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Biochemical Engineering Journal (v.65, #)
Biosynthesis of ethyl butyrate by immobilized recombinant Rhizopus oryzae lipase expressed in Pichia pastoris by Marina Guillén; Maria Dolors Benaiges; Francisco Valero (pp. 1-9).
► Recombinant Rhizopus oryzae lipase (rROL) catalyzes the synthesis of ethyl butyrate. ► Determination of the influence of different parameters on synthesis reaction. ► rROL immobilized on EP100 showed the highest initial synthesis rate. ► rROL immobilized on Sepabeads showed the best operational stability.A recombinant Rhizopus oryzae lipase expressed in Pichia pastoris has been immobilized in three different kinds of supports: EP100, Eupergit®CM and Octadecyl-Sepabeads. These immobilized derivatives have been used to catalyze the synthesis of ethyl butyrate. The effect of different parameters on initial reaction rate and yield has been studied for each biocatalyst. The variables selected for the study were: temperature, water content, shaking speed, enzyme loading, type of solvent and substrates molar ratio. The results showed the EP100 derivative as the best choice in terms of initial rate and yield but, when the residual activity as well as the reutilization capacity was studied, Octadecyl-Sepabeads exhibits the highest stability, so it was finally chosen as the best biocatalyst.
Keywords: Pichia pastoris; Rhizopus oryzae; Recombinant lipase; Flavor synthesis; Ethyl butyrate; Immobilized lipase
Utilisation of waste bread for fermentative succinic acid production by Cho Chark Joe Leung; Anaxagoras Siu Yeung Cheung; Andrew Yan-Zhu Zhang; Koon Fung Lam; Carol Sze Ki Lin (pp. 10-15).
► This paper presents a novel process to convert waste bread to succinic acid. ► Enzymatic hydrolysis yielded 0.47g glucose/g bread at 100% starch conversion. ► Enzymatic hydrolysis yielded 2.55mg FAN/g waste bread. ► Bacterial fermentation on the hydrolysate yielded 1.16g succinic acid/g glucose. ► This is the highest substrate to succinic acid yield among other food-derived media.A novel biorefinery concept of utilising waste bread as a sole nutrient source for the production of a nutrient rich feedstock for the fermentative succinic acid production by Actinobacillus succinogenes has been developed. Waste bread was used in the solid-state fermentations of Aspergillus awamori and Aspergillus oryzae that produce enzyme complexes rich in amylolytic and proteolytic enzymes, respectively. The resulting fermentation solids were added directly to a bread suspension to generate a hydrolysate containing over 100g/L glucose and 490mg/L free amino nitrogen (FAN). A first-order kinetic model was used to describe the effect of initial bread mass ratio on glucose and FAN profiles. The bread hydrolysate was used as the sole feedstock for A. succinogenes fermentations, which led to the production of 47.3g/L succinic acid with a yield and productivity of 1.16g SA/g glucose and 1.12g/Lh. This corresponds to an overall yield of 0.55g succinic acid per g bread. This is the highest succinic acid yield compared from other food waste-derived media reported to date. The proposed process could be potentially utilised to transform no-value food waste into succinic acid, one of the future platform chemicals of a sustainable chemical industry.
Keywords: Aspergillus awamori; Aspergillus oryzae; Actinobacillus succinogenes; Biorefinery; Food waste
A self-assembled nano-cluster complex based on cytochrome c and nafion: An efficient nanostructured peroxidase by Jun Hong; Wei Wang; Kun Huang; Wei-Yun Yang; Ying-Xue Zhao; Bao-Lin Xiao; Yun-Fei Gao; Zainab Moosavi-Movahedi; Shahin Ahmadian; Mousa Bohlooli; Ali Akbar Saboury; Hedayatollah Ghourchian; Nader Sheibani; Ali Akbar Moosavi-Movahedi (pp. 16-22).
► Highly efficient nanostructured peroxidase. ► Self-assembled nano-cluster complex. ► 39% catalytic efficiency of native HRP. ► Good stability to high concentration of H2O2.A nano-cluster complex with highly efficient peroxide activity was constructed by self-assembly of nafion (NF) and cytochrome c (Cyt c) in 50mM phosphate buffer at 25°C. The UV–vis spectrometry, circular dichroism (CD) and transmission electron microscopy (TEM) methods were utilized for additional characterization of the nanostructured enzyme or artificial peroxidase (AP). The nano-cluster was composed of Chain-Ball structure, with an average ball size of approximately 40nm detected by TEM method. The Michaelis–Menten ( Km) and catalytic rate ( kcat) constants of the AP were obtained in 50mM phosphate buffer solution at pH 7.0 which are equal to 2.5±0.4μM and 0.069±0.001s−1 respectively. The catalytic efficiency of AP was evaluated to be 0.028±0.005μM−1s−1, which was 39±5% as efficient as the native HRP. The AP generated here can be used in place of HRP with high stability and activity.
