Pub Date : 2024-10-11DOI: 10.1016/j.bej.2024.109522
Shengding Wang, Yuanhui Mao, Fangwei Song, Shuli Liang, Ying Lin
Rebaudioside D (Reb D) is a zero-calorie, high-intensity sweetener favored for its superior taste profile compared to other steviol glycosides such as Stevioside (ST) and Rebaudioside A (Reb A). However, Reb D naturally accounts for only about 0.5% of the dry leaf mass of stevia, creating a production challenge. To address this, a mutated glycosyltransferase PgUGT (M8) (named PgM8) from Panax ginseng and sucrose synthase mbSUS from Vigna radiata were co-expressed in Pichia pastoris. We enhanced the system by fusing PgM8 with the GPI-anchored protein GCW61 for cell surface display, achieving enzyme immobilization. Optimizing the PgM8 copy number increased catalytic activity by 82.56%. This innovation enabled continuous whole-cell catalysis for Reb D synthesis, eliminating the need for cell disruption and purification while improving strain reusability. The yield of Reb D reached 48.2 g/L (42.7 mM) in a 50 mL batch within 33 hours, suggesting that this whole-cell catalyst has great potential for large-scale industrial production.
{"title":"Surface display of glycosyltransferase PgM8 and whole-cell catalysis for efficient Rebaudioside D biosynthesis in Pichia pastoris","authors":"Shengding Wang, Yuanhui Mao, Fangwei Song, Shuli Liang, Ying Lin","doi":"10.1016/j.bej.2024.109522","DOIUrl":"10.1016/j.bej.2024.109522","url":null,"abstract":"<div><div>Rebaudioside D (Reb D) is a zero-calorie, high-intensity sweetener favored for its superior taste profile compared to other steviol glycosides such as Stevioside (ST) and Rebaudioside A (Reb A). However, Reb D naturally accounts for only about 0.5% of the dry leaf mass of stevia, creating a production challenge. To address this, a mutated glycosyltransferase PgUGT (M8) (named PgM8) from <em>Panax ginseng</em> and sucrose synthase mbSUS from <em>Vigna radiata</em> were co-expressed in <em>Pichia pastoris</em>. We enhanced the system by fusing PgM8 with the GPI-anchored protein GCW61 for cell surface display, achieving enzyme immobilization. Optimizing the PgM8 copy number increased catalytic activity by 82.56%. This innovation enabled continuous whole-cell catalysis for Reb D synthesis, eliminating the need for cell disruption and purification while improving strain reusability. The yield of Reb D reached 48.2<!--> <!-->g/L (42.7<!--> <!-->mM) in a 50<!--> <!-->mL batch within 33<!--> <!-->hours, suggesting that this whole-cell catalyst has great potential for large-scale industrial production.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"212 ","pages":"Article 109522"},"PeriodicalIF":3.7,"publicationDate":"2024-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142425035","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-11DOI: 10.1016/j.bej.2024.109529
Xiaoyun Ye , Xin Zhang , Xiurong Chen , He Cui , Lei Dong , Xiao Yu
The accumulation of residual sludge as process waste from water treatment engineering needs to be addressed urgently. Tetradesmus obliquus is an important algal species in the field of wastewater treatment. In this study, T. obliquus was cultured in different sludge extract to determine its ability to utilize wastes from the liquid phase and convert them into biomass, and to analyze the response of the microalgae to toxic stress using proteomics. The results showed that the sludge extract medium was superior to the BG11 medium in accumulating biomass, with dry weights, proteins and polysaccharides at least 1.09, 1.12 and 1.28 times higher than those of BG11 medium. In toxic group, T. obliquus reduced TOC from an initial 426.8±20.0 mg/L to 180.4±8.5 mg/L with a simultaneous 48.4 % reduction in toxicity. Toxic sludge extract produced greater damage to the photosystem of T. obliquus compared to the blank, significantly inhibiting the expression of two photosystem II core proteins, A0A383VSL5 (0.290 down) and A0A383V2Z3 (0.308 down), on day 5. However, these impairments were reversible, and at day 20, the expression of A0A383VSL5 was not inhibited, the inhibitory effect of A0A383V2Z3 (0.575 down) was attenuated. These results fill a gap on the treatment of various types of residual sludge by T. obliquus and provide promising strategies for microalgae treatment of residual sludge, whether non-toxic or toxic.
