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Biochemical Engineering Journal (v.58-59, #)
Glycerol utilisation for the production of chemicals: Conversion to succinic acid, a combined experimental and computational study by Anestis Vlysidis; Michael Binns; Colin Webb; Constantinos Theodoropoulos (pp. 1-11).
► We explore the bioconversion of biodiesel byproduct glycerol to succinic acid (SA). ► We use A. succinogenes and pure glycerol in batch lab-scale and bench-top bioreactors. ► We obtain yield and final SA concentration of 1.23g-SA/g-GLR and 29.3g-SA/L. ► We develop a model of lab-scale experiments considering substrate/product inhibition. ► We validate the model, by simulating fermentations in bench-top reactors.An effective method for the valorisation of the main by-product of biodiesel production, i.e. glycerol is investigated in this work. It involves the biological conversion of glycerol to succinic acid, a top added-value material, which can be used as a building block for the production of various commodity and specialty chemicals. Our aim is to give new insights into this bioprocess, which has so far received little attention and is open for further investigation, through a combination of experimental and computational studies. The microorganism employed here was Actinobacillus succinogenes in batch bioreactors where glycerol was used as the sole carbon source.The highest obtained product yield, final succinate concentration and productivity were found to be equal to 1.23g-succinate/g-glycerol, 29.3g-succinate/L and 0.27g-succinate/L/h, respectively. Furthermore, an unstructured model of the batch experiments was developed by considering both substrate and product inhibition. Kinetic parameters of the model were estimated by minimising the difference between experimental and predicted values. The corresponding optimisation problem was solved by using a combination of stochastic and deterministic methodologies, with the goal to probabilistically compute global minima and the resulting parameter values. The model developed can be utilised to successfully predict the concentration profiles of the five most important state variables (biomass, glycerol, succinic acid, formic acid, and acetic acid) with different initial glycerol concentrations. Scaled-up experiments in larger-scale bioreactors were used for further validation purposes. Our model can be further used to compute optimal operating/parametric conditions, which maximise yield, productivity and/or the final succinic acid concentration.
Keywords: Abbreviations; USP glycerol; pure glycerol according to the specifications of United States Pharmacopeia; PHB; polyhydroxybutyrate a polymer belonging to polyesters class; US DOE; United States Department of Energy; SARs; Small Anaerobic Reactors; ATCC; American type culture collection; vvm; volume gas/volume liquid/minute; UV; ultra violet; RI; Refractive Index; OD; 660; optical density at 660; nm; GLR; glycerol; DCW; dry cell weight; TSB; trypticase soy broth; ODEs; ordinary differential equations; SQP; Successive Quadratic Programming (deterministic optimisation method); SA; succinic acid; FA; formic acid; AA; acetic acid; PEP; phosphoenolpyruvate (branch intermediate in the glycerol metabolism to succinate); HPLC; High Performance (Pressure) Liquid Chromatography; Abs; absorbanceGlycerol; Fermentation; Succinic acid; Actinobacillus succinogenes; Kinetic model; Biodiesel
In situ pulse respirometric methods for the estimation of kinetic and stoichiometric parameters in aerobic microbial communities by Catarina S. Oliveira; Alberto Ordaz; Eugénio C. Ferreira; Madalena Alves; Frédéric Thalasso (pp. 12-19).
► In situ estimation of stoichiometric and kinetic parameters. ► Comparison of chemostat and respirometric method. ► Comparison of ASM fitting and increasing substrate concentration pulses method. In situ pulse respirometry was applied in an activated sludge bubble column treating synthetic wastewater for the estimation of the (i) maximum specific oxygen consumption rate, (ii) substrate affinity constant, (iii) biomass growth yield, (iv) maintenance coefficient, and (v) specific endogenous respiration rate. Parameters obtained from respirometry were compared to those obtained by the chemostat method, based on substrate and biomass measurements, under several dilution rates. The low sensitivity of substrate measurement methods and the difficulties of sampling heterogeneous biomass suspension are critical issues limiting the applicability of the chemostat method. Additionally, the extensive time consuming nature of this method allows concluding that chemostat method presents several disadvantages in comparison with in situ pulse respirometric techniques. Parameters were obtained from respirograms by fitting ASM1 and ASM3 models, and from experiments performed by injecting pulses of increasing substrate concentration. The injection of pulses of increasing concentration was the most adequate method, with several advantages such as a simpler experimental data interpretation, and results with better confidence.Considering the assessment and comparison of the experimental and calculation methods presented, it is recommended that the estimation of kinetic and stoichiometric parameters in mixed aerobic cultures should preferentially be performed by using in situ respirometric techniques.
Keywords: Aerobic processes; Kinetic parameters; Heterotrophes; ASM; Pirt; In situ; respirometry; Chemostat
Effects of microwave irradiation on triglyceride transesterification: Experimental and theoretical studies by Yusuke Asakuma; Yosuke Ogawa; Kouji Maeda; Keisuke Fukui; Hidetoshi Kuramochi (pp. 20-24).
Display Omitted► Preheating and maintaining a continuous temperature using microwave irradiation improve the reactivity of triolein. ► The improved reaction rate can be explained through the use of optimized structures, dipole moments and activation energies obtained using molecular orbital calculations. ► Conformational isomer formed by microwave irradiation shows stronger vibration around the carboxyl carbon.An improved reaction rate of biodiesel fuel formation was observed under microwave irradiation. Such irradiation is effective for not only heating during the reaction but also preheating. The advantages observed in this study suggest that a continuous process, that is, the introduction of reactant preheated by microwave irradiation into reactor will be feasible. The reasons for the improved reaction rate are explained through the use of optimized structures, dipole moments, IR spectra and activation energies obtained using molecular orbital calculations. Planar triolein formed by microwave irradiation has higher reactivity, a lower dipole moment, a lower activation energy and stronger vibration around the carboxyl carbon, and is more reactive than triolein with a higher dipole moment.