Keywords: Cytochrome; c; Nafion; Nano-cluster; Nanostructured peroxidase; Self-assembled; Highly efficient
Is the CANON reactor an alternative for nitrogen removal from pre-treated swine slurry? by Mónica Figueroa; Jose Ramón Vázquez-Padín; Anuska Mosquera-Corral; Jose Luis Campos; Ramón Méndez (pp. 23-29).
► CANON system is a viable alternative for nitrogen removal from pre-treated swine slurry. ► Nitrogen removal rates of 0.46kgN/(m3d) were treated working at 20°C. ► Organic matter in the wastewater did not affect the performance of the CANON reactor. ► Anammox, ammonia oxidizing and heterotrophic bacteria supported the process.The use of the completely autotrophic nitrogen removal over nitrite (CANON) process as a post-treatment for nitrogen removal from anaerobically pre-treated swine slurry is studied in the present work. The ammonium removal, under oxygen-limited conditions, in a system with anammox bacteria mainly in the form of granules and aerobic ammonium oxidizing bacteria mainly as dispersed biomass was researched in an air pulsing sequencing batch reactor operated at room temperature. The achieved nitrogen removal rate was of 0.46kgN/(m3d) treating 300mg NH4+-N/L with values of nitrogen removal efficiencies around 75%.The presence of slowly or non-biodegradable organic matter (from 260 to 45mgCOD/L) did not affect the operation of the process. By means of the FISH technique, Nitrosomonas were detected as the majority of ammonia oxidizing bacteria in the sample, and Candidatus “ Brocadia fulgida” and Candidatus “ Brocadia anammoxidans” as the anammox bacteria.The comparison of this aerobic process with other post-treatments for effluents from anaerobic digesters showed that the CANON process is a promising alternative to remove nitrogen from effluents generated in pig farms.
Keywords: Anammox; CANON; Nitrogen removal; Swine slurry; Pulsing sequencing batch reactor
Microbial conversion of biodiesel byproduct glycerol to triacylglycerols by oleaginous yeast Rhodosporidium toruloides and the individual effect of some impurities on lipid production by Jingyang Xu; Xuebing Zhao; Wencong Wang; Wei Du; Dehua Liu (pp. 30-36).
► Crude glycerol from biodiesel industry as carbon source for microbial lipid production. ► Rhodosporidium toruloides could well convert crude glycerol to triacylglycerols. ► The impurities of the crude glycerol exerted positive effects on lipid production.With the development of biodiesel industry, the byproduct glycerol will become a considerable resource as feedstock for production of many other chemicals. In present work, microbial conversion of crude glycerol to triacylglycerols (microbial lipid) was proposed and investigated using the oleaginous yeast Rhodosporidium toruloides ( R. toruloides) by one-stage batch fermentation. Compared with glucose and refined glycerol, the crude glycerol could obtain significantly higher biomass concentration and lipid yield. The highest biomass concentration of R. toruloides obtained from crude glycerol in a 5L fermenter reached 26.7g/L with an intracellular lipid content of 70%. Further study was performed to investigate the individual effect of five representative compounds which were present in crude glycerol as impurities. It was found that within the general concentration ranges, only methanol displayed somewhat inhibitive effect, while others showed positive influence on lipid production. These results indicated that crude glycerol could be directly converted to triacylglycerols by R. toruloides without purification. Contrarily, certain amount of salt and soap could promote the accumulation of biomass and lipid.
Keywords: Batch processing; Bioconversion; Glycerol; Yeast; Microbial lipid; Biodiesel
Enhanced production of astaxanthin in Paracoccus sp. strain N-81106 by using random mutagenesis and genetic engineering by Teruhiko Ide; Megumi Hoya; Toru Tanaka; Shigeaki Harayama (pp. 37-43).
