixson-Crowell kinetic model described the dissolution profiles of the material. The method of preparation is novel, simple, and environmentally friendly. The prepared biomaterial may be used in several biomedical applications, including wound healing and the controlled release of drugs in the antibiotic delivery system.
Keywords: Antibiotics; Biomedical; Biokinetics; Microcarriers; Drug delivery; Biomaterial;

Microwave pretreatment of lignocellulosic material in cholinium ionic liquid for efficient enzymatic saccharification by Kazuaki Ninomiya; Takashi Yamauchi; Chiaki Ogino; Nobuaki Shimizu; Kenji Takahashi (90-95).
We demonstrated that the enzymatic hydrolysis of cellulose after microwave pretreatment of lignocellulosic material in ionic liquids (ILs) is drastically enhanced compared with that after conventional thermal pretreatment in ILs. Three types of cholinium ILs, choline formate (ChFor), choline acetate (ChOAc), and choline propionate (ChPro), were examined. The cellulose saccharification percentage was approximately 20% for kenaf powders pretreated in ChFor, ChOAc, and ChPro by conventional heating at 110 °C for 20 min. In contrast, approximately 60–90% of cellulose was hydrolyzed to glucose after microwave pretreatment in the same ILs at 110 °C for 20 min.
Keywords: Ionic liquid; Microwave, Lignin; Cellulose; Cellulase; Bioconversion;

4-Chloro-2-methylphenoxyacetic acid (MCPA) is a selective systemic herbicide which is absorbed by leaves and roots. MCPA esters are preferred due to their low water solubility and environmental friendliness. Esterification of MCPA with n-butanol was investigated as a model reaction using immobilized enzymes under the influence of microwave irradiation. Different immobilized enzymes such as Novozym 435, Lipozyme TL IM, Lipozyme RM IM and Lipase AYS Amano were studied under microwave irradiation amongst which Novozym 435 (immobilized Candida antarctica lipase B) was the best catalyst. Effects of various parameters were systematically studied on rates and conversion. Under microwave irradiation, the initial rates were observed to increase up to 2-fold. Under optimized conditions of 0.1 mmol MCPA and 0.3 mmol n-butanol in 15 mL 1,4-dioxane as solvent, Novozym 435 showed a conversion of 83% at 60 °C in 6 h. Based on initial rate and progress curve data, the reaction was shown to follow the Ping Pong bi–bi mechanism with inhibition by MCPA and n-butanol. Esterification of MCPA was also studied with different alcohols such as isopropyl alcohol, n-pentanol, n-hexanol, benzyl alcohol and 2-ethyl-1-hexanol.
Keywords: Enzyme biocatalysis; Immobilized enzymes; Biotransformation; Kinetic parameters; 4-Chloro-2-methylphenoxyacetic acid esters; Microwave irradiation;

Enzymatic biodiesel production of microalgae lipids under supercritical carbon dioxide: Process optimization and integration by Hanifa Taher; Sulaiman Al-Zuhair; Ali H. Al-Marzouqi; Yousef Haik; Mohammed Farid (103-113).
Enzymatic biodiesel production in supercritical CO2 (SC-CO2) has recently received an increasing attention, as an alternative to the conventional chemical processes. In this study, enzymatic production of biodiesel from microalgal lipids was investigated in batch and integrated extraction-reaction systems. In the batch system, the effect of enzyme loading (15–50 wt%), temperature (35–55 °C) and methanol to lipid molar ratios (3–15:1) were studied, and response surface methodology was employed to optimize selected factors effect. The optimum transesterification yield of 80% was obtained at 47 °C, 200 bar, 35% enzyme loading, and 9:1 molar ratio after 4 h reaction in the batch system. The experimental results were also used to determine the kinetics parameters of the Ping-Pong Bi Bi model, with methanol inhibition, suggested to describe the reaction.In the continuous integrated extraction-reaction system, the effect of methanol to lipids molar ratio was investigated, and enzyme operational stability and reusability were tested. Bed regeneration by tert-butanol washing was also assessed. The optimum methanol to lipid ratio was found to be 10:1. At this ratio, the enzyme was able to attain 78% of its original activity when reused for 6 continuous cycles, and the bed was successfully reused by washing with tert-butanol.
Keywords: Lipase; Biodiesel; Microalgae lipid; Supercritical CO2; Production kinetics; Process integration;

Biological souring of crude oil under anaerobic conditions by Yasunori Tanji; Kazuya Toyama; Ryo Hasegawa; Kazuhiko Miyanaga (114-120).
Seawater injection into oil reservoirs for purposes of secondary oil recovery is frequently accompanied by souring (increased sulfide concentrations). Production of hydrogen sulfide causes various problems, such as microbiologically influenced corrosion (MIC) and deterioration of crude oil. Sulfate-reducing bacteria (SRB) are considered to be major players in souring. Volatile fatty acids (VFAs) in oil-field water are believed to be produced by microbial degradation of crude oil. The objective of this research was to investigate mechanisms of souring, focusing specifically on VFA production via crude oil biodegradation. To this end, a microbial consortium collected from an oil–water separator was suspended in seawater; crude oil or liquid n-alkane mixture was added to the culture medium as the sole carbon source, and the culture was incubated under anaerobic conditions for 190 days. Physicochemical analysis showed that preferential toluene degradation and sulfate reduction occurred concomitantly in the culture containing crude oil. Sulfide concentrations were much lower in the alkane-supplemented culture than in the crude oil-supplemented culture. These observations suggest that SRB are related to the toluene activation and VFA consumption steps of crude oil degradation. Therefore, the electron donors for SRB are not only VFA, but many components of crude oil, especially toluene. Alkanes were also degraded by microorganisms, but did not contribute to reservoir souring.
Keywords: Acetic acid; Anaerobic processes; Biodegradation; Bioreactions; Souring; Sulfate-reducing bacteria;