Keywords: Biodiesel fuel; Microwave; Transesterification; Molecular orbital calculations; FT-IR; Activation energy
High-level production of uricase containing keto functional groups for site-specific PEGylation by Hai Chen; Yingfei Lu; Zhengzhi Fang; Jingxian Liu; Hong Tian; Xiangdong Gao; Wenbing Yao (pp. 25-32).
. A p15A-based single-plasmid system compatible with most E. coli expression vectors and strains was developed for incorporating p-acetylphenylalanine into uricase at the desired sites with high efficiency and fidelity. Under optimized expression conditions, 24mg/L mutant uricase was produced, corresponding to 40% of the yield of wild-type uricase. Full-length mutant uricase was only produced in our optimized system when all three orthogonal components were present. This efficient system provides reactive handles for a rational PEGylation to manipulate uricase structure and function and will be beneficial for enhancing the incorporation of other unnatural amino acids into proteins.Display Omitted► A suppressor tRNA was optimized to highly improve the efficiency of pAcF incorporation. ► The efficiency was further enhanced by optimizing suppressor tRNA/aaRS copy number. ► The suppressor tRNA/aaRS pair had no adverse effects on the growth of host cells. ► The versatility of pAC- pAcF produced high-yield of mutant uricase with full activity. ► The keto groups provide reactive handles for a rational PEGylation of uricase.We describe an E. coli-based optimized system for the production of uricase with keto functional groups incorporated efficiently and site-specifically. In the process, the orthogonal suppressor tRNA/aminoacyl-tRNA synthetase (aaRS) pair specific for p-acetylphenylalanine ( pAcF) was optimized to be effective at pAcF incorporation, showing no toxicity to the host cells. The efficiency of pAcF incorporation was further improved by coupling five copies of the T-stem mutant suppressor tRNA gene omitted the 3′ terminal CCA with two constitutive copies of the D286R mutant aaRS gene in a single-plasmid construct. To assay the utility of the optimized system, we incorporated pAcF in response to three independent amber nonsense codons (Lys21TAG, Phe170TAG, Lys248TAG) into uricase. Under optimized expression conditions, 24mg/L mutant uricase was produced, corresponding to 40% of the yield of wild-type uricase (UOXWT). The desired specificity for incorporation of pAcF into uricase was confirmed. Kinetic measurements and spectroscopic study performed by CD did not show any relevant differences in the substrate affinity, the catalytic activity and protein secondary structure between native and mutant uricase. Additionally, the mutant uricase was site-specifically modified with methoxy-PEG-oxyamine (mPEG5K-ONH2). This efficient system provides reactive handles for a rational PEGylation to manipulate uricase structure and function and will be beneficial for enhancing the incorporation of other unnatural amino acids into proteins.
Keywords: Orthogonal tRNA/aminoacyl-tRNA synthetase; Optimization; Fermentation; Enzyme production; Site-specific PEGylation; Recombinant DNA
A comparison of the kinetic properties of free and immobilized Aspergillus oryzae β-galactosidase by Fernanda F. Freitas; Líbia D.S. Marquez; Gustavo P. Ribeiro; Gabriela C. Brandão; Vicelma L. Cardoso; Eloízio J. Ribeiro (pp. 33-38).
► We compare the properties of free and immobilized lactase from Aspergillus oryzae. ► Lactase was entrapped in alginate–gelatin beads and stabilized with glutaraldehyde. ► Temperature and pH effects on the enzymatic activity and kinetics were compared. ► Free and immobilized enzyme presented competitive inhibition by galactose. ► The immobilized enzyme presented a low resistance to substrate transfer.The objective of this work was to compare the properties of free and immobilized β-galactosidase ( Aspergillus oryzae), entrapped in alginate–gelatin beads and cross-linked with glutaraldehyde. The free and immobilized forms of the enzyme showed no decrease in enzyme activity when incubated in buffer solutions in pH ranges of 4.5–7.0. The kinetics of lactose hydrolysis by the free and immobilized enzymes were studied at maximum substrate concentrations of 90g/L and 140g/L, respectively, a temperature of 35°C and a pH of 4.5. The Michaelis–Menten model with competitive inhibition by galactose fit the experimental results for both forms. The Km and Vm values of the free enzyme were 52.13±2.8mM and 2.56±0.3gglucose/Lminmgenzyme, respectively, and were 60.30±3.3mM and 1032.07±51.6glactose/minm3catalyst, respectively, for the immobilized form. The maximum enzymatic activity of the soluble form of β-galactosidase was obtained at pH 4.5 and 55°C. Alternatively, the immobilized form was most active at pH 5.0 at 60°C. The free and immobilized enzymes presented activation energies of 6.90±0.5kcal/mol and 7.7±0.7kcal/mol, respectively, which suggested that the immobilized enzyme possessed a lower resistance to substrate transfer.
Keywords: Immobilized enzymes; β-galactosidase; Alginate; Glucose; Kinetic parameters; Lactose
Operation of a fixed-bed bioreactor in batch and fed-batch modes for production of inulinase by solid-state fermentation by Viviane Astolfi; Jaderson Joris; Ricardo Verlindo; J. Vladimir Oliveira; Francisco Maugeri; Marcio A. Mazutti; Debora de Oliveira; Helen Treichel (pp. 39-49).