{"title":"Unraveling the residual sludge-mediated waste transformation and physiological regulation mechanism of Tetradesmus obliquus","authors":"Xiaoyun Ye , Xin Zhang , Xiurong Chen , He Cui , Lei Dong , Xiao Yu","doi":"10.1016/j.bej.2024.109529","DOIUrl":"10.1016/j.bej.2024.109529","url":null,"abstract":"<div><div>The accumulation of residual sludge as process waste from water treatment engineering needs to be addressed urgently. <em>Tetradesmus obliquus</em> is an important algal species in the field of wastewater treatment. In this study, <em>T. obliquus</em> was cultured in different sludge extract to determine its ability to utilize wastes from the liquid phase and convert them into biomass, and to analyze the response of the microalgae to toxic stress using proteomics. The results showed that the sludge extract medium was superior to the BG11 medium in accumulating biomass, with dry weights, proteins and polysaccharides at least 1.09, 1.12 and 1.28 times higher than those of BG11 medium. In toxic group, <em>T. obliquus</em> reduced TOC from an initial 426.8±20.0 mg/L to 180.4±8.5 mg/L with a simultaneous 48.4 % reduction in toxicity. Toxic sludge extract produced greater damage to the photosystem of <em>T. obliquus</em> compared to the blank, significantly inhibiting the expression of two photosystem II core proteins, A0A383VSL5 (0.290 down) and A0A383V2Z3 (0.308 down), on day 5. However, these impairments were reversible, and at day 20, the expression of A0A383VSL5 was not inhibited, the inhibitory effect of A0A383V2Z3 (0.575 down) was attenuated. These results fill a gap on the treatment of various types of residual sludge by <em>T. obliquus</em> and provide promising strategies for microalgae treatment of residual sludge, whether non-toxic or toxic.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"212 ","pages":"Article 109529"},"PeriodicalIF":3.7,"publicationDate":"2024-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142445124","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-11DOI: 10.1016/j.bej.2024.109527
Chunlei Zhu , Jian Zhang , Guangfeng Huang , David Z. Zhu
The adsorption of Cd(Ⅱ) in sewage by single-modified biochar systems have limitations, whereas composite modification can enhance the efficiency. In this study, reed straw biochar and Bacillus subtilis were used as raw materials. UV radiation was employed to modify the biochar, and subsequently, Bacillus subtilis was loaded onto the biochar by adsorption, creating modified biochar composites. The Cd(II) adsorption performance and removal efficiency of these composites were then investigated. It was characterized by BET, SEM-EDS, FT-IR, XRD and ZETA potential analysis. Adsorption experiments were conducted under varying conditions (initial Cd(Ⅱ) concentration, UV radiation time, initial pH, etc.), with adsorption isotherms and kinetic models used. Results indicated that 24 hours UV radiation significantly enhanced adsorption performance, increasing the biochar’s surface area by 40 % and pore volume by 20 %, and introducing numerous pores and oxygen-containing functional groups to the biochar's surface. Significantly enhancing the saturation adsorption capacity for Cd(II) from 23.98 mg/g to 49.93 mg/g after UV- Modified biochar was loaded with Bacillus. Modified biochar composites performed better compared to single-modified biochar across different initial Cd(Ⅱ) concentrations, particularly in slightly alkaline environments. The primary adsorption mechanisms were chemical adsorption, such as ion exchange and surface precipitation. The synergistic effect of UV radiation and microbial loading significantly enhanced Cd(Ⅱ) adsorption efficiency. This study demonstrates that composite modification is a more efficient method, aiding in the removal of heavy metal ion Cd(Ⅱ) from water.