Complementary elementary modes for fast and efficient analysis of metabolic networks by Md. Bahadur Badsha; Ryo Tsuboi; Hiroyuki Kurata (121-130).
Metabolic pathway analysis facilitates understanding or designing a complex metabolic system and enables prediction of steady-state metabolic flux distributions. A serious problem of elementary mode (EM) or extreme pathway (Expa) analysis is that the computational time increases exponentially with an increase in network sizes, which makes the computation of the EMs/Expas expensive and infeasible for large-scale networks. To overcome such problems, we proposed a fast and efficient algorithm named complementary EM (cEM) analysis. To achieve the computational time improvement, we employ the EM decomposition method that explores EMs or linear combinations of them which are responsible for the metabolic flux distributions. Flux balance analysis (FBA) is used to determine possible ranges of metabolic flux distributions as the input data necessary for the EM decomposition method. The maximum entropy principle (MEP) is employed as an objective function for estimating the coefficients of cEMs. To demonstrate the feasibility of cEM analysis, we compared it with EM/Expa analysis by using two medium-scale metabolic networks of Escherichia coli and a genome-scale metabolic network of head and neck cancer cells. The cEM analysis greatly reduces the computational time and memory cost, exposing a new window for large-scale metabolic network analysis.
Keywords: Bioreactors; Elementary mode; Metabolic network; Metabolite over production; Microbial growth; Modelling;

Sheep pox virus initially adapted to replicate in primary lamb kidney cells was adapted to Vero cells by serial passages in monolayer cultures. After nine passages the virus was able to correctly replicate in Vero cells, virus titer achieved was 105.875 TCID50 (median tissue culture infective dose) ml−1.To optimize the production process, the effects of MOI (multiplicity of infection), TOI (time of infection) and the culture medium were investigated. Cell infection at a MOI of 0.005 concurrently with cell seeding showed the best results in terms of specific virus productivity. The effect of MEM enrichment with several components was investigated using the experimental design approach. 67 experiments were performed in 6-well plates to select the best combination. The highest titer was achieved when MEM was supplemented with 5 mM glucose, 5 mM fructose and 25 mM sucrose. Spinner culture confirms these data; virus titer was 107.375 TCID50  ml−1.In addition Vero cells were cultivated in a 7-l bioreactor in batch mode on 3 g l−1 Cytodex1, and infected at cell seeding at a MOI of 0.005. Maximal virus titer was 107.275 TCID50  ml−1. This corresponds to 44-fold factor enhancement compared to spinner cultures conducted in MEM + 2% FCS.
Keywords: Animal cell culture; Bioprocess design; Production kinetics; Optimization; Sheep pox vaccine; Vero cells;

High biological activity and volumetric productivity are considered as prerequisites for efficient bioprocesses, extreme halophilic Archaea have, however, lower growth rates, for which reason halophilic Archaea are so far not used in industrial bioprocesses. To overcome this physiological limit and to achieve increased volumetric productivity, the produced biomass must be retained in a bioreactor, for example equipped with an external cell retention system. In this study, the characterization and parameterization of a bioreactor setup with cell retention was carried out with an extreme halophilic archaeon. Bioprocess quantification was used to demonstrate the process controllability. Focussing on maximizing the volumetric productivity; 10-fold productivity increase was achieved compared to chemostat continuous cultures. Circulation of the broth between the bioreactor and the membrane unit can be however challenging from physiological points-of-view. Hence, operating the system with external cell retention at optimal cross flow rate is physiologically essential: at lower cross flow rates, higher extracellular protein concentrations were measured due to oxygen limitation. In turn, at higher cross flow rates, shear stress reasoned higher concentrations of DNA fragments. This work contributes in a pioneering way to the bioprocess development of extreme halophilic Archaea by optimizing continuous laboratory scale processes regarding robustness and scalability.
Keywords: Halophilic Archaea; Microfiltration for cell retention; Integrated processing; Bioprocess design; Bioprocess monitoring; Ultrafiltration;

Efficient conversion from cheese whey to lipid using Cryptococcus curvatus by Yeong Hwan Seo; Ilgyu Lee; Seok Hwan Jeon; Jong-In Han (149-153).
Cheese whey, which is a byproduct of the cheese-making process, was pretreated by means of hydrodynamic cavitation (HC) under an alkaline condition and then was converted to lipid using our newly isolated oleaginous yeast Cryptococcus curvatus. The growth rate (7.2 g/L/day) and lipid content (65%) were nearly double those obtained using commercial types of sugars such as glucose, galactose, and lactose. The lipid productivity level (4.68 g/L/day) was the highest ever reported. HC-pretreated cheese whey under alkaline condition can serve as an excellent complete nutrient source and/or as a stimulant of lipid production.
Keywords: Bioconversion; Bioprocess design; Food engineering; Microbial growth; Whey; Yeast;

