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Biochemical Engineering Journal (v.64, #)
pH-stat photoheterotrophic cultivation of indigenous Chlorella vulgaris ESP-31 for biomass and lipid production using acetic acid as the carbon source by Kuei-Ling Yeh; Chun-Yen Chen; Jo-Shu Chang (pp. 1-7).
► Effects of carbon sources on photoheterotrophic growth of a microalga were studied. ► pH control is a key factor affecting the performance of photoheterotrophic growth. ► pH-stat culture with acetic acid enhanced the lipid content and lipid productivity. ► Composition of the microalgal lipids is suitable for biodiesel production.The growth and lipid content of an isolated microalga Chlorella vulgaris ESP-31 were investigated under photoheterotrophic cultivation with different carbon sources (glucose, fructose, sucrose, glycerol, sodium acetate and acetic acid). In the absence of pH control, growing C. vulgaris ESP-31 on glucose obtained the highest biomass concentration (3.5g/L) and lipid content (26%). By controlling pH at 8.5, the growth on fructose and sodium acetate was improved, obtaining a biomass concentration of 3.2–3.6g/L and a lipid content of 24–25%. Moreover, a fed-batch operation with pH-stat feeding of acetic acid was employed to enhance biomass and lipid production. When the pH-stat culture was conducted at pH 7.0–7.5 with acetic acid feeding, the best photoheterotrophic growth performance was obtained, resulting in the highest biomass yield, lipid content, and lipid productivity of 0.68g/g CH3COOH, 50%, and 78mg/L/d, respectively. Regardless of the carbon sources used, the fatty acid profile of the microalgal lipid did not change significantly, as the lipid comprises over 60–80% of saturated fatty acids (mainly palmitic acid (C16:0) and stearic acid (C18:0)) and monounsaturated acids (mainly oleic acid (C18:1)). This lipid composition is suitable for the use in biodiesel synthesis.
Keywords: Microalgae; Fed-batch culture; Acetic acid; Bioreactors; Chlorella vulgaris; Photoheterotrophic growth
Stable expression of barley α-amylase in S. cerevisiae for conversion of starch into bioethanol by B. Liao; G.A. Hill; W.J. Roesler (pp. 8-16).
► An industrial strain of S. cerevisiae was engineered to stably secrete barley α-amylase 1. ► Multiple integration of the expression cassette into the yeast chromosome was achieved. ► The integrated genes show high mitotic stability during batch fermentation studies. ► The engineered yeast strain can ferment on raw wheat starch.An industrial strain of Saccharomyces cerevisiae, NRRL Y-132, was genetically engineered to stably secrete barley α-amylase by introducing an expression cassette containing the α-amylase cDNA and a dominant selectable marker into the ribosomal DNA loci of the yeast chromosome. Batch fermentation studies with this strain showed that the integrated expression cassette was mitotically stable for over 100 generations of continuous culture. In addition, mutation of cysteine 95 to alanine in barley α-amylase was found to increase the recombinant strain's starch hydrolyzing ability. The integrated strain showed higher starch hydrolyzing ability and ethanol production on both soluble and raw starch than the strain transformed with an episomal-based yeast expression plasmid. The results indicate that integration of the gene cassette into multiple copy loci should be given consideration when designing amylolytic yeast strains.
Keywords: Abbreviations; LB; Luria-Bertani; TB; terrific broth; YPD; yeast extract, peptone and dextrose; NTS; non-transcribed spacerBarley α-amylase; Multiple integration; Recombinant yeast; Fermentation; Bioethanol
Giant dextran particles formed by dextransucrase immobilized on a tube surface in a laminar flow by Yoichi Miyagawa; Hirokazu Seto; Keisuke Ohto; Hidetaka Kawakita (pp. 17-21).
► Dextransucrase was immobilized on to the surface of the tube in a laminar flow. ► Sucrose substrate solution was flown to the dextransucrase-immobilized tube to produce dextran. ► The produced dextran was colorized and the distribution along the tube length was determined.Complexation of dextran with the active sites of dextransucrase was used to produce dextran on a tube in a laminar flow. A dextransucrase solution was passed through a Teflon tube at flow rates of 10 and 50mL/h to immobilize the enzyme on the inner surface of the tube, and then sucrose solution was passed through the enzyme-immobilized tube to form giant dextran particles. The Reynolds number during the procedure was 4 or 20. The distribution of dextran along the flow direction in the tube was determined colorimetrically and showed that the dextran formed was distributed such that the amounts of dextran were higher near the ends than in the middle section of the tube. Direct observation of the dextran after colorization with phloxine showed that giant dextran particles, up to approximately 20μm in size, were formed. The distribution of dextran produced by the enzymatic reaction in a laminar flow was determined quantitatively.
