Pub Date : 2026-07-01Epub Date: 2026-03-06DOI: 10.1016/j.bej.2026.110150
Eunyoung Cho, Kyumin Park, Jung Heon Lee
Microreactor-based biocatalytic processes offer significant advantages, including continuous operation, precise residence-time control, and enhanced process stability. In this study, a membrane-based microreactor incorporating a tannic acid–iron(III) (TA–Fe³⁺)–functionalized anodic aluminum oxide (AAO) membrane was developed for the continuous production of L-DOPA (3,4-dihydroxy-L-phenylalanine) using immobilized tyrosinase (Tyr). SEM and FTIR analyses confirmed that the TA–Fe³⁺ coating was deposited on the membrane surface and within its nanopores, providing a robust scaffold for enzyme attachment. Among the immobilization strategies investigated, enzyme adsorption–precipitation–crosslinking (EAPC) yielded the highest catalytic performance by maximizing enzyme loading and preventing leaching under flow conditions. The resulting microreactor demonstrated exceptional long-term stability, retaining 65% of its initial activity and maintaining L-DOPA production above 0.06 mM after 30 days of continuous operation. These results highlight the effectiveness of TA–Fe³⁺–mediated surface functionalization for enzyme immobilization and demonstrate the feasibility of a continuous, enzyme-based microreactor system for sustainable biochemical synthesis.
基于微反应器的生物催化工艺具有显著的优势，包括连续操作、精确的停留时间控制和增强的工艺稳定性。在本研究中，采用单宁酸-铁（III） （TA-Fe³）+功能化阳极氧化铝（AAO）膜构建了一种膜基微反应器，用于固定化酪氨酸酶（Tyr）连续生产L-DOPA（3,4-二羟基- l -苯丙氨酸）。SEM和FTIR分析证实，TA-Fe +涂层沉积在膜表面和纳米孔内，为酶附着提供了坚固的支架。在所研究的固定策略中，酶吸附-沉淀-交联（EAPC）在流动条件下通过最大化酶载量和防止浸出而获得了最高的催化性能。由此产生的微反应器表现出优异的长期稳定性，在连续运行30天后，保持了65%的初始活性，L-DOPA产量保持在0.06 mM以上。这些结果突出了TA-Fe³+介导的表面功能化用于酶固定化的有效性，并证明了连续的、基于酶的微反应器系统用于可持续生化合成的可行性。
{"title":"Enhanced continuous production of L-DOPA using tyrosinase immobilized on a tannic acid–iron(III) functionalized anodic aluminum oxide (AAO) membrane microreactor","authors":"Eunyoung Cho, Kyumin Park, Jung Heon Lee","doi":"10.1016/j.bej.2026.110150","DOIUrl":"10.1016/j.bej.2026.110150","url":null,"abstract":"<div><div>Microreactor-based biocatalytic processes offer significant advantages, including continuous operation, precise residence-time control, and enhanced process stability. In this study, a membrane-based microreactor incorporating a tannic acid–iron(III) (TA–Fe³⁺)–functionalized anodic aluminum oxide (AAO) membrane was developed for the continuous production of <span>L</span>-DOPA (3,4-dihydroxy-<span>L</span>-phenylalanine) using immobilized tyrosinase (Tyr). SEM and FTIR analyses confirmed that the TA–Fe³⁺ coating was deposited on the membrane surface and within its nanopores, providing a robust scaffold for enzyme attachment. Among the immobilization strategies investigated, enzyme adsorption–precipitation–crosslinking (EAPC) yielded the highest catalytic performance by maximizing enzyme loading and preventing leaching under flow conditions. The resulting microreactor demonstrated exceptional long-term stability, retaining 65% of its initial activity and maintaining <span>L</span>-DOPA production above 0.06 mM after 30 days of continuous operation. These results highlight the effectiveness of TA–Fe³⁺–mediated surface functionalization for enzyme immobilization and demonstrate the feasibility of a continuous, enzyme-based microreactor system for sustainable biochemical synthesis.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"231 ","pages":"Article 110150"},"PeriodicalIF":3.7,"publicationDate":"2026-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147387823","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}
Numerical simulation of biological methanation remains challenging due to the strong coupling between mass transfer, hydrodynamics, and bioreaction in such gas-fed bioreactors. This work presents an extension of a 1D spatio-temporal gas–liquid model for bubble columns to the case of biological methanation. To this end, three major improvements are added to the previously published model: (i) the consideration of water production due to the biological methanation reaction, (ii) the implementation of an additional equation for the liquid height, (iii) the introduction of a variable maintenance model based on the spatial heterogeneity of the H2 mass transfer. The model was validated with experimental data from literature. Results indicate that water production acts as a dilution term, as revealed by simulations performed under both constant and variable height conditions. This led to a reduced biomass concentration while preserving methane production owing to a redistribution of H2 consumption between maintenance and growth. These intriguing results were confirmed by analytical solutions at steady-state. The variable maintenance model further allows connecting the decrease in performances through scale-up to increased local deviations between H2 mass transfer and cell demand. Also, a basic moving mesh method now extends the gas–liquid dynamic 1D model capabilities to any fed-batch (bio) reactor.