The present study investigated the metabolism of different acetate:propionate ratios (0.25, 0.33, 0.5, 1.0, 2.0, 3.0, 4.0) in equimolar carbon concentration during an anaerobic decomposition process under defined laboratory conditions and evaluated the engaged methanogenic community. Significant differences on a metabolic level (gas production, gas composition, volatile fatty acid (VFA) concentration) were observed between acetate:propionate ratios ≤1 and ≥2. Generally ratios ≥2 resulted in a faster methane production and VFA decomposition compared to ratios ≤1. Regarding the composition of the methanogenic community as well as the abundance of Methanosarcinales these differences were not reflected in an appropriate manner when DNA based methods (dHPLC and qPCR) were applied. However, by a culture based approach these differences could be documented showing a significant difference in the number of cultivable methanogens between initial acetate:propionate ratios ≤1 and ≥2.
Keywords: Acetate; Propionate; Methanogenic community; Methanogens; Acetate propionate ratio; Biogas;

Improving the carbon balance of fermentations by total carbon analyses by Jens Buchholz; Michaela Graf; Bastian Blombach; Ralf Takors (162-169).
Carbon balancing of microbial fermentations is a valuable tool for the evaluation of the process performance and to identify the presence of undesired by-products. In this study, we demonstrate the relevance of total carbon (TC) analysis for carbon balancing in fermentations with the wild-type of Corynebacterium glutamicum by (i) quantifying significant amounts of dissolved inorganic carbonic species (TIC) in the culture medium and (ii) determining the effective (mass) carbon content of the biomass fraction (M C,X ). In principle, TC based carbon balancing yielded at fully matching carbon balances. Thus, the application of our TC approach for the accurate detection of TIC and M C,X increased the total carbon recovery in standard batch fermentations with C. glutamicum on glucose from about 76% to carbon closures of 94–100% in contrast to conventional approaches. Besides, the origin of the missing 6%-gap could be attributed to incomplete quantification of all carbon sources in the liquid phase. To conclude this study, the concept of TC-based balancing was transferred to an l-lysine production process, successfully quantifying relevant system carbon fractions, which resulted in matched carbon recoveries.
Keywords: Bioprocess monitoring; Bioreactions; Carbon balance; Corynebacterium glutamicum; Fermentation; Instrumentation;

Direct affinity immobilization of recombinant heparinase I fused to maltose binding protein on maltose-coated magnetic nanoparticles by Jingjun Wu; Lilin Zhou; Huijuan Zhang; Jun Guo; Xiang Mei; Chong Zhang; Jinying Yuan; Xin-Hui Xing (170-177).
Hybrid magnetic Fe3O4@SiO2-poly(ethylene oxide)-maltose (Fe3O4@SiO2-PEO-mal) nanoparticles synthesized by our group can be used as affinity adsorption carriers for direct separation of maltose binding protein-fused Hep I (MBP-Hep I) from a crude enzyme solution in a magnetic field. In this work, different PEO molecular weights for Fe3O4@SiO2-PEO-mal nanoparticles were used for characterizing of MBP-Hep I immobilization. The results showed that all four kinds of Fe3O4@SiO2-PEO-mal magnetic nanoparticles (6k, 20k, 35k and 100k for PEO) exhibited excellent adsorption capacities and the adsorption ratio increased as the PEO molecular weight increased from 6k to 100k. All four kinds of immobilized MBP-Hep I exhibited significantly improved stability at 30 °C compared with free MBP-Hep I and their half-lives were 20–50 times that of the free MBP-Hep I. Fe3O4@SiO2-PEO-mal nanoparticles with a PEO molecular weight of 100k were best able to immobilize MBP-Hep I (Fe3O4@SiO2-PEO100k-mal-MBP-Hep I). The molecular weight distribution profiles and anticoagulant activities, obtained from heparin depolymerization by free Hep I, free MBP-Hep I and Fe3O4@SiO2-PEO100k-mal-MBP-Hep I were the same. Furthermore, Fe3O4@SiO2-PEO100k-mal-MBP-Hep I exhibited reasonable reusability during enzymatic production of low molecular weight heparins (LMWHs).
Keywords: Affinity; Adsorption; Heparinase I; Immobilization; Magnetic nanoparticles; Enzyme biocatalysis;