Keywords: Dextran; Dextransucrase; Circular tube; Laminar flow
Modeling bacteriophage attachment using adsorption efficiency by Zachary J. Storms; Logan Smith; Dominic Sauvageau; David G. Cooper (pp. 22-29).
► Demonstrates robustness of adsorption efficiency model. ► Shows the applicability of adsorption efficiency model to different phages. ► Gives experimental support for assuming reversible step in adsorption is negligible. ► Demonstrates better fit of model compared to common sequential adsorption model. ► Shows limits to model, e.g. it does not apply to phages without side tail fibers adsorption.Typically, models of bacteriophage adsorption consider the process in two steps: reversible and irreversible attachment. In this study, a recently introduced one-step adsorption model, the adsorption efficiency model, is used to describe the adsorption of T-series bacteriophages to Escherichia coli. The adsorption efficiency model simplifies phage attachment to a single step: irreversible binding. The adsorption efficiency ( ɛ) is used to account for unadsorbed phages. The model accurately describes T-series phage adsorption (T2, T4, T5, T6, and T7) under a variety of conditions. In addition, the model is compared to a commonly used two-step adsorption model, the sequential model. Experimental data support the assumptions of the adsorption efficiency model and suggest that the reversible first step of T-series phage adsorption is equivalent to irreversible attachment under the conditions tested. The adsorption efficiency model was not appropriate for a phage λ strain lacking side tail fibers. However, the model did agree with data previously published for a strain of phage λ possessing side tail fibers, as is the case of all T-series strains tested. This suggests that the adsorption efficiency model applies to phages containing side tail fibers
Keywords: Bacteriophage; Adsorption; Adsorption efficiency; Modeling; Kinetic parameters; Dynamic simulation
Kinetics and equilibrium studies on removal of zinc(II) by untreated and anionic surfactant treated dead biomass of yeast: Batch and column mode by Devlina Das; Geetanjali Basak; Lakshmi V; Nilanjana Das (pp. 30-47).
► Biosorption of zinc(II) by two yeast species has been investigated. ► Remarkable increase of zinc(II) removal was noted by SDS treatment. ► Kinetics was predicted satisfactorily by fractional power model. ► Equilibrium data was analyzed using two, three and four parameter models. ► Column data was fitted in BDST model.The present study was carried out using dead biomass of isolated yeast species viz. Candida rugosa and Candida laurentii as biosorbents for the removal of Zn(II) from aqueous environment. C. rugosa and C. laurentii exhibited 65.4% and 54.8% removal of zinc at pH 6.0 in presence of 90mgL−1 Zn(II) at 30°C in batch system. Remarkable increase in Zn(II) removal was noted using dead yeast biomass treated with anionic surfactant sodium dodecyl sulphate (SDS) which was confirmed through SEM analysis. Kinetic studies based on various models were carried out and the results showed a very good compliance with the fractional power model. The experimental data were analyzed using two, three and four parameter isotherm models. The most appropriate equation for describing the isotherm profile was Freundlich model. The biosorbent performance was evaluated in column mode packed with SDS treated dead biomass of C. rugosa entrapped in sodium alginate beads. FT-IR analysis showed the involvement of –NH, –CO and –COOH functional groups in the binding of Zn(II) by yeast. The present study confirmed that immobilized SDS treated dead biomass of C. rugosa may serve as potential and eco-friendly biosorbent for removal of Zn(II) ions from aqueous solution.
Keywords: Biosorption; Zn(II); Kinetic parameters; Modelling; Wastewater treatment; Yeast
The elicited two-stage submerged cultivation of Antrodia cinnamomea for enhancing triterpenoids production and antitumor activity by Ching-Jen Liu; Chien-Chi Chiang; Been-Huang Chiang (pp. 48-54).