{"title":"An extended 1D dynamic model of biological methanation considering water production, variable height and maintenance heterogeneity","authors":"Julian Federico Sanchez Caldas, Arnaud Cockx, Carlos Robles Rodriguez, Jérôme Morchain","doi":"10.1016/j.bej.2026.110109","DOIUrl":"10.1016/j.bej.2026.110109","url":null,"abstract":"<div><div>Numerical simulation of biological methanation remains challenging due to the strong coupling between mass transfer, hydrodynamics, and bioreaction in such gas-fed bioreactors. This work presents an extension of a 1D spatio-temporal gas–liquid model for bubble columns to the case of biological methanation. To this end, three major improvements are added to the previously published model: (i) the consideration of water production due to the biological methanation reaction, (ii) the implementation of an additional equation for the liquid height, (iii) the introduction of a variable maintenance model based on the spatial heterogeneity of the H<sub>2</sub> mass transfer. The model was validated with experimental data from literature. Results indicate that water production acts as a dilution term, as revealed by simulations performed under both constant and variable height conditions. This led to a reduced biomass concentration while preserving methane production owing to a redistribution of H<sub>2</sub> consumption between maintenance and growth. These intriguing results were confirmed by analytical solutions at steady-state. The variable maintenance model further allows connecting the decrease in performances through scale-up to increased local deviations between H<sub>2</sub> mass transfer and cell demand. Also, a basic moving mesh method now extends the gas–liquid dynamic 1D model capabilities to any fed-batch (bio) reactor.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"230 ","pages":"Article 110109"},"PeriodicalIF":3.7,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146196855","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 : 2026-06-01Epub Date: 2026-02-02DOI: 10.1016/j.bej.2026.110096
Okyanus Yazgin , Martin F. Luna , Peter Neubauer , Ernesto C. Martinez , M. Nicolas Cruz Bournazou
Bioprocess development can benefit significantly from the use of mathematical models for prediction and optimization, yet the uncertainty in these models can hinder reliable early-stage decision-making for industrial-scale processes. This study introduces a telescopic model-based design of experiments approach that directly targets the reduction of uncertainty in key performance indicators (KPIs) at the optimum process conditions rather than focusing solely on model parameter precision. Using a sugarcane-to-ethanol biorefinery use case, the proposed approach is benchmarked against a traditional parameter-focused approach. Results demonstrate that the proposed strategy reduces KPI uncertainty more efficiently, identifies economically favorable process conditions faster, and prioritizes the estimation of parameters most influential on the KPI.