► Evaluation of the performance of inulinases production by SSF using strategies of batch and fed-batch fermentation (FBF). ► Inulinase production in bioreactors pilot scale production and kinetic behavior of the process in terms of temperature and CO2 production. ► The partial characterization of the crude enzymatic extract obtained by FBF was also carried out.This work is focused on the inulinase production by solid-state fermentation (SSF) in a fixed-bed reactor (34cm diameter and 50cm height) with working capacity of 2-kg of dry substrate operated in batch and fed-batch modes. It was investigated different strategies for feeding the inlet air in the bioreactor (saturated and unsaturated air) as alternative to remove the metabolic heat generated during the microbial growth by evaporative cooling. The kinetic evaluation of the process carried out in batch mode using unsaturated air showed that the evaporative cooling decreasing the mean temperature of the solid-bed, although the enzyme production was lower than that obtained using saturated air. Results showed that maximum enzyme activity (586±63Ugds−1) was obtained in the fed-batch mode using saturated air after 24h of fermentation. The enzymatic extract obtained by fed-batch mode was characterized and presented optimum temperature and pH in the range of 52–57°C and 4.8–5.2, respectively. For a temperature range from 40 to 70°C the enzyme presented decimal reduction time, D-value, ranging from 5748 to 47h, respectively. For a pH range from 3.5 to 5.5 the enzyme showed good stability, presenting D-values higher than 2622h. In terms of Michaelis–Mentem parameters were demonstrated that the crude inulinase activity presented higher affinity for substrate sucrose compared to inulin.
Keywords: Fed-batch fermentation; Agroindustrial residues; Kluyveromyces marxianus; NRRL Y-7571; Inulinase; Partial characterization; Solid-state fermentation
Mathematical modeling of biological sulfide removal in a fed batch bioreactor by Aliakbar Roosta; Abdolhossein Jahanmiri; Dariush Mowla; Ali Niazi (pp. 50-56).
Display Omitted► Biological removal of hydrogen sulfide was studied in a fed batch bioreactor. ► A mathematical model was developed for this system using obtained experimental data. ► Rate of sulfur production ( r1) is independent of DO value except at very low DO. ► Rate of elemental sulfur oxidation into sulfate ( r2) increases with increase of DO value. ► Increase of HS− load leads to increase of elemental sulfur selectivity.In this study, biological sulfide removal is investigated in a fed batch bioreactor. In this process, sulfide is converted into elemental sulfur particles as an intermediate in the oxidation of hydrogen sulfide to sulfate. The main product is sulfur at low dissolved oxygen or at high sulfide concentrations and also more sulfates are produced at high dissolved oxygen. According to the carried out reactions, a mathematical model is developed. The model parameters are estimated and the model is validated by comparing with some experimental data. The results show that, the proposed model is in a good agreement with experimental data. According to the experimental result and mathematical model, sulfate and sulfur selectivity are sensitive to the concentration of dissolved oxygen. For sulfide concentration 0.2 (mM) in the bioreactor and dissolved oxygen of 0.5ppm, only 10% of sulfide load is converted to sulfate, while it is 60% at the same sulfide concentration and dissolved oxygen of 4.5ppm. At high sulfide load to the bioreactor, the concentration of uneliminated sulfide increases; it leads to more sulfur particle selectivity and consequently, less sulfate selectivity.
Keywords: Bioreactors; Dissolved oxygen; Dynamic modeling; Fed-batch culture; Biological sulfide removal; Thiobacillus thioparus
Development and characterization of a polyampholyte-based reactor immobilizing soybean seed coat peroxidase for analytical applications in a flow system by Lisandro R. Denaday; M. Victoria Miranda; Rosa M. Torres Sánchez; Juan M. Lázaro Martínez; Lucía V. Lombardo Lupano; Viviana Campo Dall’ Orto (pp. 57-68).
► A polyampholyte was tested as support for a non-purified enzyme. ► The polyampholyte can be obtained by a one-step synthetic strategy. ► Immobilized soybean seed coat peroxidase was used for analytical purposes. ► Kinetic parameters and diffusional restrictions were evaluated for the flow reactor. ► The reactor based on peroxidase was used for food discrimination.The aim of this work was the development of a reusable enzymatic reactor based on soybean peroxidase for analytical purposes in a flow system. The innovation consisted in the application of a polyampholyte as a support for the immobilization of the enzyme extracted from soybean seed coat, without previous purification.A polyampholyte with high loading capacity for proteins was tested as a sorbent, reaching a maximum adsorption capacity ( q0) of 0.10mgg−1 and an equilibrium dissociation constant ( K d) of 1.3×10−8M.The catalytic activities of free and immobilized enzyme were first compared in batch. The Michaelis constant ( K m) for phenol at saturating H2O2 decreased upon immobilization.The reactor was then coupled to an amperometric detector in a flow system. The effects of the immobilization on kinetics and equilibrium parameters were evaluated using catechol and H2O2 as substrates. The values for the apparent Michaelis constant for catechol (K′m) were higher than the intrinsic parameter of the immobilized enzyme ( K m, 0.08mM) due to external mass-transport limitations. Internal diffusional restrictions were absent since the normalized Eadie–Hofstee plots did not exhibit sigmoidal curves.The reactor was tested as an analytical tool for food analysis. The enzymatic activity was stable over two months.
Keywords: Polyampholyte; Peroxidase; Enzymatic reactor; Kinetic parameters; Red wines; Bioadsorption
Evolution of bioaggregate strength during aerobic granular sludge formation by Junfeng Wan; Irene Mozo; Ahlem Filali; Alain Liné; Yolaine Bessière; Mathieu Spérandio (pp. 69-78).