► Using the airlift reactor with dual net draft tube increased k L a and biomass content. ► Chitosan and CaCl2 as the chemical elicitation. ► Oxygen limitation and temperature fluctuation as physical elicitation. ► Elicitation by two-stage fermentation will increase triterpenoids content. ► The triterpenoids effectively inhibited proliferation of HeLa cells.A two-stage submerged fermentation with elicitation was used to increase triterpenoids production of Antrodia cinnamomea in an airlift bioreactor with dual net draft tube. The dual net draft tube bioreactor had higher k L a value (7.3h−1) than the solid tube bioreactor (5.5h−1). The fungus was cultured at 25°C for 12days, then elicited with chemicals and physical factors. It was found that comparing to calcium chloride, chitosan was a more potent chemical elicitor. At dosage level of 100mg/L, it not only resulted in more biomass production but also yielded higher crude triterpenoids production. The triterpenoids production could be significantly enhanced by means of the oxygen limitation and temperature fluctuation strategies. The maximum triterpenoids production could be achieved by using static culture (oxygen limitation) with temperature fluctuation between 25°C (for 10h) and 10°C (for 14h). The in vitro anticancer activity assay indicated that the crude triterpenoids produced by A. cinnamomea using the developed elicited two-stage cultivation protocol effectively inhibited proliferation of human cervical epithelioid carcinoma HeLa cells in a dosage dependent manner. The IC50 of the crude triterpenoids from the mycelium of A. cinnamomea after 21days cultivation for HeLa cells was 25μg/mL.
Keywords: Antrodia cinnamomea; Elicitation; Airlift bioreactors; Bioprocess design; Submerged culture; Viability
Purification and characterization of an organic solvent-stable lipase from Pseudomonas stutzeri LC2-8 and its application for efficient resolution of (R, S)-1-phenylethanol by Yan Cao; Yu Zhuang; Changjin Yao; Bin Wu; Bingfang He (pp. 55-60).
► An organic solvent-stable lipase from Pseudomonas stutzeri was purified. ► This is the first report on the characteristics of the lipase from P. stutzeri. ► Lipase LC2-8 showed high tolerance in the presence of various organic solvents. ► This lipase showed high chiral selectivity towards (R, S)-1-phenylethanol.An organic solvent-stable lipase from newly isolated solvent-tolerance bacterium Pseudomonas stutzeri LC2-8 was purified by acetone precipitation and anion exchange chromatography. The apparent molecular mass of the purified lipase was estimated by SDS-PAGE to be 32kDa. The open reading frame (ORF) of lipase LC2-8 encodes 311 amino acids with 287 amino acid residues in the mature lipase which shared 96% homology at the amino acid level with the putative lipase LipC from Pseudomonas stutzeri A1501. The optimum pH and temperature for lipase activity were 8.0 and 30°C, respectively. Its hydrolytic activity was found to be highest towards p-nitrophenyl caproate (C8). Lipase LC2-8 showed high tolerance in the presence of various organic solvents. Most of the hydrophilic solvents tested strikingly enhanced the activity and stability of lipase LC2-8. The half-life of lipase LC2-8 was extended to 10-fold in the presence of isopropanol, acetone, ethanol and methanol. The transesterification resolution of (R, S)-1-phenylethanol by lipase LC2-8 was carried out with the yield of 47.6%, the enantiomeric excess of residual substrate ( ees) was 99.9%, giving an E-value over 200. The solvent-stable lipase LC2-8 showed an attractive potency for application in biocatalysis in non-aqueous systems.
Keywords: Organic solvent-stability; Lipase; Purification; Pseudomonas stutzeri; Enantioseparation; Biocatalysis
In-depth characterization of Lactobacillus delbrueckii subsp. lactis 313 for growth and cell-envelope-associated proteinase production by Dominic Agyei; Michael K. Danquah (pp. 61-68).
► Anaerobic, rather than microaerophilic conditions, give high growth rate and cell biomass for Lactobacillus delbrueckii subsp. lactis 313 (LDL 313). ► Temperatures of 37–45°C and initial pH of 6 to 7 are optimum for growth and cell-envelope-associated proteinase (CEP) production. ► CEP activity profiles for bound and released CEP differ for anaerobic and microaerophilic cultures. ► LDL 313 produces CEPI type proteinase.The effect of process conditions on the growth and production of cell-envelope-associated proteinase (PrtL) by Lactobacillus delbrueckii subsp. lactis ATCC® 7830™ (LDL 313) was studied. Cell growth was profuse under the temperature conditions of 37°C, 40°C and 45°C, with cell growth rates, μmax increasing with temperatures. Initial culture pH of 6 displayed the highest PrtL yield of 2.33U. For each growth temperature the cell growth rates under anaerobic conditions were markedly higher than microaerophilic conditions, attributable to efficient sugar utilization under anaerobic fermentation. Overall, the proteolytic activities of cell-bound PrtL (bPrtL) were found to be higher than that of released PrtL (rPrtL). Higher rPrtL activities were displayed by anaerobic cultures over the entire fermentation period whereas the converse was true for bPrtL with microaerophilic cultures in the mid-late exponential phase displaying higher bPrtL activities than anaerobic cultures. Further, the proteinases had caseinolytic specificity for β-casein and κ-casein placing them in the cell-envelope-associated proteinases (CEPs) class I (CEPI) of the lactococcal CEPs grouping. This study provides insights into conditions for profuse growth and proteinase production by LDL 313 for subsequent technological applications in fermented foods, the dairy industry and bioactive peptide production.