{"title":"A KPI-based experimental design strategy for bioprocess development","authors":"Okyanus Yazgin , Martin F. Luna , Peter Neubauer , Ernesto C. Martinez , M. Nicolas Cruz Bournazou","doi":"10.1016/j.bej.2026.110096","DOIUrl":"10.1016/j.bej.2026.110096","url":null,"abstract":"<div><div>Bioprocess development can benefit significantly from the use of mathematical models for prediction and optimization, yet the uncertainty in these models can hinder reliable early-stage decision-making for industrial-scale processes. This study introduces a telescopic model-based design of experiments approach that directly targets the reduction of uncertainty in key performance indicators (KPIs) at the optimum process conditions rather than focusing solely on model parameter precision. Using a sugarcane-to-ethanol biorefinery use case, the proposed approach is benchmarked against a traditional parameter-focused approach. Results demonstrate that the proposed strategy reduces KPI uncertainty more efficiently, identifies economically favorable process conditions faster, and prioritizes the estimation of parameters most influential on the KPI.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"230 ","pages":"Article 110096"},"PeriodicalIF":3.7,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146196856","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 : 2026-05-01Epub Date: 2026-01-25DOI: 10.1016/j.bej.2026.110098
YangTao Yuan , JiaJia Mi , QiWei Li , JianPing Shi
The development of portable and reliable sensors for monitoring phenolic pollutants in water remains a significant challenge. Nanozyme-based colorimetric assays offer a promising alternative to conventional methods. However, their practical application is often hindered by poor portability and stability in liquid-phase systems. To address this, we developed a novel hydrogel-based colorimetric sensor. This sensor is empowered by a dual-enzyme active nanozyme. It is designed for the on-site detection of hydroquinone (HQ) and catechol (CC). The carbon-coated MnFe oxide (MnFe-O/C) nanoparticles, derived from a MnFe-Prussian blue analogue precursor, exhibits good intrinsic oxidase- and peroxidase-like activities. Leveraging the inhibitory effect of HQ and CC on the nanozyme-catalyzed chromogenic reaction, a sensitive solution-phase colorimetric assay was established. This assay achieved detection limits of 5.78 μM for HQ and 10.43 μM for CC. Furthermore, a portable and standalone sensor was fabricated by embedding the MnFe-O/C nanozyme within a borax-crosslinked carboxymethyl cellulose hydrogel network. When coupled with a smartphone for RGB analysis, the hydrogel sensor enables on-site and quantitative detection of HQ and CC in real water samples. The recovery rates in spiked water samples ranged from 94.80 % to 99.14 % for HQ and 97.32 %-108.80 % for CC, demonstrating high accuracy and reliability. This work not only offers a viable approach for constructing dual-enzyme active nanozymes, but also provides a practical tool for detecting phenolic pollutants in water.
{"title":"Hydrogel-based colorimetric sensor using dual-enzyme active MnFe-O/C nanozymes for on-site detection of phenolic pollutants in water","authors":"YangTao Yuan , JiaJia Mi , QiWei Li , JianPing Shi","doi":"10.1016/j.bej.2026.110098","DOIUrl":"10.1016/j.bej.2026.110098","url":null,"abstract":"<div><div>The development of portable and reliable sensors for monitoring phenolic pollutants in water remains a significant challenge. Nanozyme-based colorimetric assays offer a promising alternative to conventional methods. However, their practical application is often hindered by poor portability and stability in liquid-phase systems. To address this, we developed a novel hydrogel-based colorimetric sensor. This sensor is empowered by a dual-enzyme active nanozyme. It is designed for the on-site detection of hydroquinone (HQ) and catechol (CC). The carbon-coated MnFe oxide (MnFe-O/C) nanoparticles, derived from a MnFe-Prussian blue analogue precursor, exhibits good intrinsic oxidase- and peroxidase-like activities. Leveraging the inhibitory effect of HQ and CC on the nanozyme-catalyzed chromogenic reaction, a sensitive solution-phase colorimetric assay was established. This assay achieved detection limits of 5.78 μM for HQ and 10.43 μM for CC. Furthermore, a portable and standalone sensor was fabricated by embedding the MnFe-O/C nanozyme within a borax-crosslinked carboxymethyl cellulose hydrogel network. When coupled with a smartphone for RGB analysis, the hydrogel sensor enables on-site and quantitative detection of HQ and CC in real water samples. The recovery rates in spiked water samples ranged from 94.80 % to 99.14 % for HQ and 97.32 %-108.80 % for CC, demonstrating high accuracy and reliability. This work not only offers a viable approach for constructing dual-enzyme active nanozymes, but also provides a practical tool for detecting phenolic pollutants in water.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"229 ","pages":"Article 110098"},"PeriodicalIF":3.7,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146075985","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}
Acidification frequently impairs anaerobic digestion by causing volatile fatty acid (VFA) accumulation and process collapse. Here, we report a material–microbe integrated strategy for acidification control based on mixed-valence iron oxides–encapsulated hollow carbon spheres (FeOx@HCS), which couple redox-active Fe(II/III) interfaces with a conductive carbon framework. To overcome this limitation, FeOx@HCS were synthesized via an aerosol-assisted method and introduced to a glucose-fed digester intentionally operated under acidification-prone conditions. The amendment effectively suppressed VFA accumulation (<500 mg L⁻¹ vs. >800 mg L⁻¹ in the control), maintained near-neutral pH, and consequently stabilized methane production. Rather than relying on external buffering, FeOx@HCS mitigated acidification by reshaping microbial community structure and improving syntrophic metabolism. Metagenomic analysis demonstrated that FeOx@HCS reshaped the microbial community by enriching methanogens such as Methanoregula (22.8 %) and Methanothrix (1.3 %), while limiting the proliferation of acidogenic bacteria. Correspondingly, the relative abundance of key methanogenic genes (POR, Ftr, Mch, Mtd, Mcr, Hdr) increased by 19–35 %. Metagenome-assembled genomes (MAG) confirmed complementary metabolic roles, with Microbacter facilitating acidogenesis and Methanoregula/Methanosarcina driving hydrogenotrophic and acetoclastic methanogenesis. Notably, genes associated with intracellular electron transfer in methanogens (Hdr, Frh) were enriched, whereas genes encoding conductive pili or cytochromes were not, suggesting a material-mediated enhancement of electron transfer rather than classical biologically driven DIET. Collectively, this study provides genome-resolved mechanistic evidence that FeOx-based carbon interfaces stabilize anaerobic digestion by coordinating acidification control, syntrophic metabolism, and electron transfer, thereby extending current Fe–C material studies beyond performance enhancement toward mechanistic interpretation.