► High SAV and low settling time are not necessary to produce granules. ► Size of conventional sludge flocs is linked to the turbulence Kolmogorov microscale. ► First step of granules formation is an increase of cohesion and a decrease of SVI. ► When no hydraulic selection is performed, bimodal particle size distribution is observed. ► High energy dissipation on mature granules leads to surface detachment (50μm).This work investigated the modification of aggregate properties during the formation of granular sludge in a sequencing batch airlift reactor (SBAR). The cohesion of biological aggregates was quantified by subjecting sludge samples to two different controlled shear stresses in a stirred reactor. For reference sludge (without granules), flocs broke and reformed easily, indicating that floc size was controlled by the turbulence micro-scale (Kolmogorov scale, here from 17μm to 62μm). In contrast, granules showed high strength which enabled them to resist turbulence and their size was no longer imposed by the Kolmogorov micro-scale. Different steps were observed during the granulation process: a first increase of aggregate cohesion associated with a decrease in sludge volume index (SVI), a growth of aggregates with detachment of fragile particles from the surface and, finally, an increase in the sizes of small and large granules to reach a pseudo-stable size distribution. Results suggest that small particles could have formed the seeds for new granules, as they were maintained in the bioreactor. Here, granular sludge was formed in an SBAR with a conventional settling time (30min), i.e. without particle washout, and with a low superficial air velocity (SAV=0.6cms−1): it is thus demonstrated that high SAV and low settling time are not necessary to produce granules, but probably only accelerate the accumulation of granules. It is shown that the increase of cohesion is the initial phenomenon explaining the granule formation concomitantly with bacterial aggregates densification. It seems important, in the future, to investigate the reasons for this cohesion increase, which is possibly explained either by bacterial bounding interactions or the excretion of extracellular polymeric substances (EPS).
Keywords: Aerobic granular sludge; Aggregate strength; Shear; Cohesion
Comparison of reactive porous media for sulfur-oxidizing denitrification of high nitrate strength wastewater by Grace M. Nisola; Mark C.F.R. Redillas; Eulsaeng Cho; Midoek Han; Namjong Yoo; Wook-Jin Chung (pp. 79-86).
► Porous sulfur-oxidizing denitrification media were synthesized via thermal fusion. ► Synthesized media provided sulfur and alkalinity sources. ► Media properties influenced denitrification performances. ► Macrovoidal media structure promoted biomass attachment and substrate supply. ► Porous sulfur/crushed oyster shell was the most effective media for denitrification.Three packing materials for sulfur oxidizing denitrification packed bed systems seeded with acclimated anoxic sludge were evaluated. Two porous media were prepared via thermal fusion with sodium bicarbonate as porogen: sulfur fused with powdered (1) calcium carbonate (CaCO3) (SCa) and (2) oyster shell (SCr). Randomly packed sulfur and limestone granules (S+L) media were used as the control. Results revealed that SCr is the most suitable media as it exhibited the highest nitrate removals and lowest nitrite accumulation. It has macrovoidal pores which facilitated microbial attachment. Additionally, SCr had the highest CaCO3 loading per unit volume and highest media dissolution rate which was favorable to avert pH decrease. But due to high denitrification activity, high sulfate levels in SCr may necessitate a post-treatment step prior to effluent discharge. Due to poor biomass attachment, S+L is most sensitive to change in fluid flow condition. As hydraulic retention time is decreased, S+L exhibited intensive and irreversible performance decline. Inferior denitrification performance of SCa was mainly due to low CaCO3 loading per unit volume, low dissolution kinetics and low alkalinity consumption by denitrifiers. Using modified Stover–Kincannon kinetic model, overall performance and denitrification capacities can be arranged as SCr>S+L>SCa.
Keywords: Limestone; Denitrification; Nitrate removal; Sulfur oxidation; Packed bed; Oyster shells
Regime analysis of a Baeyer–Villiger bioconversion in a three-phase (air–water–ionic liquid) stirred tank bioreactor by R. Melgarejo-Torres; D. Torres-Martínez; M. Gutiérrez-Rojas; A. Gómez de Jesús; G.J. Lye; S. Huerta-Ochoa (pp. 87-95).
► We conduct a regime analysis on a three-phase stirred tank bioreactor. ► We studied a bioconversion process using [MeBuPyrr][BTA] as the dispersed phase. ► Regime analysis was based on characteristic times of different mechanisms involved. ► Oxygen transfer rate under certain operating conditions could be a limiting step.The aim of this work was to conduct a regime analysis on a three-phase (air–water–ionic liquid) stirred tank bioreactor of the Baeyer–Villiger bioconversion process, using [MeBuPyrr][BTA] ionic liquid as the dispersed phase. The regime analysis based on characteristic times of the different mechanisms involved (mixing, mass transfer, reaction) can yield a quantitative estimate of bioreactor performance. The characteristic time obtained for oxygen uptake rate (54s−1) was among the characteristic times determined for oxygen transfer (13–129s−1) under different operating conditions, suggesting that the oxygen transfer rate under certain operating conditions could be a limiting step in the bioconversion process. Further enhancement of oxygen transfer rates requires proper selection of the bioreactor operational conditions, and improved design of the ionic liquid used as oxygen transfer vector.
Keywords: Regime analysis; Multiphase bioreactor; Ionic liquid; Bioconversion; Mass transfer; Oxygen transfer
Fermentation strategies for the production of lipase by an indigenous isolate Burkholderia sp. C20 by Chien-Hung Liu; Chun-Yen Chen; Yao-Wen Wang; Jo-Shu Chang (pp. 96-102).