► We improved the productivity of astaxanthin in Paracoccus sp. strain by a combination of (i) random mutagenesis and (ii) gene cloning/overexpression of astaxanthin-biosynthetic genes. ► The recombinant strain produced astaxanthin at 58mg/l—56 times the production of the original strain. ► The yield was further increased by fed-batch fermentation to reach 480mg/l culture fluid.Astaxanthin is the most powerful antioxidant of all carotenoids and has great commercial value for use in the aquacultural, pharmaceutical, and food industries. The marine bacterium Paracoccus sp. strain N-81106 is one of the natural producers of astaxanthin, but its astaxanthin productivity is too low for economically feasible industrial production. We tried to improve the productivity of astaxanthin by this strain by a combination of (i) random mutagenesis and (ii) gene cloning/overexpression of astaxanthin-biosynthetic genes. In the first step, we isolated astaxanthin-overproducing mutants after random mutagenesis of strain N-81106. The mutants isolated, NG5 and NG9, showed greater production of astaxanthin than did the wild-type strain N-81106. The yield of astaxanthin in the mutants was about 17 times that in strain N-81106. In the second step, we cloned the astaxanthin-biosynthesis genes of strain N-81106 in Escherichia coli by using a broad-host-range vector, and we then mobilized the cloned genes from E. coli to strains N-81106, NG5, and NG9. The recombinant NG5 produced astaxanthin at 58mg/l—56 times the production of the original strain. The yield was further increased by fed-batch fermentation to reach 480mg/l culture fluid.
Keywords: Abbreviations; IPP; C; 5; isopentenyl pyrophosphate; NTG; N; -methyl-; N; ′-nitro-; N; -nitrosoguanidine; GGPP; geranylgeranyl diphosphate Paracoccus; Carotenoids; Biosynthesis; Fermentation; Fed-batch culture; Recombinant DNA
Reduction of bromate in a biological activated carbon filter under high bulk dissolved oxygen conditions and characterization of bromate-reducing isolates by Juan Liu; Jianwei Yu; Dong Li; Yu Zhang; Min Yang (pp. 44-50).
Display Omitted► A BAC filter showed significant removal of bromate in the presence of high DO. ► Rhodocyclaceae/ Comamonadaceae-related bacteria may play a role in BrO3− reduction. ► Two isolated strains exhibited BrO3− reducing ability in the presence of NO3−.A biological activated carbon (BAC) filter was constructed using BAC from a pilot system that exhibited the ability to reduce bromate (BrO3−) and two BrO3−-reducing bacteria were isolated and characterized. The BAC filter could almost completely reduce BrO3− (60μgBr/L) to bromide (Br−) at an influent dissolved oxygen (DO) level of approximately 8.0mg/L and an empty bed contact time of 30±2min using acetate as the electron donor shortly after the start-up. Phylogenetic analysis of the 16S rRNA gene sequences of a biological sample from the BAC filter showed that among the six detected orders, Rhodocyclales- and Burkholderiales-related microorganisms were dominant and Rhodocyclaceae- and Comamonadaceae-related microorganisms may play a role in BrO3− reduction. Two isolated pure cultures, i.e. Sphingomonas sp. 4721 and Deinococcus sp. 4710, exhibited the ability to reduce BrO3− in the presence of NO3−. The result of this study clearly indicated that DO was a competitor of BrO3− as an electron acceptor while NO3− was not. Construction of a BAC filter which could restrict oxygen transfer within a biofilm still remains to be a challenge.
Keywords: Biofilters; Bromate-reducing bacteria; Dissolved oxygen; Biofilms; Biotransformation; Characterization
PHB production by Methylocystis hirsuta from natural gas in a bubble column and a vertical loop bioreactor by Fereshteh Rahnama; Ebrahim Vasheghani-Farahani; Fatemeh Yazdian; Seyed Abbas Shojaosadati (pp. 51-56).
► Methylocystis hirsuta was used to produce PHB from natural gas in loop bioreactors. ► The effect of methane and nitrogen content on PHB production was determined. ► PHB production was found to be a growth associated metabolite. ► PHB content was 51.6% of cell dry weight in a VTLB.Biosynthesis of poly-3-hydroxybutyrate (PHB) by Methylocystis hirsuta from natural gas in two different media was studied for the first time. After selection of the suitable medium, the effect of two key parameters such as methane to air ratio and nitrogen content on the PHB production was determined in a bubble column bioreactor using a full factorial design. It was found that both of these factors had a significant effect on the PHB accumulation (42.5% w/w of cell dry weight). PHB production by M. hirsuta, unlike other methanotrophic bacteria, was found to be a growth associated metabolite. Subsequently, the PHB production was carried out in a forced-liquid vertical tubular loop bioreactor (VTLB) at optimum condition determined in the bubble column bioreactor. The PHB content of biomass was 51.6% w/w of cell dry weight (CDW) in the VTLB. These results indicated that the loop bioreactors specially are suitable candidates for PHB production from natural gas.