Enhanced accumulation of individual ganoderic acids in a submerged culture of Ganoderma lucidum by the overexpression of squalene synthase gene by Jiang-Sheng Zhou; Sen-Lin Ji; Meng-Fei Ren; Yi-Long He; Xiao-Ran Jing; Jun-Wei Xu (178-183).
Ganoderic acids (GAs) produced by Ganoderma lucidum exhibit antitumor and antimetastasis activities. This study aimed to improve the production of individual GAs by engineering the biosynthetic pathway of GAs in G. lucidum through the overexpression of squalene synthase (SQS) gene. SQS catalyzes the first enzymatic step from the mevalonate pathway toward triterpene biosynthesis. The effects of SQS gene overexpression on the accumulation of individual GAs and their intermediates (squalene and lanosterol) by a submerged culture of G. lucidum and on the transcription levels of GA biosynthesis genes in this mushroom were investigated. The maximum contents of GA-Mk, GA-T, GA-Me, and GA-S in G. lucidum overexpressing the SQS gene were 16, 40, 43, and 53 μg/100 mg dry cell weight, respectively, which were 2.86-, 2.67-, 1.95-, and 1.25-fold of those obtained in wild-type strain (WT). The maximum contents of squalene and lanosterol in the SQS gene-overexpression strain were 1.55- and 1.68-fold higher than those of the WT strain. The transcription levels of the biosynthetic genes encoding SQS and lanosterol synthase were up-regulated by 15.6- and 1.93-fold, respectively, in G. lucidum overexpressing the SQS gene, suggesting that increased GA biosynthesis may result from a higher expression of those genes.
Keywords: Submerged culture; Metabolite over production; Filamentous fungi; Fermentation; Individual ganoderic acid; Squalene synthase (SQS);

Sunflower oil modification for production of semisolid fats was carried out via acidolysis using palmitic and stearic acids (P + St), hexane and a developed biocatalyst from Rhizomucor miehei lipases. Its kinetic behavior was studied by employing three mathematical models proposed in the literature. Furthermore, a new model was proposed to describe not only the variation of triacylglycerols (TAG), diacylglycerols (DAG), and free fatty acids groups but also the acyl migration reaction occurrence. The effect of the reaction temperature on the kinetic and equilibrium parameters, as well as TAG and reaction intermediates profiles was analyzed. Increasing reaction temperature generated major changes in the overall composition of acylglycerols and gave rise to the highest composition of P + St in the obtained structured lipids (58%, 70 h, 60 °C). P + St incorporation was successfully adjusted by an empirical model (Model I) and a lumped parameter model (Model II) for all the studied reaction times, while the model based on a Ping Pong Bi Bi mechanism (Model III) was only able to describe the kinetics behavior (through the variation of reactant saturated fatty acids concentration) until 24 h. Experimental data were fit satisfactorily by the proposed model (Model IV), showing that the increment in the disaturated TAG formation achieved by the increment in temperature was principally related to the favored DAG formation from triunsaturated TAG.
Keywords: Enzyme biocatalysis; Modeling; Lipase; Kinetic parameters; Structured lipids; Sunflower oil;

Pyrosequencing reveals the inhibitory impact of chronic exposure to erythromycin on activated sludge bacterial community structure by Ilke Pala-Ozkok; Ateequr Rehman; Emine Ubay-Cokgor; Daniel Jonas; Derin Orhon (195-205).
The study investigated changes in the microbial population structure sustained at two different sludge ages of 10 d and 2 d under chronic impact of erythromycin. It intended to observe the experimental correlation between variable process kinetics and changes in the composition of the microbial community induced by erythromycin. Samples from two fill/draw reactors operated with continuous erythromycin dosing of 50 mg/L were collected for the analysis of microbial population structure using high-throughput sequencing of 16SrRNA gene. Significant changes were observed in the composition of microbial community during the exposure period. Richness analysis for slower growing culture indicated that most microbial fractions were inactivated and eliminated in favor of fewer more resistant species in different phyla. Sludge age appeared to control the impact of erythromycin on microbial composition. At a sludge age of 2 d, erythromycin appeared to generate richer community with faster growing and more compatible species. For slower growing culture, elimination of vulnerable species was supported by decrease in the number of shared level OTUs. For faster growing culture, shared species level OTUs also decreased significantly upon exposure to erythromycin, suggesting rapid washout and replacement by more resistant species. Resistance gene analysis yielded positive results for mph(A) gene indicating presence of erythromycin-resistant components in the microbial community.
Keywords: Aerobic processes; Antibiotic; Chronic inhibition; DNA; Kinetic parameters; 454-Pyrosequencing;

A novel recycling process using the treated citric acid wastewater as ingredients water for citric acid production by Hong-Jian Zhang; Jian-Hua Zhang; Jian Xu; Lei Tang; Zhong-Gui Mao (206-213).
In this study, an integrated process coupling citric acid and methane fermentations was proposed to solve severe wastewater pollution problem in cassava-based citric acid production. The accumulation patterns of the potential and major inhibitors in this process, including organic compounds, volatile fatty acids (VFAs), total ions and pigments were investigated. Both simulation and experimental results indicated that these inhibitors could reach their equilibrium levels after 3–7 fermentation runs when reutilizing the treated citric acid wastewater. As a result, the proposed citric acid fermentation process by recycling the wastewater treated in methane fermentation could be stably operated for more than 15 runs, which could save a large amount of fresh water and relieve the severe wastewater pollution in citric acid production potentially.
Keywords: Anaerobic processes; Citric acid; Process integration; Recycling; Waste-water treatment;

