Biochemical Engineering Journal (v.54, #3)

BEJ Keywords (IV).

Ultrasound-assisted fermentation enhances bioethanol productivity by Ahmad Ziad Sulaiman; Azilah Ajit; Rosli Mohd Yunus; Yusuf Chisti (141-150).
► Macro kinetic model screening for water gas shift reaction carried out with CFD. ► The results are validated against experimental literature data. ► Langmuir Hinshelwood model have been found to better predict the CO conversion.Production of ethanol from lactose by fermentation with the yeast Kluyveromyces marxianus (ATCC 46537) under various sonication regimens is reported. Batch fermentations were carried out at low-intensity sonication (11.8 W cm−2 sonication intensity at the sonotrode tip) using 10%, 20% and 40% duty cycles. (A duty cycle of 10%, for example, was equivalent to sonication for 1 s followed by a rest period (no sonication) of 10 s.) Fermentations were carried out in a 7.5 L (3 L working volume) stirred bioreactor. The sonotrode was mounted in an external chamber and the fermentation broth was continuously recirculated between the bioreactor and the sonication chamber. The flow rate through the sonication loop was 0.2 L min−1. All duty cycles tested improved ethanol production relative to control (no sonication). A 20% duty cycle appeared to be optimal. With this cycle, a final ethanol concentration of 5.20 ± 0.68 g L−1 was obtained, or nearly 3.5-fold that of the control fermentation. Sonication at 10% and 20% cycles appeared to stimulate yeast growth compared to the control fermentation, but 40% duty cycle had a measureable adverse impact on cell growth. Sonication at 10% and 20% cycles enhanced both the extracellular and the intracellular levels of β-galactosidase enzyme. Although at the highest duty cycle sonication reduced cell growth, cell viability remained at ≥70% during most of the fermentation. Sonication at a controlled temperature can be used to substantially enhance productivity of bioethanol fermentations.
Keywords: Sonobioreactors; Ultrasound; Kluyveromyces marxianus; β-galactosidase; Bioethanol; Ethanol; Fermentation;

► Elementary mode analysis was used for micro-aerobic PDO production by Klebsiella pneumoniae. ► PP pathway and TCA cycle was the most efficient for generating reducing equivalent. ► An endogenous HOR gene was cloned from K. pneumoniae ACCC10082 and overexpressed. ► The activities of HOR and total PDO dehydrogenase increased 5.8-fold and 1.1-fold. ► Increased carbon flux flowed from glycerol to PDO and to PP pathway provided coenzyme.Coenzyme limitation is one of the most important issues for 1,3-propanediol (PDO) production. Elementary mode analysis indicated that pentose phosphate pathway and TCA cycle were the most efficient pathways for generating reducing equivalent NADPH and NADH. Under the optimal condition for PDO production, 0.542 mol NADPH/(mol glycerol), accounting for 61.7% of the total reducing equivalent would be produced, which requires the fast conversion of NADPH for PDO synthesis. Based on the above analysis, an endogenous NADPH-dependent alcohol dehydrogenase (HOR) was cloned and overexpressed for NADPH usage in Klebsiella pneumoniae ACCC10082. The activities of HOR and total 1,3-propanediol dehydrogenase (PDOR) increased 5.8-fold and 1.1-fold than that of the wild type strain. In the fed-batch fermentation, the PDO concentration and yield of the constructed strain increased 10.4% and 9.4% while the highest 3-hydroxypropionaldehyde accumulation reduced 35.1% compared with that of the wild type strain. Metabolic flux analysis suggested that the increase of PDO yield was due to the enhanced carbon flux flowed to pentose phosphate pathway which provided coenzyme for HOR utilization. This work is helpful for the further understanding of PDO metabolism in K. pneumoniae and also useful for the strain improvement of PDO production.
Keywords: Metabolic pathway analysis; Elementary mode analysis; HOR; 3-Hydroxypropionaldehyde; 1,3-Propanediol; Overxpression;