Keywords: Poly-3-hydroxybutyrate (PHB); Methylocystis hirsuta; Biosynthesis; Bioreactor; Bubble columns; Optimization
A kinetic and thermodynamic study of the lipase-catalyzed remote resolution of a chiral tertiary alcohol by Shi-Zhen Wang; Jian-Ping Wu; Gang Xu; Li-Rong Yang (pp. 57-62).
► Novozym 435 catalyzed remote asymmetric alcoholysis of a tertiary alcohol. ► The chiral discrimination was achieved four bonds away from the center. ► A kinetic model based on the reversible ping-pong bi-bi mechanism was proposed. ► The product inhibition and substrate inhibition were also considered. ► The kinetic and thermodynamic analysis were investigated using kinetic parameters.The kinetics and thermodynamics of Candida antarctica lipase B-catalyzed remote asymmetric alcoholysis of the citalopram intermediate 3-[(acetyloxy)methyl]-4-[4-(dimethylamino)-1-(4-fluorophenyl)-1-hydroxybutyl]-benzonitrile (diol monoacetate) have been studied. A kinetic model based on the reversible ping-pong bi-bi mechanism with competitive enantiomer substrates was proposed. The product inhibition by each diol enantiomer and the substrate inhibition by isobutyl alcohol were also considered. The diffusion limitation was proven to be negligible. By reducing the degree of freedom in parameter estimation by model discrimination and Haldane equations, 14 free parameters were successfully identified. The model parameters were simulated by the Matlab program using time–concentration curves of different diol monoacetate concentrations; the simulated values fit the experimental values well, with an average relative error of 9.6%. The reaction activity and enantioselectivity of C. antarctica lipase B toward the tertiary alcohol were investigated by kinetic and thermodynamic analysis using simulated kinetic parameters.
Keywords: Dynamic modeling; Kinetic parameters; Lipase; Immobilized enzymes; Tertiary alcohol; Asymmetric alcoholysis
Power input correlation to characterize the hydrodynamics of cylindrical orbitally shaken bioreactors by Wolf Klöckner; Stéphanie Tissot; Florian Wurm; Jochen Büchs (pp. 63-69).
► Introduction of a new power input correlation for disposable orbitally shaken bioreactors. ► Application validated using 200 L and 2000 L orbitally shaken bioreactors. ► Prediction of power input and hydrodynamics in cylindrical shaken bioreactors of all scales.Disposable cylindrical shaken bioreactors using plastic bags or vessels represent a promising alternative to stainless steel bioreactors, because they are flexible, cost-effective and can be pre-sterilized. Unlike conventional well-established steel bioreactors, however, such disposable bioreactor systems have not yet been precisely characterized. Thus, the aim of this current work is to introduce a new power input correlation as a potential means to characterize the hydrodynamics of these new systems. A set of relevant power input variables was defined and transformed into dimensionless numbers by using the Buckingham's π-Theorem. These numbers were then experimentally varied to quantify the relationship among the numbers. A simple correlation was generated for the power input with seven variables. The application of this new correlation was validated using 200 L and 2000 L orbitally shaken bioreactors. In conclusion, the proposed correlation is a useful tool to predict the power input and hydrodynamics during cell cultivation in cylindrical shaken bioreactors of all scales.
Keywords: Power input; Disposable bioreactors; Single use; orbitally shaken; Hydrodynamics
Review on production and medical applications of ɛ-polylysine by Swet Chand Shukla; Amit Singh; Anand Kumar Pandey; Abha Mishra (pp. 70-81).
Display Omitted► ɛ-Polylysine is naturally occurring biodegradable and nontoxic towards human. ► A comprehensive report on biosynthetic mechanism and molecular genetics of ɛ-PL production were discussed. ► Strain isolation, microbial production and purification of ɛ-PL were also discussed. ► ɛ-PL have various biomedical application like dietary agent, drug carriers, anticancer agent enhancer, biochip coatings.ɛ-Polylysine (ɛ-PL) is a homopolyamide linked by the peptide bond between the carboxylic and epsilon amino group of adjacent lysine molecules. It is naturally occurring biodegradable and nontoxic towards human. This review article gives an insight about the various ɛ-PL producing strains, their screening procedures, mechanism of synthesis, characterization, and its application in the medical field. The poly cationic nature of ɛ-PL at physiological pH makes it as one of the potential candidates in the field of drug delivery. Most of the biomedical applications till date use synthetic α-PLL as a raw material. However, it is believed that naturally occurring ɛ-PL would be an ideal substitute.