{"title":"UV-modified biochar-Bacillus subtilis composite: An effective method for enhancing Cd(II) adsorption from water","authors":"Chunlei Zhu , Jian Zhang , Guangfeng Huang , David Z. Zhu","doi":"10.1016/j.bej.2024.109527","DOIUrl":"10.1016/j.bej.2024.109527","url":null,"abstract":"<div><div>The adsorption of Cd(Ⅱ) in sewage by single-modified biochar systems have limitations, whereas composite modification can enhance the efficiency. In this study, reed straw biochar and <em>Bacillus subtilis</em> were used as raw materials. UV radiation was employed to modify the biochar, and subsequently, <em>Bacillus subtilis</em> was loaded onto the biochar by adsorption, creating modified biochar composites. The Cd(II) adsorption performance and removal efficiency of these composites were then investigated. It was characterized by BET, SEM-EDS, FT-IR, XRD and ZETA potential analysis. Adsorption experiments were conducted under varying conditions (initial Cd(Ⅱ) concentration, UV radiation time, initial pH, etc.), with adsorption isotherms and kinetic models used. Results indicated that 24 hours UV radiation significantly enhanced adsorption performance, increasing the biochar’s surface area by 40 % and pore volume by 20 %, and introducing numerous pores and oxygen-containing functional groups to the biochar's surface. Significantly enhancing the saturation adsorption capacity for Cd(II) from 23.98 mg/g to 49.93 mg/g after UV- Modified biochar was loaded with <em>Bacillus</em>. Modified biochar composites performed better compared to single-modified biochar across different initial Cd(Ⅱ) concentrations, particularly in slightly alkaline environments. The primary adsorption mechanisms were chemical adsorption, such as ion exchange and surface precipitation. The synergistic effect of UV radiation and microbial loading significantly enhanced Cd(Ⅱ) adsorption efficiency. This study demonstrates that composite modification is a more efficient method, aiding in the removal of heavy metal ion Cd(Ⅱ) from water.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"212 ","pages":"Article 109527"},"PeriodicalIF":3.7,"publicationDate":"2024-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142432357","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-10DOI: 10.1016/j.bej.2024.109528
Chaoguang Wang , Xiaohan Hui , George Marshall , Wenhan Xiao , Xiaomei Zhang , Jianying Qian , Jinsong Gong , Guoqiang Xu , Jinsong Shi , Zhenghong Xu
β-Nicotinamide mononucleotide is recognized as a significant bioactive nucleotide, which is can be used in the fields of health industries. Many studies on the synthesis of NMN have involved Escherichia coli and the current methods are limited by safety problems as well as the expense of the substrate. Herein, GRAS-grade Saccharomyces cerevisiae was chosen as chassis cells to synthesize NMN using the substrates glucose and nicotinamide. First, the gene for the key enzyme nicotinamide phosphoribosyltransferase (Nampt) was screened from different sources, and site-directed mutation was performed to improve the synthesis of NMN. The concentration of intracellular NMN in yeast expressing the D83N-Nampt mutant derived from Chitinophaga pinensis reached 413.4 mg/L, which was 3.7 times higher than that of yeast expressing wild enzymes. The synthesis of NMN was further enhanced by overexpressing Nampt combined with weakening of the further metabolism of NMN. Subsequently, the supply of precursor phosphate ribose pyrophosphate (PRPP) was increased by overexpressing the PRPP synthase mutant, which led to the concentration of intracellular NMN increased to 775.9 mg/L from 537.8 mg/L. Finally, the concentration of intracellular NMN reached 1.2 g/L at 6 h after whole-cell catalytic optimization, which is the highest titer achieved by S. cerevisiae from inexpensive substrate glucose and nicotinamide. This study provides the synthesis of NMN by S. cerevisiae with a new and promising method.