► Fermentation technology was developed to produce lipase from indigenous isolate Burkholderia sp. C20. ► Adjusting the aeration rate from 0.5 to 2vvm led to an increase in the overall lipase productivity from 0.057 to 0.076U/(mlh), which was further improved to 0.09U/(mlh) by adjusting the agitation speed to 100rpm. ► Production of Burkholderia lipase followed mixed growth-associated kinetics with a yield coefficient of 524U/g-dry-cell-weight. ► Stepwise addition of carbon substrate was effective in enhancing lipase production in the fermentor. Burkholderia sp. C20 strain isolated from food wastes produces a lipase with hydrolytic activities towards olive oil. Fermentation strategies for efficient production of this Burkholderia lipase were developed using a 5-L bench top bioreactor. Critical factors affecting the fermentative lipase production were examined, including pH, aeration rate, agitation rate, and incubation time. Adjusting the aeration rate from 0.5 to 2vvm gave an increase in the overall lipase productivity from 0.057 to 0.076U/(mlh), which was further improved to 0.09U/(mlh) by adjusting the agitation speed to 100rpm. The production of Burkholderia lipase followed mixed growth-associated kinetics with a yield coefficient of 524U/g-dry-cell-weight. The pH optimum for cell growth and lipase production was different at 7.0 and 6.0, respectively. Furthermore, stepwise addition of carbon substrate (i.e., olive oil) enhanced lipase production in both flask and bioreactor experiments.
Keywords: Bioreactor design; Burkholderia; sp.; Fermentation; Lipase; Olive oil; Product kinetics
Enhanced phenolic antioxidants production in submerged cultures of Inonotus obliquus in a ground corn stover medium by Xiangqun Xu; Jinwei Zhu (pp. 103-109).
► Extra- and intra-cellular phenols production and antioxidant activity by I. obliquus. ► Effects of lignocellulosic material (corn stover) in shake flask and fermenter cultures. ► A significant increase in phenols production by lignocellulose decomposition. ► Enhanced antioxidant activity of phenols by lignocellulose decomposition.The medicinal mushroom Inonotus obliquus has been a folk remedy for a long time in East-European and Asian countries. It is currently ascribed to a number of phenolic compounds as well as triterpenoids and polysaccharides responsible for significant biological and pharmacological properties. A study was conducted to determine the effects of inclusion of lignocellulosic material, in this case corn stover on production and antioxidant activity of extracellular (EPC) and intracellular phenolic compounds (IPC) by Inonotus obliquus in submerged fermentation. The corn stover medium contained 3% ground corn stover and 3.5% corn flour but the control medium contained 5% corn flour without corn stover. All of the other components were same in the two media. Decomposition rates of cellulose, hemicellulose, and lignin in the corn stover substrate were 20.9%, 17.9%, and 19.8% through 288h of submerged cultivation. Lignocellulose decomposition in the corn stover-containing medium yielded significantly higher EPC (118.9/135.7mg GAE (gallic acid equivalents)) and IPC (21.2/23.7mg GAE) than in the control medium (34.7/42.5mg GAE of EPC and 12.5/13.5mg GAE of IPC) per liter of culture broth (EPC) and per gram of mycelia (IPC) in shake flask cultures/10L fermenter runs. Both EPC and IPC from the corn stover medium showed a higher scavenging activity against hydroxyl radicals and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals than those from the control medium during the later fermentation period. In dose-dependent experiments, EPC from the corn stover medium at 216h demonstrated a significantly stronger free radical scavenger activity against DPPH and hydroxyl radicals, shown as much lower IC50 values, than that from the control medium and IPC from the two media.
Keywords: Fermentation; Inonotus obliquus; Microbial growth; Optimisation; Phenolic compounds; Submerged culture
Experimental and theoretical analysis of a novel deep-bed solid-state bioreactor for cellulolytic enzymes production by Khushal Brijwani; Praveen V. Vadlani; Keith L. Hohn; Dirk E. Maier (pp. 110-123).
► A novel deep bed solid-state bioreactor was designed and fabricated. ► Performance of bioreactor was evaluated during cellulolytic enzymes production. ► Two-phase heat and mass transfer model was developed. ► Model showed better agreement with observed experimental data. ► Model explicitly predicted the bioreactor performance in various scenarios.A novel deep-bed solid-state bioreactor was designed and fabricated for cellulolytic enzymes production using mixed fungal cultures. Better temperature and moisture control was achieved through a unique bioreactor design comprising an outer wire-mesh frame with internal air distribution along with near-saturation conditions within the cabinet. Without airflow through the internal distributors, maximum temperatures of 48°C and 52°C were observed during half- and full-capacity operation. These were reduced to 44°C and 43°C on resumption of airflow. In terms of cellulolytic enzyme production, no significant differences occurred in filter paper activity with depth in half-capacity operation; however, in full-capacity operation, top-level filter paper activity (5.39FPU/g-solids) was significantly different from middle- and bottom-level activity. Top level beta-glucosidase, endocellulase, and xylanase activities were significantly ( P<0.05) different from middle and bottom levels in both half- and full-capacity operation. A two-phase coupled heat and mass transfer model was developed that predicted the experimental trends reasonably well. Model predictions confirmed that cabinet temperature of 30°C and distributor airflow rate of 3.42kgh−1 during operation enabled effective temperature control.