Knowing the distribution of the density of radiant energy within a photo-bio-reactor and its impact on the growth of microalgae through the local rate of absorption of radiant energy is essential for the analysis, modeling and design of photo-bio-reactors. In this work we develop a physical model and a computer simulation algorithm, in order to accurately predict the rate of absorption of photons in microalgal cultures at each point of a reactor irradiated with a light source made of different arrangements of LEDs emitting in blue and red spectral regions. Results showed that the average absorption rates in the culture irradiated with blue LEDs were higher than that irradiated with red LEDs. However, the radiation emitted by red LEDs rendered greater energy efficiency for biomass production compared to that emitted by blue LEDs. The development of a computational model based on a Monte Carlo method allowed the determination of the local volumetric rate of photon absorption at each point inside the PBR at different culture times. The information obtained with this tool allowed a detailed assessment of the effect of different radiation conditions on growth of microalgae, regardless of the PBR and the light source used.
Keywords: Microalgae; Modeling; Bioreactors; Light Emitting Diode; Monte Carlo; Scale Up;

The role of volumetric power input in the growth, morphology, and production of a recombinant glycoprotein by Streptomyces lividans in shake flasks by Luz D. Marín-Palacio; Ramsés A. Gamboa-Suasnavart; Norma A. Valdez-Cruz; Luis Servín-González; Ma. Soledad Córdova-Aguilar; Enrique Soto; Wolf Klöckner; Jochen Büchs; Mauricio A. Trujillo-Roldán (224-233).
The impact of flask geometry on Streptomyces lividans growth and morphology, production and O-mannosylation of a recombinant O-glycoprotein (APA from Mycobacterium tuberculosis) was described and associated to the evolution of the volumetric power input (P/V) in three shake flask geometries. During the exponential growth, the highest P/V was found in baffled flasks (BF) with 0.51 kW/m3, followed by coiled flasks (CF) with 0.44 kW/m3 and normal Erlenmeyer flasks (NF) with 0.20 kW/m3 (flasks volume of 250 mL, filling with 50 mL and agitated at 150 rpm). During the stationary phase, P/V decreased 20% in BF and CF, but increased two times in NF, surely due to changes in mycelial morphology and its effects on rheology. Also, NF cultures were carried out at a filling volume and agitation of 15 mL, 150 rpm (15 mL-NF), and 25 mL, 168 rpm (25 mL-NF), in order to raise P/V closely to the values obtained in CF. However, different growth, morphology and recombinant protein productivity were obtained. These data indicate that P/V is not a definitive parameter that can determine bacteria growth and morphology, not even glycoprotein production. But it can be proposed that the oxygen transfer in the center of the pellets and hydromechanical stress might be the more relevant parameters than P/V.
Keywords: Shaken bioreactors; Morphology; Orbital shaking; Power dissipation; Suspension rheology; Recombinant glycoproteins;

Effects of operating parameters on hydrogen production from raw wet steam-exploded cornstalk and two-stage fermentation potential for biohythane production by Zhidan Liu; Qian Li; Chong Zhang; Linjun Wang; Bing Han; Baoming Li; Yuanhui Zhang; Hongzhang Chen; Xin-Hui Xing (234-238).
Biohythane (biohydrogen + biomethane) production from agricultural residue is a win–win solution for the supply of renewable energy and valorization of waste biomass. This study reported the first investigation on hydrogen fermentation directly using raw wet steam-exploded cornstalk (SC) without any further processing for drying or detoxification. The effects of key operating parameters (feedstock concentration, initial pH and heat treatment of seed sludge) were systematically studied. The suitable conditions for hydrogen fermentation from the wet SC were the feedstock concentration at 200 g L−1 (TS, 6–8%), pH at 6.5 and seed sludge without heat treatment. In addition, compared to one-stage biomethane fermentation, the two-stage biohythane fermentation by integrating hydrogen fermentation with biomethane production from SC led to the hydrogen and methane yields of 12 and 195 L kg−1  TS−1, respectively, corresponding to an increased energy recovery of 26%, reduced fermentation time and facilitated conversion of volatile fatty acids. These results demonstrated the feasible energy-efficient biohydrogen or biohythane production from the wet steam-exploded cornstalk, implying the promising potential of this method for harvesting clean hythane vehicle fuel from agricultural biomass.
Keywords: Anaerobic processes; Biohydrogen; Bioconversion; Biogas; Fermentation; Steam-exploded cornstalk;

Thermobifida fusca not only produces cellulases, hemicellulases and xylanases, but also excretes butyric acid. In order to achieve a high yield of butyric acid, the effect of different carbon sources: mannose, xylose, lactose, cellobiose, glucose, sucrose and acetates, on butyric acid production was studied. The highest yield of butyric acid was 0.67 g/g C (g-butyric acid/g-carbon input) on cellobiose. The best stir speed and aeration rate for butyric acid production were found to be 400 rpm and 2 vvm in a 5-L fermentor. The maximum titer of 2.1 g/L butyric acid was achieved on 9.66 g/L cellulose. In order to test the production of butyric acid on lignocellulosic biomass, corn stover was used as the substrate, on which there was 2.37 g/L butyric acid produced under the optimized conditions. In addition, butyric acid synthesis pathway was identified involving five genes that catalyzed reactions from acetyl-CoA to butanoyl-CoA in T. fusca.
Keywords: Fermentation; Batch processing; Cellulase; Cellulose; Butyric acid;