Keywords: Process conditions; Microbial growth; Lactobacilli; Proteinase; Bioactive peptides
Isolation and characterization of phenol degrading bacteria immobilized onto cyclodextrin-hydrogel particles within a draft tube spouted bed bioreactor by Bárbara Safont; Ana Isabel Vitas; Francisco Javier Peñas (pp. 69-75).
► A draft tube spouted bed bioreactor with an hydrogel support for phenol removal. ► Comamonas acidovorans was dominant during high performance of spouted bed reactor. ► Maximum elimination capacity of 2.79kg-phenol/m3d in the spouted bed reactor. ► Phenol, system design, oxygen and intermediates as bacterial selection factors.A draft-tube spouted bed bioreactor was developed to investigate the microbial degradation of aqueous phenol using a cyclodextrin-based support material. Bacteria from activated sludge were acclimated to phenol in a continuous stirred tank bioreactor, and then immobilized onto the hydrogel particles within the spouted bed bioreactor. Microorganisms obtained under different operating conditions in both bioreactors were isolated and characterized. Batch phenol degradation assays performed on isolated dominant strains showed that Acinetobacter baumannii was the most resistant to phenol. Microbial population distribution in bioreactors was not only affected by phenol concentration, but also by oxygen availability, the system configuration and the presence of intermediates formed during phenol metabolization. A maximum elimination capacity of 2.79kg-phenol/m3d was achieved in the spouted bed bioreactor, with Comamonas acidovorans being the dominant strain during high degradation periods.
Keywords: Phenol degradation; Airlift Bioreactors; Bubble Columns; Aerobic Processes; Immobilized Cells; Hydrogel Support
Preparation and characterization of a novel 3D scaffold from poly(ɛ-caprolactone)/biphasic calcium phosphate hybrid composite microspheres adhesion by Trinh-Quang Bao; Rose Ann Franco; Byong-Taek Lee (pp. 76-83).
Display Omitted► In this study, experimental conditions were varied to obtain the a novel PCL/BCP-HCM 3D scaffold. ► NH4HCO3 was used as a key to create the high porous scaffold. ► Morphology, structure and components of scaffold, mechanical, and pore size distribution were characterized. ► The cytotoxicity result, SEM cell morphology and confocal image showed cell adhesion and proliferation very well on scaffold. ► This scaffold keeps a promise apply for bone tissue engineering.The use of scaffold-based strategies in tissue engineering applications requires that the micro-architecture of the scaffold satisfies key micro-structural and biological requirements. In this study, we examined the effects of experimental fabrication conditions on the synthesis of a scaffold formed by a poly(ɛ-caprolactone)/biphasic calcium phosphate (PCL/BCP) hybrid composite microspheres adhesion. The goal of our study was to identify the optimal fabrication conditions. An optimized poly(ɛ-caprolactone)/biphasic calcium phosphate hybrid composite microspheres (PCL/BCP-HCM) 3D scaffold was prepared using the gas foaming and spontaneous emulsion droplets adherence (GF-SEDA) technique. Optical microscopy, μCT, SEM, mercury porosimeter, EDS, XRD, a universal testing machine, and MTT assays were used to characterize the assembled scaffolds. When the GF-SEDA technique was used under optimal conditions, the resulting novel PCL/BCP-HCM 3D scaffold had a micro-porous homogenous structure composed of PCL and BCP with a large distribution of pore sizes (ranging from 0.01 to 1000μm) and highly interconnected micro-pores. In addition, the total porosity of the scaffold was 74% with a compressive strength of 0.82MPa. Furthermore, an in vitro study of cell-scaffold interaction demonstrated that cell adhesion, growth, and proliferation were significant over the course of 5 days in culture.
Keywords: PCL/BCP-HCM 3D scaffold; GF-SEDA; In vitro; study; Composite; Adhesion; Porous
Development of a biofilm technology for the production of 1,3-propanediol (1,3-PDO) from crude glycerol by Silvia Casali; Mine Gungormusler; Lorenzo Bertin; Fabio Fava; Nuri Azbar (pp. 84-90).