Production of recombinant EGFP via surface display of ice nucleation protein and self-cleavage intein by Jiun-Yan Wu; Tsung-Yu Tsai; Tzu-Tsen Liu; Chia-Chi Lin; Jiann-Hwa Chen; Shih-Chun Yang; Chwen-Jen Shieh; Yung-Chuan Liu (158-163).
Display Omitted► In this study we develop a novel recombinant EGFP production approach. ► We construct a plasmid with INP, INT and the EGFP genes. ► The protein chimera of INP–INT–EGFP can be induced in Escherichia coli cell surface. ► By holding the cell pellets in Tris-buffer, EGFP is released from cell surface. ► EGFP is harvested via centrifugation, no cell disruption is needed.In this study, a novel recombinant protein production system was developed by constructing a protein chimera with a self-cleavage segment along with a cell surface display segment to finally produce a model protein entity (i.e., enhanced green fluorescent protein, EGFP). In the plasmid construction, the EGFP gene was fused to genes of a self-cleaving intein (INT) and an ice nucleation protein (INP) which can anchor on the cell membrane. In the cultivation of recombinant Escherichia coli, the cells expressed high green florescence after the addition of isopropyl-β-d-thiogalactopyranoside (IPTG) as the inducer. By simply holding the cell pellets in Tris–HCl (pH 10.0) buffer at room temperature, EGFP was solubilized from the INP–INT segment embedded on the cell surface via intein's self-cleavage function. The EGFP concentration of 273 mg/L and recovery of 88% were obtained at day 5, whereas the best EGFP productivity of 187 mg/L/d was obtained at day 1. The EGFP can be harvested only via centrifugation, and no cell disruption process is required. This simplified approach is expected to be applicable for obtaining recombinant functional proteins for academic and industrial use.
Keywords: Recombinant protein production; Surface display; Ice nucleation protein; Intein; Enhanced green fluorescent protein; Protein purification;

A three-dimensional discrete lattice-based system for modeling the growth of aerial hyphae of filamentous fungi on solid surfaces: A tool for investigating micro-scale phenomena in solid-state fermentation by Juliana Hey Coradin; Aline Braun; Graciele Viccini; Luiz Fernando de Lima Luz Jr; Nadia Krieger; David Alexander Mitchell (164-171).
► We developed a model for describing the growth of aerial hyphae of filamentous fungi. ► The model is lattice-based but has a flexibility similar to lattice-free models. ► The model can describe experimental profiles for the density of aerial hyphae. ► It can be used to explore micro-scale phenomena in solid-state fermentation.In solid-state fermentation systems, the growth of aerial hyphae into the interparticle spaces increases the pressure drop through forcefully aerated beds. Aerial hyphae can also bind particles into agglomerates, restricting the transfer of O2 to the particle surfaces. Despite these important effects, to date relatively little effort has been made to understand the growth patterns of these hyphae. In the current work we present a discrete lattice-based model that can be used to simulate the growth of the aerial hyphae of filamentous fungi. In the model, the elongation of hyphae involves the successive addition of 10 μm cubes, with random numbers being used to choose the direction of growth. The model was able to describe profiles available in the literature for the density of the aerial hyphae, as a function of height above the surface, for a situation in which the filamentous fungus Rhizopus oligosporus was grown on potato dextrose agar. The model can be modified to describe various different situations involving the growth of filamentous fungi in solid-state fermentation systems, such as the growth of penetrative hyphae and the growth of hyphae within the wet mycelial layer that often forms at the surfaces of particles. It therefore represents a useful tool for investigating phenomena that occur at the micro-scale in solid-state fermentation systems.
Keywords: Solid-state fermentation; Rhizopus oligosporus; Filamentous fungi; Discrete model; Lattice-based model; Aerial hyphae;