Inherent antioxidant activity and high yield production of antioxidants in Phanerochaete chrysosporium by Liang Liu; Piao Xu; Guangming Zeng; Danlian Huang; Meihua Zhao; Cui Lai; Ming Chen; Ningjie Li; Chao Huang; Cong Wang; Min Cheng; Xiaoxiao He; Mingyong Lai; Yibin He (245-254).
Incremental reactive oxygen species (ROS) under adversity environment could cause oxidative damages to microorganisms during environmental applications. In this study, we highlight a novel role of P. chrysosporium as antioxidants. The P. chrysosporium extracts possessed remarkable antioxidant activity, expressing a dose-dependent total antioxidant activity and accompanied with high reactive oxygen radical (O2, •OH and H2O2) scavenging capacity. In addition, it was established that, various compounds, such as antioxidant enzymes (catalase, superoxide dismutase and glutathione peroxidase) and low-molecular-weight antioxidant components (glutathione, phenolics and flavonoids) existed steadily in P. chrysosporium. Linear regression analysis and Pearson correlation coefficient analysis demonstrated that enzymatic and non-enzymatic antioxidants took part in processes and acted as “antioxidant network” in P. chrysosporium by confirming with the significant correlation coefficients among the tested antioxidants. Furthermore, those tested antioxidants were found sensitive response to Cd exposure, a 2.78-fold and 2.35-fold of stimulation has been found in SOD and phenolics, respectively, which provide a novel dimension to the involvement of antioxidants in the antioxidant defense system of P. chrysosporium. The paper proposes a new validation procedure to specifically validate the admirable tolerance and high efficiency of P. chrysosporium in environmental treatment application, taking advantage of the remarkable antioxidant activity.
Keywords: Filamentous fungi; Antioxidant capacity; Reactive oxygen radical scavenging capacity; Enzymes; Oxygen transfer; Biophysical chemistry;

The economics of virus-like particle and capsomere vaccines by Yap P. Chuan; Nani Wibowo; Linda H.L. Lua; Anton P.J. Middelberg (255-263).
Effective control of infectious diseases relies on new vaccine technologies that can quicken and broaden vaccine delivery. Novel modular virus-like particle (VLP) and capsomere technologies have been recently reported. These technologies utilize murine polyomavirus (MuPyV) VLPs and capsomeres as potent delivery systems to carry and display antigenic modules consisting of heterologous peptides, in the form of modular constructs capable of inducing high levels of specific antibodies against bacterial or viral antigens. These constructs are prepared using high-yield microbial synthesis, potentially enabling low-cost, rapid and scalable manufacture of new vaccines. To evaluate this potential, this study analyzes the economics of capsomere and VLP production using process simulation. Data here show that the unit production cost (UPC) for capsomere is up to 69% lower than that for VLP at the comparison scale (500 L fermentor), due to a simpler downstream process and a higher product yield. For VLP production, reactive diafiltration assembly was shown to have a UPC 30% lower than dilution assembly. Sensitivity analysis of uncertain process inputs with Monte Carlo simulations revealed a significant influence of final biomass concentration on UPC, contributing up to 50% of variance observed in the UPC probability distribution. Despite such process variability, optimized capsomere or VLP vaccine production, using a 500-L or 1500-L fermentor respectively, has more than 80% chance of producing vaccine at a cost less than 1 cent per dose based on a conservative assumption of 50 μg protein per vaccine dose. With a 10-kL fermentor, both the capsomere and VLP processes have productivity that could allow manufacture of 320 million vaccine doses in 2.3 and 4.7 days, respectively. This study confirms with quantitative data the possible economic, speed and scale benefits of the modular capsomere and VLP vaccine technologies, which can potentially redefine current vaccine distribution landscape and time-scale benchmarks.
Keywords: Biosynthesis; Bioprocess design; Process integration; Microbial growth; Vaccine; Virus-like particleX;

Oxygen transfer (k La) is one of the most essential factors affecting microbial cell growth and metabolite formation. In this study, low-cost methods capable of effectively enhancing oxygen transfer were investigated by adding oxygen vectors into the fermentation broth. Upon addition of n-dodecane and n-hexadecane, the k La of the fermentation medium reached remarkably higher values than that of the control. In response to the enhanced oxygen transfer, welan gum concentration and yield reached maxima of 33.9 ± 0.56 g/L and 0.761 ± 0.010 g/g, respectively. The DCW reached a maximum of 7.82 ± 0.18 g/L with n-dodecane addition. Welan gum productivity and yield were significantly affected by k La under n-hexadecane addition because of the promotion of the highest transcriptional levels of genes in the metabolic flux for welan gum biosynthesis. In addition, transcription levels of TCA and electron transfer chain (ETC) genes exhibited maximum up-regulation under n-dodecane, which resulted in enhanced cell growth. These results suggest a mechanism for enhanced k La using oxygen vectors to improve welan gum biosynthesis through regulation of transcriptional levels of ETC, TCA, and metabolic pathways for welan gum biosynthesis genes during fermentation.
Keywords: Welan gum; Biosynthesis; Dissolved oxygen; Oxygen transfer; Viscosity; Electron transfer chain;