{"title":"Whole-cell synthesis of nicotinamide mononucleotide by recombinant Saccharomyces cerevisiae from glucose and nicotinamide","authors":"Chaoguang Wang , Xiaohan Hui , George Marshall , Wenhan Xiao , Xiaomei Zhang , Jianying Qian , Jinsong Gong , Guoqiang Xu , Jinsong Shi , Zhenghong Xu","doi":"10.1016/j.bej.2024.109528","DOIUrl":"10.1016/j.bej.2024.109528","url":null,"abstract":"<div><div>β-Nicotinamide mononucleotide is recognized as a significant bioactive nucleotide, which is can be used in the fields of health industries. Many studies on the synthesis of NMN have involved <em>Escherichia coli</em> and the current methods are limited by safety problems as well as the expense of the substrate. Herein, GRAS-grade <em>Saccharomyces cerevisiae</em> was chosen as chassis cells to synthesize NMN using the substrates glucose and nicotinamide. First, the gene for the key enzyme nicotinamide phosphoribosyltransferase (<em>Nampt</em>) was screened from different sources, and site-directed mutation was performed to improve the synthesis of NMN. The concentration of intracellular NMN in yeast expressing the D83N-<em>Nampt</em> mutant derived from <em>Chitinophaga pinensis</em> reached 413.4 mg/L, which was 3.7 times higher than that of yeast expressing wild enzymes. The synthesis of NMN was further enhanced by overexpressing <em>Nampt</em> combined with weakening of the further metabolism of NMN. Subsequently, the supply of precursor phosphate ribose pyrophosphate (PRPP) was increased by overexpressing the PRPP synthase mutant, which led to the concentration of intracellular NMN increased to 775.9 mg/L from 537.8 mg/L. Finally, the concentration of intracellular NMN reached 1.2 g/L at 6 h after whole-cell catalytic optimization, which is the highest titer achieved by <em>S. cerevisiae</em> from inexpensive substrate glucose and nicotinamide. This study provides the synthesis of NMN by <em>S. cerevisiae</em> with a new and promising method.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"212 ","pages":"Article 109528"},"PeriodicalIF":3.7,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142441714","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Downstream separation and purification of poly(3-hydroxybutyrate) has been developed via combined crude enzyme bacterial cell lysis followed by solvent extraction and circular utilisation of bacterial lysate as nutrient supplement for PHB production. Enzymatic lysis of Paraburkholderia sacchari cells was initially evaluated using crude enzymes produced via solid state fermentation of Aspergillus oryzae. The optimum protease activity (156 U per g cell dry weight) resulted in 66.9 % lysis of residual cell weight. PHB purification with 1,3-dioxolane, dimethyl carbonate, anisole, ammonium laurate and chloroform were evaluated at different processing parameters (extraction duration, temperature) using wet and dry bacterial cells as well as enzymatically lysed wet bacterial cells. The highest recovery yield (94.5 %) and purity (98.1 %) were obtained with 1,3-dioxolane at 80°C and 6 h processing time using enzymatically disrupted bacterial cells. The bacterial cell lysate was used as nutrient supplement for circular PHB production in fed-batch P. sacchari bioreactor culture leading to the production of 78.7 gPHB/L, 56.9 % (w/w) PHB content and 2.3 g/(Lꞏh) productivity. Low global warming potential (1.3 CO2-eq/kgPHB) and abiotic depletion fossil (14.4 MJ/kgPHB) were estimated for PHB purification via enzymatic cell lysis and PHB extraction with 1,3-dioxolane demonstrating the development of a sustainable and circular process for PHB production.
{"title":"Process development and environmental impact assessment of sustainable poly(3-hydroxybutyrate) separation and purification via Paraburkholderia sacchari cell lysis using crude enzymes","authors":"Olga Psaki , Lina Zoghbi , Drosoula Despoina Galani , Anastasios Giannoulis , Ioanna-Georgia I. Athanasoulia , Eleni Stylianou , Demetres Briassoulis , Apostolis Koutinas , Dimitrios Ladakis","doi":"10.1016/j.bej.2024.109526","DOIUrl":"10.1016/j.bej.2024.109526","url":null,"abstract":"<div><div>Downstream separation and purification of poly(3-hydroxybutyrate) has been developed via combined crude enzyme bacterial cell lysis followed by solvent extraction and circular utilisation of bacterial lysate as nutrient supplement for PHB production. Enzymatic lysis of <em>Paraburkholderia sacchari</em> cells was initially evaluated using crude enzymes produced via solid state fermentation of <em>Aspergillus oryzae</em>. The optimum protease activity (156 U per g cell dry weight) resulted in 66.9 % lysis of residual cell weight. PHB purification with 1,3-dioxolane, dimethyl carbonate, anisole, ammonium laurate and chloroform were evaluated at different processing parameters (extraction duration, temperature) using wet and dry bacterial cells as well as enzymatically lysed wet bacterial cells. The highest recovery yield (94.5 %) and purity (98.1 %) were obtained with 1,3-dioxolane at 80°C and 6 h processing time using enzymatically disrupted bacterial cells. The bacterial cell lysate was used as nutrient supplement for circular PHB production in fed-batch <em>P. sacchari</em> bioreactor culture leading to the production of 78.7 g<sub>PHB</sub>/L, 56.9 % (w/w) PHB content and 2.3 g/(Lꞏh) productivity. Low global warming potential (1.3 CO<sub>2</sub>-eq/kg<sub>PHB</sub>) and abiotic depletion fossil (14.4 MJ/kg<sub>PHB</sub>) were estimated for PHB purification via enzymatic cell lysis and PHB extraction with 1,3-dioxolane demonstrating the development of a sustainable and circular process for PHB production.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"213 ","pages":"Article 109526"},"PeriodicalIF":3.7,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142535062","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-10DOI: 10.1016/j.bej.2024.109525