► The oxidative stress in Blakeslea trispora in a bubble column reactor was studied. ► Enhanced aeration caused high oxidative stress in the fungus. ► The high oxidative stress increased significantly the production of carotenes.The oxidative stress and the morphological changes in Blakeslea trispora induced by enhanced aeration during carotene production in a bubble column reactor was investigated. Enhanced aeration caused changes of the morphology of microorganism from aggregates with large projected area to aggregates with small projected area. This morphological differentiation of the fungus was associated with high oxidative stress as evidenced by increase of the specific activities of superoxide dismutase (SOD) and catalase (CAT). The oxidative stress in B. trispora resulted in a significant increase in carotene production. The highest concentration of carotenes (55.0 ± 2.5 mg/g dry biomass) was obtained at aeration rate of 4 vvm. Very high oxidative stress in B. trispora caused a change in the biosynthesis of carotenes resulting an increase in γ-carotene concentration. The maximum proportion of β-carotene (91.68%), γ-carotene (44.67%), and lycopene (11.54% of total carotenes) was observed at aeration rates of 4, 5, and 5 vvm, respectively. The external addition of butylated hydroxytoluene (BHT) and hydrogen peroxide in the medium did not improve the production of carotenes.
Keywords: Carotenes; Blakeslea trispora; Aeration rate; Oxidative stress; Bubble column reactor;

► Chemical and biological characterizations of sludge thermal treatment (≤100 °C). ► Highlight the use of flow cytometry to investigate cell lysis during heat treatment. ► Low thermal treatment induces cell lysis and a partial floc destructuration. ► Such treatment conditions do not enhance total biogas production. ► Low thermal treatment (≤100 °C) is enough to improve global biogas production rate.Although many information is currently available about sludge minimization processes in wastewater treatment plant (WWTP), few data are available about their fundamental mechanisms especially microbial changes. In order to clarify the relationship between sludge reduction efficiency and both chemical and biological modifications, the effects of thermal treatment on activated sludge were investigated by combining the monitoring of cell lysis using flow cytometry (FCM), organic matter solubilization, floc structure and biodegradability. For the maximal temperature (95 °C) applied, COD, proteins, HLS and sugars solubilization degrees reached 12.4 (±1.3)%, 18.6 (±1.8)%, 9.6 (±1)% and 7.4 (±1.9)%, respectively, showing clearly the transfer of organic matter from the particulate to the soluble fraction of the sludge. The results from FCM analysis showed that thermal treatment induces a progressive cell lysis when increasing temperature from 50 to 95 °C. However, the impact on floc structure seemed to be limited as floc destructuration was limited to the temperature elevation at 50 °C. Above 50 °C floc size distribution remained almost constant. The results from biodegradability tests did not show any improvement of the intrinsic biodegradability after the thermal treatment. Only the increase of the specific digestion rate was observed. This study which provides a complete investigation of chemical, physical and biological effects of thermal treatment allows a better knowledge of fundamental mechanisms involved during heat treatment to improve sludge reduction processes.
Keywords: Sludge disintegration; Thermal treatment; Solubilization; Cell lysis; Biodegradability;

Batch culture of Acidithiobacillus caldus on tetrathionate by D.W. Shiers; D.E. Ralph; H.R. Watling (185-191).
► Growth of At. caldus on a defined tetrathionate medium is reported. ► Growth is accompanied by the formation and consumption of polythionates ► No trithionate was detected amongst the polythionates ► An analysis of the efficiency of conversion of substrate into biomass is attempted. Acidithiobacillus caldus (DSM 8584) grew aerobically in minimal medium at 45 °C with potassium tetrathionate as the sole energy source. Oxidation of tetrathionate during batch culture involved the production of sulfite, thiosulfate, penta- and hexathionate which were then consumed after the tetrathionate was exhausted. Average growth yields over the batch were 3.5 g(dry wt.) mol(S4O6)−1, somewhat less than yields reported for continuous growth on the same substrate. Thiosulfate was unstable under sterile culture conditions and reacted spontaneously to give tetra-, penta- and hexathionate. It is suggested that the occurrence of polythionates during growth of A. caldus on tetrathionate is due to formation of thiosulfate as the first step in tetrathionate oxidation. Observed growth yields were compared with a thermodynamic framework which suggested a growth efficiency of ca. 10%. The pattern of growth yield and thermodynamic analysis suggest the formation of elemental sulfur although this was not observed.
Keywords: Acidithiobacillus caldus; Tetrathionate oxidation; Cell yields; Polythionates;