Split focal adhesion kinase for probing protein–protein interactions by Yidan Ma; Teruyuki Nagamune; Masahiro Kawahara (272-278).
Since protein–protein interactions (PPIs) regulate a variety of cellular processes, the detection of PPIs is crucial for elucidating the underlying molecular mechanisms as well as developing therapeutics. In this study, we propose a novel system to detect PPIs using the distinct domains of focal adhesion kinase (FAK). In this system named “split FAK”, the linker and kinase domains in native FAK are tethered separately to two target proteins of interest. The interaction between the target proteins brings the linker and kinase domains into proximity, which leads to phosphorylation at Y397 of the linker domain, recruitment of another tyrosine kinase Src, and phosphorylation at Y576 of the kinase domain. PPIs are readily detected by probing phosphorylation at Y397 and Y576 of these domains. To demonstrate this system, we designed a series of split FAK chimeras with different domain structures. Consequently, dimerizer-induced interaction between FK506-binding protein 12 (FKBP) and the T2098L mutant of FKBP12-rapamycin binding domain (FRB) was clearly detected by probing phosphorylation at the specific tyrosine residues of most of the split FAK chimeras. This is a novel PPI detection system based on a mechanism-inspired design of a trans-activated split kinase.
Keywords: Protein–protein interaction; Focal adhesion kinase; Animal cell culture; Biomedical; Protein; Recombinant DNA;

Besides potential applications in the agriculture field as natural nitrogen fertilizer, N2-fixing cyanobacteria have recently gained some attentions for new applications linked to the potential production of biologically active molecules or biohydrogen. Ammonium bioproduction is also gaining attention with the potential use of microalgae in biofuels production and the concerns about the increasing needs for nitrogen substrates. This study has investigated some phenotypic traits linked to biomass production and ammonium release in multicellular cyanobacteria, Anabaena variabilis PCC 7937. It confirms that this wild-type strain has no natural ability for ammonium excretion under diazotrophic conditions. A mutant strain, A. variabilis PCC 7937-C9, was obtained after double random mutagenesis treatments with ethyl methane–sulfonate and screening in batch cultures for resistance to the effect of a glutamine synthetase inhibitor, l-methionine-d,l-sulfoximine (MSX). Although significantly characterized by shorter cell filaments, the growth parameters in photobioreactors of the mutant strain cultures were in the same range of values than those of the wild type. In the presence of MSX this strain was shown to produce extracellular ammonium, with specific rates up to 4.9 μmol NH4 +  mg Chl a−1  h−1. The efficiency of this strain, estimated by its specific rate of ammonium excretion, was shown to be improved after consecutive batch cultures with increasing concentrations of MSX. Such mutant strains are of potential use for investigating ways to improve extracellular ammonium bioproduction.
Keywords: Anabaena variabilis; Nitrogen fixing cyanobacteria; Heterocyt; Ammonium excretion; Mutant; Repetitive cultivations;

Olive stones are an agro-industrial by-product abundant in the Mediterranean area that is regarded as a potential lignocellulosic feedstock for sugar production. Statistical modeling of dilute-sulphuric acid hydrolysis of olive stones has been performed using a response surface methodology, with treatment temperature and process time as factors, to optimize the hydrolysis conditions aiming to attain maximum d-xylose extraction from hemicelluloses. Thus, solid yield and composition of solid and liquid phases were assessed by empirical modeling. The highest yield of d-xylose was found at a temperature of 195 °C for 5 min. Under these conditions, 89.7% of the total d-xylose was recovered from raw material. The resulting solids from optimal conditions were assayed as substrate for enzymatic hydrolysis, while fermentability of hemicellulosic hydrolysates was tested using the d-xylose-fermenting yeast Pachysolen tannophilus. Both bioprocesses were considerably influenced by enzyme loading and inoculum size. In the enzymatic hydrolysis step, about 56% of cellulose was converted into d-glucose by using an enzyme/solid ratio of 40 FPU g−1, while in the fermentation carried out with a cell concentration of 2 g L−1 a yield of 0.44 g xylitol/g d-xylose and a global volumetric productivity of 0.11 g L−1  h−1 were achieved.
Keywords: Olive stones; Acid hydrolysis; d-Xylose; Fermentation; Xylitol; Response surface methodology;

Evaluation and enhanced operational performance of microbial fuel cells under alternating anodic open circuit and closed circuit modes with different substrates by Surajbhan Sevda; Xochitl Dominguez-Benetton; Heleen De Wever; Karolien Vanbroekhoven; T.R. Sreekrishnan; Deepak Pant (294-300).
The present study evaluates the performance of air-cathode microbial fuel cells (MFCs) under alternating open circuit/closed circuit (OC/CC) modes and its effect on independent-electrode and full-cell potentials, power output (at different external resistances) and the polarization behaviour of the electrodes. Three different types of feeds were evaluated using this approach: (1) phosphorus buffer solution (PBS) with acetate as carbon source, (2) glucose-rich synthetic wastewater, and (3) sewage from wastewater treatment plant enriched with fermented molasses. When MFCs were suddenly switched to CC from OC and then again back to OC from CC, the behaviour of the anodes vs reference electrode (Ag/AgCl, 3 M KCl) was monitored. When electric circuit of the MFCs was switched from open to closed circuit, for all cases: (a) the anode potential-shift (vs Ag/AgCl) reallocated in the positive direction in about 200–400 mV, (b) the air-cathode potential-shift (vs Ag/AgCl) reallocated in the negative direction in about 10–25 mV, and (c) the cell-potential difference started at around 0 mV and progressively increased as the MFC reached stability. This behaviour was consistently reproduced during different OC/CC cycles. The systems studied delivered good performance with both controlled media and industrial wastewater. Additionally, this study provides insightful characterization of the independent-electrode behaviours.
Keywords: Microbial fuel cell; Open circuit; Closed circuit; Bioelectricity; Anode; Wastewater;