Loc Ton-That , Thi-Phuong-Tu Nguyen , Bich-Ngoc Duong , Duy-Khoi Nguyen , Ngoc-An Nguyen , Thien‑Hoang Ho , Van-Phuc Dinh
Understanding adsorption mechanisms plays an instrumental role in designing and operating adsorption-based wastewater treatment systems. This research systematically demonstrated the comprehensive adsorption mechanism of Pb(II) ions onto biochar synthesized from jackfruit peel in an aqueous solution using theoretical adsorption models and cutting-edge analytical techniques, such as FT-IR, TG-DSC, and SEM-EDX. The results showed that the adsorption process followed the Redlich-Peterson isothermal model and the intraparticle diffusion kinetic model under optimized conditions. The key mechanisms contributing to effective Pb(II) adsorption include complexation, ion - exchange, and intradiffusion. Furthermore, using the hydrothermal method to active biochar improves the surface area of JPT, leading to the maximum Pb(II) adsorption capacity of jackfruit peel-derived biochar to be 83.86 mg/g, higher than biochars from other parts of jackfruit waste (seeds and stems) and some materials from different agricultural residues in previous studies. These findings contribute to narrowing the gap in understanding heavy metal adsorption using biomass residues, theoretical models, and their mechanisms. Additionally, these indicate that agricultural by-products such as jackfruit peel are environmentally friendly and economical materials for implementing strategies aimed at mitigating heavy metal pollution in wastewater.
{"title":"Insights into Pb (II) adsorption mechanisms using jackfruit peel biochar activated by a hydrothermal method toward heavy metal removal from wastewater","authors":"Loc Ton-That , Thi-Phuong-Tu Nguyen , Bich-Ngoc Duong , Duy-Khoi Nguyen , Ngoc-An Nguyen , Thien‑Hoang Ho , Van-Phuc Dinh","doi":"10.1016/j.bej.2024.109525","DOIUrl":"10.1016/j.bej.2024.109525","url":null,"abstract":"<div><div>Understanding adsorption mechanisms plays an instrumental role in designing and operating adsorption-based wastewater treatment systems. This research systematically demonstrated the comprehensive adsorption mechanism of Pb(II) ions onto biochar synthesized from jackfruit peel in an aqueous solution using theoretical adsorption models and cutting-edge analytical techniques, such as FT-IR, TG-DSC, and SEM-EDX. The results showed that the adsorption process followed the Redlich-Peterson isothermal model and the intraparticle diffusion kinetic model under optimized conditions. The key mechanisms contributing to effective Pb(II) adsorption include complexation, ion - exchange, and intradiffusion. Furthermore, using the hydrothermal method to active biochar improves the surface area of JPT, leading to the maximum Pb(II) adsorption capacity of jackfruit peel-derived biochar to be 83.86 mg/g, higher than biochars from other parts of jackfruit waste (seeds and stems) and some materials from different agricultural residues in previous studies. These findings contribute to narrowing the gap in understanding heavy metal adsorption using biomass residues, theoretical models, and their mechanisms. Additionally, these indicate that agricultural by-products such as jackfruit peel are environmentally friendly and economical materials for implementing strategies aimed at mitigating heavy metal pollution in wastewater.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"212 ","pages":"Article 109525"},"PeriodicalIF":3.7,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142445125","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-09DOI: 10.1016/j.bej.2024.109523
Fernando Cantarero-Rivera , Doris H. D’Souza , Madhu Dhar , Jiajia Chen
Computational fluid dynamics (CFD) models have been developed to simulate cell culturing bioreactors but assume water-like viscosity properties due to significant data gaps. This study characterized the dynamic viscosity of HEK-293 cell cultures and evaluated its effect on mixing performance in a spinner flask bioreactor. Viscosity measurements indicated that the cell culture media, media with microcarriers, and cell cultures presented shear thinning behaviors within the measured shear rate range of 1 to 100 s-1. The viscosity also increased with the microcarrier concentrations and growth of cell culture. The CFD model, incorporating dynamic viscosity data, showed that shear stress and Kolmogorov length profiles are significantly influenced by microcarrier concentrations and cell culture growth. Higher microcarrier concentrations led to higher average shear stress and Kolmogorov values. The cultured HEK-293 cells after seven days of growth also had higher average shear stress and Kolmogorov values than at the day of seeding, indicating an impact caused by the cells’ metabolism and biomass. Overall, the results indicated that assuming water-like properties underestimates shear stress and Kolmogorov length scales, especially at zones of lower shear rates due to the observed shear thinning behavior. Thus, careful monitoring of dynamic viscosity of cell cultures and proper control of mixing parameters are critical to deliver the desired mixing conditions for optimized cell growth especially during scale-up production operations.