Keywords: Abbreviations; Pls; polylysine synthetase; NaSCN; sodium thiocynate; FTIR; Fourier transform infrared spectroscopy; NMR; nuclear magnetic resonance spectroscopy; MION; monocrystalline iron oxide nanoparticle; NPs; nanoparticles; IgM; immunoglobulin Mɛ-Polylysine; Homopolyamide; S. albulus Lysinopolymerus; Conjugate; Drug carrier; Targeting
Synthetic biology devices as tools for metabolic engineering by Eric Shiue; Kristala L.J. Prather (pp. 82-89).
► Synthetic biology devices may be useful tools for metabolic engineering. ► The widespread application of synthetic biology devices is hindered by the complexity of biology. ► Continued work with these devices may help elucidate design rules for future device engineering.Metabolic engineering focuses on controlling cellular metabolism and heterologous pathway flux to maximize the production of a product of interest. In recent years, various “devices” have begun to emerge from synthetic biology which could find widespread application in the field of metabolic engineering. In this review, we describe devices from synthetic biology and discuss the difficulties that may be encountered when using these devices as tools for metabolic engineering.
Keywords: Synthetic biology devices; Metabolic engineering; Process integration; Large scale cultivation; Sensors; Scale-up
Pectinase production from lemon peel pomace as support and carbon source in solid-state fermentation column-tray bioreactor by Héctor A. Ruiz; Rosa M. Rodríguez-Jasso; Raúl Rodríguez; Juan C. Contreras-Esquivel; Cristóbal N. Aguilar (pp. 90-95).
► Solid-state fermentation by Aspergillus niger Aa-20. ► Effect of particle size on growth and invasion ability by Aspergillus niger. ► Screening of different Aspergillus species for pectinase enzyme production. ► Pectinase production in a column-tray bioreactor.Pectinase is an important enzyme that finds application in many food processing industries and solid-state fermentation (SSF) is an attractive technology for enzyme production. In this work, a SSF process is described for the production of pectinase by Aspergillus niger Aa-20 and lemon peel pomace (LPP) as support and carbon source in a solid-state bioreactor. The process consists of three steps. (1) Selection of microorganism for SSF. Eight different fungal strains from the genus Aspergillus and Penicillium were screened for invasion ability on LPP; (2) Selection of particle size. Invasion ability of selected fungal strain was analyzed on four particle sizes of LPP; (3) SSF process was operated in a column-tray bioreactor at 30°C and 70% moisture content, 194mL/min of air flow rate and substrate particle size (2–0.7mm) of LPP for 96h. Results showed, that high levels of pectinase activities were obtained. The maximum pectinase activity obtained was 2181U/L. Maximum biomass and maximum specific growth rate of A. niger Aa-20 were Xmax=8mgglucosamine/g of LPP and μmax=0.1271/h. The LPP and the use of A. niger Aa-20 in SSF suggest as a very promising process for pectinase production.
Keywords: Solid-state fermentation; Bioreactor design; Pomace; A. niger; Pectinase
Non-supplemented aqueous extract from dry olive mill residue: A possible medium for fungal manganese peroxidase production by Inmaculada Sampedro; Alessandro D’Annibale; Federico Federici; Inmaculada Garcia Romera; Josè Antonio Siles; Maurizio Petruccioli (pp. 96-99).
► Aqueous dry olive mill residue is a valuable growth medium for MnP production. ► In a column reactor, Lentinus tigrinus and Phlebia sp. produced 43 and 39nkatalmL−1 MnP. ► MnP activities and productivities obtained in this study are very promising.The low moisture content of the dry olive mill residue (DOR), solid waste from the two-phase olive oil extraction process, enables its long-term storage and its ready reconstitution with water thus leading to a candidate liquid growth medium for the microbial production of enzymes of commercial interest. To test this hypothesis, two white-rot fungi (WRF), namely Lentinus tigrinus CBS 577.79 and Phlebia sp. DABAC 9, were grown in shaken cultures on non-supplemented aqueous extracts of the dry olive mill residue (ADOR) and compared for their ability to grow therein and produce Mn-dependent peroxidase (MnP). Then, in order to assess the feasibility of scale transfer to the reactor level, the two fungi were grown in a 3-L bubble-column reactor on 25% (w/v) ADOR at 28°C and an aeration rate of 0.3vvm. Under these conditions, L. tigrinus and Phlebia sp. produced 42.60±1.74 and 38.88±1.68 nkatal mL−1 MnP with respective mean volumetric productivities of 222.9 and 324.1nkatalL−1h−1. These results clearly show that the use of non-supplemented ADOR as a production medium enables a significant MnP production thus representing an excellent basis for process optimization.
Keywords: Dry olive mill residue; Aqueous extract; White-rot fungi; Submerged fermentations; Mn-dependent peroxidase