Keywords: Trichoderma reesei; Aspergillus oryzae; Solid-state bioreactor; Cellulolytic enzymes; Heat and mass transfer; N-tank in series model
Enhanced production of periplasmic interferon alpha-2b by Escherichia coli using ion-exchange resin for in situ removal of acetate in the culture by Joo Shun Tan; Ramakrishnan Nagasundara Ramanan; Tau Chuan Ling; Mustafa Shuhaimi; Arbakariya B. Ariff (pp. 124-132).
Display Omitted► Application of anion-exchange resins to remove acetate in fermentation broth. ► Increasing anion-exchange resin will inhibit the growth of E. coli. ► Increasing anion-exchange resin will increase aggregation of inclusion bodies. ► Optimal concentration of anion-exchange resins maximized growth and production.The possibility of using in situ addition of anion-exchange resin for the removal of acetate in the culture aimed at improving growth of E. coli and expression of periplasmic human interferon-α2b (PrIFN-α2b) was studied in shake flask culture and stirred tank bioreactor. Different types of anion-exchange resin were evaluated and the concentration of anion-exchange resin was optimized using response surface methodology. The addition of anion-exchange resins reduced acetate accumulation in the culture, which in turn, improved growth of E. coli and enhanced PrIFN-α2b expression. The presence of anion-exchange resins did not influence the physiology of the cells. The weak base anion-exchange resins, which have higher affinity towards acetate, yielded higher PrIFN-α2b expression as compared to strong anion-exchange resins. High concentrations of anion-exchange resin showed inhibitory effect towards growth of E. coli as well as the expression of PrIFN-α2b. The maximum yield of PrIFN-α2b in shake flask culture (501.8μg/L) and stirred tank bioreactor (578.8μg/L) was obtained at ion exchange resin (WA 30) concentration of 12.2g/L. The production of PrIFN-α2b in stirred tank bioreactor with the addition of ion exchange resin was about 1.8-fold higher than that obtained in fermentation without ion exchange resin (318.4μg/L).
Keywords: Acetic acid; Adsorption; Anion-exchange resins; Bioreactors; Fermentation; Periplasmic interferon-alpha2b
On-line monitoring of recombinant bacterial cultures using multi-wavelength fluorescence spectroscopy by Garima Jain; Guhan Jayaraman; Öznur Kökpinar; Ursula Rinas; Bernd Hitzmann (pp. 133-139).
► We present results from the production of recombinant hFGF-2 by Escherichia coli. ► PLS models are presented to estimate biomass, glucose and CPR. ► Extrapolation of PLS models possible. ► Changes in the fluorescence spectra are presented depending on growth rate. ► Growth rate influences concentration of flavin-like molecules and NAD(P)H.Multi-wavelength fluorescence spectroscopy was evaluated as a tool for on-line monitoring of recombinant Escherichia coli cultivations expressing human basic fibroblast growth factor (hFGF-2). The data sets for the various combinations of the excitation and emission spectra from batch cultivations were analyzed using principal component analysis. Chemometric models (the partial least squares method) were developed for correlating the fluorescence data and the experimentally measured variables such as the biomass and glucose concentrations as well as the carbon dioxide production rate. Excellent correlations were obtained for these variables for the calibration cultivations. The predictability of these models was further tested in batch and fed-batch cultivations. The batch cultivations were well predicted by the PLS models for biomass, glucose concentrations and carbon dioxide production rate (RMSEPs were respectively 5%, 7%, 9%). However, when tested for biomass concentrations in fed-batch cultivations (with final biomass three times higher than the highest calibration data) the models had good predictability at high growth rates (RMSEPs were 3% and 4%, respectively for uninduced and induced fed-batch cultivations), which was as good as for the batch cultivations used for developing the models (RMSEPs were 3% and 5%, respectively for uninduced and induced batch cultivations). The fed-batch cultivations performed at low growth rates exhibited much higher fluorescence for fluorophores such as flavin and NAD(P)H as compared to fed-batch cultivations at high growth rate. Therefore, the PLS models tended to over-predict the biomass concentrations at low growth rates. Obviously the cells changed their concentration of biogenic fluorophores depending on the growth rate. Although multi-wavelength fluorescence spectroscopy is a valuable tool for on-line monitoring of bioprocess, care must be taken to re-calibrate the PLS models at different growth rates to improve the accuracy of predictions.
Keywords: Fluorescence spectroscopy; On-line monitoring; Escherichia coli; Cultivation; Chemometrics; Human basic fibroblast growth factor
Effect of cryopreservation and microencapsulation of lactic acid bacterium Enterococcus faecium MC13 for long-term storage by Paulraj Kanmani; R. Satish Kumar; N. Yuvaraj; K.A. Paari; V. Pattukumar; Venkatesan Arul (pp. 140-147).
Display Omitted► This is the first report on the preservation of intestinal probiotic strain Enterococcus faecium MC13 from fish ( Mugil cephalus). ► Various cryoprotective sugars and milk products were used as cryoprotectants for the preservation of probiotic E. faecium MC13. ► Maximum relative cell viability was obtained when cells were freeze dried with trehalose. ► Microencapsulation technique was used to improve the survival of probiotic cells during the course of long term storage and simulated gastric and intestinal fluid. ► In vivo analysis revealed that alginate–chitosan capsules facilitate the direct release of probiotic cells into the intestinal tract.The aim of this work was to investigate the effect of cryoprotectants on the survival of probiotic bacterium Enterococcus faecium MC13 during freeze drying and storage. The maximum relative cell viabilities were observed when cells were freeze dried and stored at −20°C, which is optimum temperature for the preservation of E. faecium. At all storage temperatures, trehalose was found to be retaining the highest relative cell viability than other cryoprotectants. In addition, alginate–chitosan capsules were produced to encapsulate E. faecium with the aim of enhancing survival of probiotic cells and keeping the probiotic during exposure to the harsh gastro-intestinal conditions. Encapsulation of probiotic into alginate–chitosan capsules found to be retaining higher survival of probiotic cells (4.342±0.26LogCFUmL−1) at −20°C for six months. Microencapsulated cells were resistant to simulated gastric (pH 2.0) and intestinal fluids (pH 7.5), resulting in significantly enhanced survival when compared with free cells. During in vivo treatment, capsules were broken and probiotic cells were directly released into the intestinal tract of rat. This result showed that microencapsulation of E. faecium MC13 with alginate and a chitosan coating offers an effective means of delivery of viable cells to the colon and maintains their survival during the adverse gastro-intestinal conditions.