{"title":"Characterization of the dynamic viscosity of cell cultures and its effect on mixing performance in a spinner flask bioreactor","authors":"Fernando Cantarero-Rivera , Doris H. D’Souza , Madhu Dhar , Jiajia Chen","doi":"10.1016/j.bej.2024.109523","DOIUrl":"10.1016/j.bej.2024.109523","url":null,"abstract":"<div><div>Computational fluid dynamics (CFD) models have been developed to simulate cell culturing bioreactors but assume water-like viscosity properties due to significant data gaps. This study characterized the dynamic viscosity of HEK-293 cell cultures and evaluated its effect on mixing performance in a spinner flask bioreactor. Viscosity measurements indicated that the cell culture media, media with microcarriers, and cell cultures presented shear thinning behaviors within the measured shear rate range of 1 to 100<!--> <!-->s<sup>-1</sup>. The viscosity also increased with the microcarrier concentrations and growth of cell culture. The CFD model, incorporating dynamic viscosity data, showed that shear stress and Kolmogorov length profiles are significantly influenced by microcarrier concentrations and cell culture growth. Higher microcarrier concentrations led to higher average shear stress and Kolmogorov values. The cultured HEK-293 cells after seven days of growth also had higher average shear stress and Kolmogorov values than at the day of seeding, indicating an impact caused by the cells’ metabolism and biomass. Overall, the results indicated that assuming water-like properties underestimates shear stress and Kolmogorov length scales, especially at zones of lower shear rates due to the observed shear thinning behavior. Thus, careful monitoring of dynamic viscosity of cell cultures and proper control of mixing parameters are critical to deliver the desired mixing conditions for optimized cell growth especially during scale-up production operations.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"212 ","pages":"Article 109523"},"PeriodicalIF":3.7,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142425032","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Azo compounds, extensively utilized across various industries, contribute to the release of toxic effluents that are detrimental to both the environment and human health. Traditional methods for azo dye removal often result in harmful byproducts or concentrated sludge, complicating disposal efforts. This study explores the potential of two yeast strains, Candida tropicalis and Pichia kudriavzevii, to effectively decolorize azo dyes (TD4, TD5, and TD6) while simultaneously accumulating lipids. The cultures achieved 80–90 % decolorization of the selected dyes during incubation, with Pichia showing higher efficiency across multiple dyes compared to Candida. Lipid profiling identified valuable fatty acids, such as palmitic acid and oleic acid, with potential applications in biofuels and other industries. Total Organic Carbon (TOC) analysis revealed a reduction in TOC, indicating degradation and mineralization of the dyes by the yeasts. Metabolic profiling via LC-MS confirmed the degradation, showing the presence of intermediates such as azoles, azolines, isoquinolines, pyridines, and benzopyrans in dye-supplemented cultures. Additionally, pathways related to energy metabolism, amino acid metabolism, drug metabolism (cytochrome P450), degradation of aromatic compounds, and steroid biosynthesis were enriched in the dye-treated cultures. Lipid output in the presence of dyes ranged from 40 % to 90 %. The study thus demonstrates a proof of concept for economically viable lipid production combined with efficient dye removal, presenting a sustainable solution to environmental and industrial challenges.