► Immobilization via attachment on polyurethane foam and Vukopor®. ► 1,3-Propanediol productivities are higher when shorter hydraulic retention times were used. ► Immobilization of microbial cells provide resistance to extreme environmental conditions.Glycerol is the main by-product of transesterification of fats in the biodiesel production. 1,3-Propanediol (1,3-PDO) is a valuable chemical that can be obtained from glycerol by microbial conversion. A number of Enterobacteriaceae species are able to produce 1,3-PDO from glycerol in stirred tank freely suspended cell bioreactors. Little is known about the use of crude glycerol in the production of 1,3-PDO and about the opportunity to intensify the process via strain immobilization in packed bed bioreactors.In this work, Citrobacter freundii, strain DSM 15979, and Pantoea agglomerans, strain DSM 30077, were tested for their ability to produce 1,3-PDO from crude glycerol in shaken flask batch conditions and in packed bed biofilm reactors operating under continuous conditions. Three different hydraulic retention times (HRT) were comparatively tested (8, 4 and 2h) in order to understand its effects on 1,3-PDO production under immobilized cell conditions. The study revealed that HRT significantly influenced the process performances. The best productivities were observed when a HRT of 2h was applied. However, both strains were found to be good candidates for 1,3-PDO production in biofilm reactors, even though P. agglomerans displayed quite higher productivities (3.6g/(Lh)) than the other strain.
Keywords: 1,3-Propanediol; Citrobacter freundii; Fermentation; Glycerol immobilized cells; Packed bed bioreactors; Pantoea agglomerans
Bioprocess engineering aspects of heterologous protein production in Pichia pastoris: A review by Gabriel Potvin; Ayla Ahmad; Zisheng Zhang (pp. 91-105).
Pichia pastoris is currently one of the most effective and versatile systems for the expression of heterologous proteins. Its success is due to its powerful methanol-inducible alcohol oxidase 1 (AOX1) promoter, capacity to perform post-translational modifications and pathways leading to recombinant products secretion. Despite its advantages, P. pastoris cultivation is plagued by high protease expression levels, high sensitivity to methanol levels, nutrient-deficiency when grown on defined media, difficulties in systematic study due to product-specific effects, and health and safety concerns associated with the storage of large quantities of methanol. Difficulties also arise that are specific to the cultivation methods and control strategies used.The present review discusses several bioprocess engineering aspects related to P. pastoris cultivation, including the different promoters available, both constitutive and inductive, on- and off-line process parameter monitoring methods, fed-batch and continuous cultivation control strategies, proteolytic degradation of products and methods to minimize associated yield reductions, and the different models devised to describe cell growth and protein production. The economics of P. pastoris cultivation, through a case-study involving recombinant phytase production, are also discussed. The study of yeast fermentation is currently very dynamic, and P. pastoris is on its way towards becoming a standard expression system.
Keywords: Continuous cultivation; Fed-batch cultivation; Fermentation; Pichia pastoris; Process modeling; Proteolytic degradation
Advances in biotechnological production of 1,3-propanediol by Guneet Kaur; A.K. Srivastava; Subhash Chand (pp. 106-118).
► “State of the art” in the biological production of 1,3-PD, its separation & key strategic bottlenecks for research has been summarized. ► Bioprocess engineering aspects for 1,3-PD production have been highlighted. ► Key role of modelling & its use for 1,3-PD process optimization has been proposed.1,3-propanediol (1,3-PD) is a chemical compound with myriad applications particularly as a monomer for the production of polyesters, polyethers and polyurethanes. It is a raw material for the production of biodegradable plastics, films, solvents, adhesives, detergents, cosmetics and medicines. Various strategies have been employed for the microbial production of 1,3-PD which include several bioprocess cultivation techniques facilitated by natural and/or genetically engineered microbes. Though 1,3-PD is produced in nature by the bioconversion of glycerol its production directly from sugars like glucose has been also made possible by the development of recombinant strains. This review presents the “state of the art” in the biotechnological production technologies of 1,3-PD particularly with respect to bioprocess engineering methods. It also highlights the significance of mathematical model-based approach for designing various bioreactor operating strategies to facilitate the improvement in 1,3-PD production. Attempt has also been made to focus on the protocols used for downstream processing of 1,3-PD and the associated problems. Finally concluding remarks on the future outlook on biobased 1,3-PD to reduce the dependence on disappearing fossil fuels are presented.
Keywords: Abbreviations; 1,3-PD; 1,3-propanediol; 3-HPA; 3-hydroxypropanaldehyde; 2,3-BD; 2,3-butanediol; DHAP; dihydroxyacetone phosphate; D; dilution rateGlycerol; 1,3-propanediol; Bioprocess design; Modelling; Polytrimethylene terephthalate; Downstream processing