Keywords: Enterococcus faecium; MC13; Freeze drying; Microencapsulation; Preservation; Probiotics; Survival
Electrochemical detection of xanthine in fish meat by xanthine oxidase immobilized on carboxylated multiwalled carbon nanotubes/polyaniline composite film by Rooma Devi; Sandeep Yadav; C.S. Pundir (pp. 148-153).
A commercial xanthine oxidase (XOD) was immobilized covalently onto carboxylated multiwalled carbon nanotubes (c-MWCNT) and polyaniline (PANI) composite film electrodeposited on the surface of a Pt electrode, using N-ethyl-N′-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) chemistry. The optimized xanthine biosensor showed linear response range of 0.6–58μM, with a detection limit of 0.6μM (S/N=3), and a correlation coefficient of 0.98.A commercial xanthine oxidase (XOD) was immobilized covalently onto carboxylated multiwalled carbon nanotubes (c-MWCNT) and polyaniline (PANI) composite film electrodeposited on the surface of a Pt electrode, using N-ethyl-N′-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) chemistry. A xanthine biosensor was fabricated using XOD/c-MWCNT/PANI/Pt electrode as a working electrode, Ag/AgCl (3M KCl) as standard electrode and Pt wire as auxiliary electrode connected through a potentiostat. The enzyme electrode was characterized by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectrophotometry and electrochemical impedance spectroscopy (EIS). The biosensor showed optimum response within 4s at pH 7.0 and 35°C, when polarized at 0.4V. The optimized xanthine biosensor showed linear response range of 0.6–58μM, with a detection limit of 0.6μM (S/N=3), and a correlation coefficient of 0.98. The biosensor was applied to determine xanthine in fish meat. The biosensor lost 50% of its initial activity after its 200 uses over a period of 100 days.
Keywords: Xanthine; Xanthine oxidase; Electrochemical biosensor; Carboxylated multiwalled carbon nanotubes; Polyaniline; Fish meat
Evaluation of a pilot-scale biotrickling filter as a VOC control technology for the plastic coating sector by F.J. Álvarez-Hornos; C. Lafita; V. Martínez-Soria; J.M. Penya-Roja; M.C. Pérez; C. Gabaldón (pp. 154-161).
Display Omitted► Emissions from coating process have been successfully treated using a BTF pilot unit. ► The EBRT to meet legal regulations depends on the paint-solvent used in the process. ► The BTF has been shown economically competitive in comparison with thermal treatment. ► A reduction of 66% in CO2 emissions has been estimated. ► The evolution of the microbial population was carried out by FISH.The performance and feasibility of a biotrickling filter (BTF) pilot unit for the treatment of exhaust gases from two robotic spray paint booths at a plastic coating facility were investigated. The volatile organic compound (VOC) concentrations in the emissions of the exhaust gases from the paint booths were relatively stable, although the VOC composition depended on the applied solvent-paint formulation in the booths. The pilot plant was operated for one year at empty bed residence times (EBRTs) ranging from 30 to 93s. The performance of the system was affected by the solvent-paint formulations. An EBRT between 30 and 40s was enough to meet legal requirements for products containing more than 60% biodegradable compounds, whereas a minimum EBRT of 80s was required for emissions mainly composed of hydrophobic VOCs. The dynamics of the microbial population was carried out by fluorescence in situ hybridisation (FISH), indicating a high microbial diversity with composition changes associated with the solvent-paint used. The feasibility of the BTF was evaluated, showing that this technology is economically and environmentally competitive in comparison with thermal treatment technology.
Keywords: Biodegradation; Biofilters; Fluorescence in situ hybridisation; Volatile organic compounds
Influence of particle size and chemical composition on the performance and kinetics of anaerobic digestion process of sunflower oil cake in batch mode by M.A. De la Rubia; V. Fernández-Cegrí; F. Raposo; R. Borja (pp. 162-167).
► We sieve Sunflower Oil Cake to analyze three different particles sizes. ► We analyze the effect of particle size and chemical composition on biogas production. ► Higher particle size, with low lignin content, increases biogas production. ► We model the effect of particle size on methane yield.Biochemical methane potential (BMP) assays of sunflower oil cake (SuOC) were carried out to research the effect of different particle sizes and their chemical composition on methane yields and kinetics. Particle size ranges of (1) 0.355–0.55mm, (2) 0.710–1.0mm and (3) 1.4–2.0mm in diameter were evaluated. The highest methane yield 213±8mLCH4g−1 VSadded was obtained for the largest particle size analyzed (3), against 186±6mLCH4g−1 VSadded obtained for particles 1 and 2. This may be attributed to the different lignocellulose compositions of the various particle size ranges studied and to organic matter removals (47.2% for 3, against ∼41.5% for 1 and 2). The evolution of propionic acid concentration was found to be fundamental for explaining the lowest rate of biogas production for the smallest (1) particle size studied, with a specific rate constant k of 0.45±0.02d−1, while values of 0.61±0.02d−1 and 0.50±0.01d−1 were obtained for particles 2 and 3, respectively.