{"title":"Integrating azo dye degradation and lipid accumulation by Candida tropicalis and Pichia kudriavzevii along with insights into underlying metabolomics for treatment of textile effluents","authors":"Sadik Dantroliya, Pooja Doshi, Ishan Raval, Chaitanya Joshi, Madhvi Joshi","doi":"10.1016/j.bej.2024.109521","DOIUrl":"10.1016/j.bej.2024.109521","url":null,"abstract":"<div><div>Azo compounds, extensively utilized across various industries, contribute to the release of toxic effluents that are detrimental to both the environment and human health. Traditional methods for azo dye removal often result in harmful byproducts or concentrated sludge, complicating disposal efforts. This study explores the potential of two yeast strains, <em>Candida tropicalis</em> and <em>Pichia kudriavzevii</em>, to effectively decolorize azo dyes (TD4, TD5, and TD6) while simultaneously accumulating lipids. The cultures achieved 80–90 % decolorization of the selected dyes during incubation, with <em>Pichia</em> showing higher efficiency across multiple dyes compared to <em>Candida</em>. Lipid profiling identified valuable fatty acids, such as palmitic acid and oleic acid, with potential applications in biofuels and other industries. Total Organic Carbon (TOC) analysis revealed a reduction in TOC, indicating degradation and mineralization of the dyes by the yeasts. Metabolic profiling via LC-MS confirmed the degradation, showing the presence of intermediates such as azoles, azolines, isoquinolines, pyridines, and benzopyrans in dye-supplemented cultures. Additionally, pathways related to energy metabolism, amino acid metabolism, drug metabolism (cytochrome P450), degradation of aromatic compounds, and steroid biosynthesis were enriched in the dye-treated cultures. Lipid output in the presence of dyes ranged from 40 % to 90 %. The study thus demonstrates a proof of concept for economically viable lipid production combined with efficient dye removal, presenting a sustainable solution to environmental and industrial challenges.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"212 ","pages":"Article 109521"},"PeriodicalIF":3.7,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142425034","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-05DOI: 10.1016/j.bej.2024.109515
Carl Dinter , David Vonester , David Flitsch , Moritz Mertens , Marc Tüschenbönner , Maximilian Hoffmann , Jochen Büchs , Jørgen Magnus
Shake flasks are widely spread in microbial process development. Characterization of the processes by manual offline sampling is time-consuming, highly laborious and a contamination risk. Online monitoring of key parameters would provide deeper insights, while saving time and effort. In this study, a device for optical online monitoring of dissolved oxygen tension (DOT), biomass, pH value and viscosity in shake flasks is presented. DOT measurement relies on fluorescent oxygen sensitive nanoparticles. The fluorescence intensity signal of the nanoparticles is used to trigger the DOT and scattered light measurements inside the rotating bulk liquid. The scattered light signal (610 – 630 nm) can be correlated to offline measured optical density OD600, even at elevated viscosity. The pH value is monitored online by using pH sensor spots, fixed inside the shake flasks. The shift of the angle of the bulk liquid Θ-Θ0 is correlated to the offline measured viscosity. Detection of the leading edge of the bulk liquid, necessary for viscosity measurement, can be performed either using the fluorescence intensity signal of the oxygen nanoparticles or the scattered light signal.