The synthesis of Cephalexin in novel recyclable aqueous two-phase systems was studied, using immobilized penicillin acylase as biocatalyst, 7-amino-3-desacetoxy cephalosporanic acid (7-ADCA) as nucleophile and phenyl glycine methyl ester (PGME) as acyl donor. The aqueous two-phase system (ATPS) was composed by two pH-response copolymers PADBA and PMDB, which could be recycled efficiently. In the ATPS, high partition coefficient (2.57) for Cephalexin and low partition coefficient (0.21) for 7-ADCA were obtained. Then the synthesis of Cephalexin in ATPS and conventional one-phase system was compared. Higher Cephalexin yield was obtained in ATPS. In addition, synthesis in ATPS was improved at higher substrates concentrations, obtaining yield of 98.2% with 50 mM 7-ADCA and 150 mM PGME at pH 5.8, 20 °C. Even reducing acyl donor to nucleophile ratio to 2, the yield can still above 90%. PADBA/PMDB recycling aqueous two-phase system is suitable for synthesis of Cephalexin.
Keywords: Aqueous two phase; Immobilized enzymes; Biocatalysis; Product inhibition; Cephalexin; pH-sensitive polymer;

Bioconversion of spent coffee grounds into carotenoids and other valuable metabolites by selected red yeast strains by Siniša Petrik; Stanislav Obruča; Pavla Benešová; Ivana Márová (307-315).
Spent coffee grounds (SCG) represent the main coffee industry residues with a great potential to be reutilized in various biotechnological processes. In this study, several carotenogenic yeasts strains were exploited for the production of vitamin-enriched biomass, cultivating in SCG-based media. The fermentation was firstly carried out in Erlenmeyer flasks in order to select the best biomass and pigment producer. Among four tested strains, Sporobolomyces roseus showed the highest potential for the accumulation of carotenoids. Maximum pigment concentration and yield was obtained when cultivating in SCG-based media, 12.59 mg l−1 and 1.26 mg g−1, respectively. Comparing both, the batch and the fed-batch cultivation modes, the strategy of sequential addition of pre-concentrated SCG media in the bioreactor gave higher biomass yield (maximum 41 g l−1 during 41–48 h after the beginning of fermentation). Thus, SCG can be considered as potentially promising industrial waste stream for economically feasible production of enriched yeasts biomass.
Keywords: Spent coffee grounds; Bioconversion; Yeast; Carotenoids; Bioreactors; Fed-batch culture;

Metabolic profiling analysis of the degradation of phenol and 4-chlorophenol by Pseudomonas sp. cbp1-3 by Yingjie Liu; Jiao Liu; Chen Li; Jianping Wen; Rui Ban; Xiaoqiang Jia (316-323).
To investigate the enhancement of phenol on the biodegradation of 4-chlorophenol (4-cp), metabolic profiling approach was performed for the first time to analyze metabolite changes of Pseudomonas sp. cbp1-3 using single substrate (succinate, phenol, and 4-cp) and dual substrate (mixtures of phenol and 4-cp). Phosphoric acid, γ-aminobutyric acid, 4-cp, 4-chlorocatechol, and catechol were shown to change significantly. Results indicated that phenols, especially 4-cp, depressed cell growth by inhibiting its primary metabolic pathway. In addition, the addition of phenol into the 4-cp-containing medium had a global influence on cells including the accumulation of amino acids, amines, saturated fatty acids, and monoacylglycerols as well as the concentration changes of metabolite participating in phenols biodegradation, thus enhancing the degradation of 4-cp. This study provided novel insights into the biodegradation of mixed phenolic compounds and the method could be used to investigate the biodegradation of complicated multi-pollutants.
Keywords: Metabolic profiling analysis; Biodegradation; Chromatography; Microbial growth; Waste treatment; Pseudomonas sp.;

CFD modeling of pretreatment reactors at very high lignocellulose solids content requires the characterization of rheological properties of this specific system. In this study, the non-Newtonian rheological properties of the corn stover–water mixture at high solid loading up to 50% with helical ribbon agitation were characterized. The rheological model was developed by introducing the power law model and measuring the torque values of the mixing system in a small reactor (5 L), and then applied to the larger reactors (50 L and 500 L). The CFD model was developed based on the determined rheological properties. The fluid dynamics experiments in three reactors with different scales were used for validation of the rheological model. The calculated power consumption and mixing efficiency by CFD modeling were in good agreements with the experimental results. This study provided a practical method for the rheology characterization and CFD model at high solids loading. The CFD model could be applied to the design and structure analysis of pretreatment reactors in lignocellulose biorefining processes.
Keywords: Corn stover; Pretreatment; Rheological properties; Computational fluid dynamics (CFD); Helical ribbon impeller; High solids content;