Keywords: Batch assay; Biochemical methane potential (BMP); Kinetics; Lignocellulose; Particle size; Sunflower oil cake (SuOC)
Wastewater treatment systems harbor specific and diverse yeast communities by Qingxiang Yang; Florent E. Angly; Zhe Wang; Hao Zhang (pp. 168-176).
Display Omitted► We detected the diversity of yeast in three full-scale wastewater treatment systems using PCR–DGGE and cultivation techniques. ► The yeast richness and diversity here were larger than reported for marine and sugar-rich soil habitats. ► Each wastewater system harbors specific and unique yeast community. ► Culturing yeasts is a powerful way to complement culture-independent approaches to study their diversity.Yeasts, as a group of single-celled fungi, are widely distributed in nature and play important roles in biotechnological applications. However, how the types of wastewater and the treatment processes influence yeast populations is not clear. In this study, both cultivation and culture-independent methods were used to investigate the distribution and diversity of yeasts in three typical full-scale plants processing biopharmaceutical, papermaking and municipal wastewater. Cultivable yeasts were very abundant ranging from 102 to 105cfug−1 sludge, and highly diverse with 48 taxons belonging to 21 different genera, thus exceeding the yeast richness reported for marine and sugar-rich soil habitats. Genera Rhodotorula, Candida, Trichosporon, Pichia and some unidentified Ascomycetes were the most frequent populations cultivated. However, the compositions of yeast community structures in the plants were dissimilar and were shaped primarily by the type of wastewater treated but also by processing conditions. Culturing yeasts is a powerful way to complement culture-independent approaches to study their diversity, since significant more yeast species, especially quite a few possible novel species were recorded and isolated by cultivation method.
Keywords: Yeast; Wastewater treatment; Diversity; Distribution; Bioreactors; Molecular imprinting
Converting crude glycerol to 1,3-propandiol using resting and immobilized Klebsiella sp. HE-2 cells by Chiao-Ling Wong; Chien-Chang Huang; Wen-Ming Chen; Jo-Shu Chang (pp. 177-183).
► Efficient 1,3-PDO fermentation technology was developed with indigenous Klebsieblla sp. ► Resting-cell operation with optimal medium and culture conditions enhanced 1,3-PDO production. ► Using immobilized cells greatly improved the operational stability and reusability. ► Crude glycerol from biodiesel manufacture plant could be converted to 1,3-PDO efficiently.In this study, 1,3-propanediol (1,3-PDO) was produced from crude glycerol through the fermentation of resting and immobilized cells of a Klebsieblla sp. HE-2 strain isolated from a hydrogen producing anaerobic sludge collected in Southern Taiwan. The Klebsieblla sp. HE-2 cells were first grown on a fermentation medium (FM medium). The medium was then switched to resting-cell medium (RC medium) tailored to improve the production of 1,3-PDO. Using a glycerol-amended FM medium, the soluble metabolites consisted of 1,3-PDO, 2,3-butanediol, and ethanol and byproducts (such as acetic acid and lactic acid) at a content of 18, 28, 49, and 5% (of total soluble metabolites), respectively. When the culture was transferred from the FM medium to the RC medium, the concentration of 1,3-PDO was doubled from 5g/L to 10g/L. Using immobilized cells of Klebsieblla sp. HE-2 greatly improved the operational stability and reusability of the cells, as the immobilized cells could be used for 6 cycles without significant activity loss. The immobilized cells were able to directly utilize non-pretreated crude glycerol obtained from a local biodiesel manufacturing plant for 1,3-PDO production with an efficiency comparable to that obtained from using pure glycerol.
Keywords: Glycerol; Immobilised cells; Growth kinetics; Bioconversion; Klebsiella; sp.; 1,3-Propanediol
Unstructured model for free and immobilized cell culture without pH control of Bifidobacterium animalis subsp. lactis Bb 12—Inhibitory effect of the undissociated organic acids by Hasan Jalili; Béatrice Balannec; Hadi Razavi; Abdeltif Amrane (pp. 184-188).
► A model was developed to describe growth and organic acids production of B. animalis. ► The Verlhust and a modified Luedeking–Piret models were considered. ► An additional term involving the undissociated form of the acids was introduced. ► Data recorded during free and immobilized cell cultures were satisfactory described. ► The part of lactic and acetic acids produced can be calculated from production data.A model was developed to describe growth and organic acids production of Bifidobacterium animalis growing without pH control in free and immobilized cell culture. The Verlhust model was considered for growth, and to account for the inhibition observed at acidic pH, the Luedeking–Piret production model was modified by introducing an additional term involving the undissociated form of the organic acids, acetic and lactic acids, the main inhibitory species. To describe the relationship between pH and both the dissociated and the undissociated forms of organic acids, the Henderson–Hasselbach equation was considered. The model was found to satisfactory describe experimental growth and production data recorded during free and immobilized cell cultures. The part of each acid produced can be deduced from the calculated production data, since a constant lactic to acetic acid mass ratio was found, 1.29 and 1.66 during free and immobilized cell cultures. Owing to the acidic pH values recorded, 4.43 at lowest, higher amounts of undissociated acetic acid were produced, leading to a higher inhibitory effect of this acid if compared to lactic acid.
Keywords: Bifidobacterium; Batch cultures; Kinetic models; Organic acids; Product inhibition