{"title":"Combined optical measurement of dissolved oxygen tension (DOT), pH value, biomass and viscosity in shake flasks","authors":"Carl Dinter , David Vonester , David Flitsch , Moritz Mertens , Marc Tüschenbönner , Maximilian Hoffmann , Jochen Büchs , Jørgen Magnus","doi":"10.1016/j.bej.2024.109515","DOIUrl":"10.1016/j.bej.2024.109515","url":null,"abstract":"<div><div>Shake flasks are widely spread in microbial process development. Characterization of the processes by manual offline sampling is time-consuming, highly laborious and a contamination risk. Online monitoring of key parameters would provide deeper insights, while saving time and effort. In this study, a device for optical online monitoring of dissolved oxygen tension (DOT), biomass, pH value and viscosity in shake flasks is presented. DOT measurement relies on fluorescent oxygen sensitive nanoparticles. The fluorescence intensity signal of the nanoparticles is used to trigger the DOT and scattered light measurements inside the rotating bulk liquid. The scattered light signal (610 – 630 nm) can be correlated to offline measured optical density OD<sub>600</sub>, even at elevated viscosity. The pH value is monitored online by using pH sensor spots, fixed inside the shake flasks. The shift of the angle of the bulk liquid Θ-Θ<sub>0</sub> is correlated to the offline measured viscosity. Detection of the leading edge of the bulk liquid, necessary for viscosity measurement, can be performed either using the fluorescence intensity signal of the oxygen nanoparticles or the scattered light signal.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"212 ","pages":"Article 109515"},"PeriodicalIF":3.7,"publicationDate":"2024-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142425031","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-05DOI: 10.1016/j.bej.2024.109520
Valentine Tingaud , Philippe Lawton , Johan Peralez , Madiha Nadri-Wolf , Isabelle Pitault , Claudia Cogné , Elisabeth Errazuriz , Eyad Al Mouazen , Claire Bordes
Scale-up of bioprocesses involving animal cell culture is hampered by the sensitivity of the cells to hydrodynamic stress, either from agitation or bubble bursting. Here, the hydrodynamic stress experienced by a recent cell line, the DuckCelt®-T17 avian cells, previously used for viral vaccine production, is investigated in shake flasks and in a 3 L bioreactor. Cell stress was assessed by monitoring the dissolved oxygen in the culture medium, which depends on Oxygen Transfer Rate (OTR) and Oxygen Uptake Rate (OUR) during cultivation. Classical parameters such as the maximum growth rate (µmax) and metabolite profiles were also determined. A dynamic model able to predict nutrient consumption, metabolic waste production, viable cell number and OUR was also developed and validated from the data measured in shake flasks. The experiments performed in the stirred tank bioreactor (STBR) show that OUR depended on both the cell growth phase and the stirring conditions. The oxygen consumption of the cells during the exponential growth phase (where there were no nutrient and O2 limitations) was significantly altered at average and maximum shear rates above 70 and 840 s−1, respectively, indicating highly shear-sensitive cells. OUR is a suitable tool to identify the hydrodynamic conditions for robust cell growth. The scale-up criteria to be favored for the DuckCelt®-T17 cell culture in STBRs would be the shear and/or the tip’s speed.
{"title":"Oxygen uptake rate analysis to evaluate the impact of hydrodynamic stress on the growth of the avian cell line DuckCelt®-T17","authors":"Valentine Tingaud , Philippe Lawton , Johan Peralez , Madiha Nadri-Wolf , Isabelle Pitault , Claudia Cogné , Elisabeth Errazuriz , Eyad Al Mouazen , Claire Bordes","doi":"10.1016/j.bej.2024.109520","DOIUrl":"10.1016/j.bej.2024.109520","url":null,"abstract":"<div><div>Scale-up of bioprocesses involving animal cell culture is hampered by the sensitivity of the cells to hydrodynamic stress, either from agitation or bubble bursting. Here, the hydrodynamic stress experienced by a recent cell line, the DuckCelt®-T17 avian cells, previously used for viral vaccine production, is investigated in shake flasks and in a 3 L bioreactor. Cell stress was assessed by monitoring the dissolved oxygen in the culture medium, which depends on Oxygen Transfer Rate (OTR) and Oxygen Uptake Rate (OUR) during cultivation. Classical parameters such as the maximum growth rate (<em>µ</em><sub>max</sub>) and metabolite profiles were also determined. A dynamic model able to predict nutrient consumption, metabolic waste production, viable cell number and OUR was also developed and validated from the data measured in shake flasks. The experiments performed in the stirred tank bioreactor (STBR) show that OUR depended on both the cell growth phase and the stirring conditions. The oxygen consumption of the cells during the exponential growth phase (where there were no nutrient and O<sub>2</sub> limitations) was significantly altered at average and maximum shear rates above 70 and 840 s<sup>−1</sup>, respectively, indicating highly shear-sensitive cells. OUR is a suitable tool to identify the hydrodynamic conditions for robust cell growth. The scale-up criteria to be favored for the DuckCelt®-T17 cell culture in STBRs would be the shear and/or the tip’s speed.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"212 ","pages":"Article 109520"},"PeriodicalIF":3.7,"publicationDate":"2024-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142425081","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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