Extractive fermentation of l-(+)-lactic acid by Pediococcus pentosaceus using electrodeionization (EDI) technique by Pailin Boontawan; Sunthorn Kanchanathawee; Apichat Boontawan (192-199).
► EDI technique promotes lactic acid production yield. ► The in situ lactate removal results in reduction of deactivaton constant. ► This membrane bioreactor system significantly reduced impurities.In this work, extractive fermentation of l-(+)-lactic acid (lactate) was carried out using an electrodeionization (EDI) technique. Pediococcus pentosaceus was used as the biocatalyst because of its homo-fermentative characteristic. The effect of initial lactate concentrations on microbial growth was initially investigated. A mathematical simulation of the product inhibition was successfully illustrated. It was found that the critical lactate concentration was approximately 80 g/L. Various operating conditions were investigated to assess the EDI performance. The system was subsequently applied for in situ removal of lactate from the fermentation broth. The specific deactivation constant (k d) was substantially reduced from 0.026 h−1 to 0.0054 h−1 resulting in a much improved half life of the biocatalyst. The highest lactate concentration in the receiving solution was 185 g/L.
Keywords: Electrodeionization; l-(+)-lactic acid; Extractive fermentation; Product inhibition;

CADLIVE Converter for constructing a biochemical network map by Kentaro Inoue; Sayaka Tomeda; Shinpei Tonami; Yuki Shimokawa; Masayo Ono; Hiroyuki Kurata (200-206).
► We develop the CADLIVE Converter as a new extension of our CADLIVE system. ► Efficient, direct connection of various databases to CADLIVE. ► Easy handling of complex, heterogeneous network data. ► High efficiency for data integration, pathway analysis and dynamic modeling. ► Fast layout and visualization of an integrated map.In systems biotechnology biochemical network maps are the essential bases for analyzing their topological features and for simulating their dynamic behaviors. Most of network data are available at public databases, relating to gene expression, protein–protein interaction, and metabolic pathways. On the other hand, since those databases use a variety of data formats, computational tools always require the conversion of such database formats into their available format. In this work, we developed a GUI-based WWW browser program, which converts different databases (KEGG, RegulonDB, DIP, etc.) into the format available to our CADLIVE system, integrates a variety of network data into a fully functional pathway map, and visualizes the integrated map. This program is named the CADLIVE Converter. We demonstrated the feasibility and availability of it by performing metabolic pathway analysis and dynamic modeling. The visualization of the network map lead to an intuitive understanding of topological architecture of complex networks.
Keywords: Bioinformatics; Systems biotechnology; Software; Simulator; Network; Integration; Design;

Downstream processing accounts for a large proportion of the manufacturing cost of biopharmaceutical products. One of the several approaches to improve the process economics include reducing the amount of contaminants and micronised cell debris in the product stream that is presented to the secondary purification steps. We have reported a detailed study on the use of hydrodynamic cavitation and its distinctive capacity to release periplasmic products with higher selectivity compared to other conventional mechanical methods of cell disruption. In the current study, the influence of orifice geometry on selective release of periplasmic proteins is presented. The amount of shear in the system is varied through changing the geometry of the orifice plate, in terms of shape, size and number of orifices used for generating cavities. For a constant intensity of cavitation, of the various shapes studied circular orifice that permits higher number of jet stream from the orifice plates and thus higher shear was found to be advantageous in releasing periplasmic products selectively from E. coli.
Keywords: Cell disruption; Hydrodynamic cavitation; Selective release; Orifice geometry;