酸化经常通过引起挥发性脂肪酸（VFA）积累和过程崩溃而损害厌氧消化。在这里，我们报告了一种基于混合价氧化铁-封装中空碳球（FeOx@HCS）的材料-微生物一体化酸化控制策略，该策略将氧化还原活性铁（II/III）界面与导电碳框架耦合在一起。为了克服这一限制，我们通过气溶胶辅助方法合成了FeOx@HCS，并将其引入了一个在容易酸化的条件下操作的葡萄糖消化器。这一修正有效地抑制了VFA的积累（500 mg L -⁻¹vs 800 mg L -⁻¹），保持了接近中性的pH值，从而稳定了甲烷的产生。FeOx@HCS不是依靠外部缓冲，而是通过重塑微生物群落结构和改善合成代谢来减轻酸化。宏基因组分析表明，FeOx@HCS通过富集产甲烷菌如Methanoregula（22.8% %）和Methanothrix（1.3 %）来重塑微生物群落，同时限制产酸菌的增殖。相应的，关键产甲烷基因（POR、Ftr、Mch、Mtd、Mcr、Hdr）的相对丰度增加了19 ~ 35% %。宏基因组组装基因组（MAG）证实了互补的代谢作用，Microbacter促进酸生成，Methanoregula/Methanosarcina驱动氢营养和醋酸裂解甲烷生成。值得注意的是，产甲烷菌中与细胞内电子转移相关的基因（Hdr, Frh）被富集，而编码导电毛或细胞色素的基因则没有富集，这表明物质介导的电子转移增强，而不是经典的生物驱动的DIET。总的来说，这项研究提供了基因组解析的机制证据，证明基于feox的碳界面通过协调酸化控制、合成代谢和电子转移来稳定厌氧消化，从而将当前的Fe-C材料研究从性能增强扩展到机制解释。
{"title":"Metagenomic mechanisms of acidification control by FeOx@HCS in stabilizing methanogenesis","authors":"Mengyue Fu, Yu Liu, Pengju Li, Xianliang Yi, Yang Liu, Jingjing Zhan, Hao Zhou","doi":"10.1016/j.bej.2026.110110","DOIUrl":"10.1016/j.bej.2026.110110","url":null,"abstract":"<div><div>Acidification frequently impairs anaerobic digestion by causing volatile fatty acid (VFA) accumulation and process collapse. Here, we report a material–microbe integrated strategy for acidification control based on mixed-valence iron oxides–encapsulated hollow carbon spheres (FeOx@HCS), which couple redox-active Fe(II/III) interfaces with a conductive carbon framework. To overcome this limitation, FeOx@HCS were synthesized via an aerosol-assisted method and introduced to a glucose-fed digester intentionally operated under acidification-prone conditions. The amendment effectively suppressed VFA accumulation (<500 mg L⁻¹ vs. >800 mg L⁻¹ in the control), maintained near-neutral pH, and consequently stabilized methane production. Rather than relying on external buffering, FeOx@HCS mitigated acidification by reshaping microbial community structure and improving syntrophic metabolism. Metagenomic analysis demonstrated that FeOx@HCS reshaped the microbial community by enriching methanogens such as <em>Methanoregula</em> (22.8 %) and <em>Methanothrix</em> (1.3 %), while limiting the proliferation of acidogenic bacteria. Correspondingly, the relative abundance of key methanogenic genes (POR, Ftr, Mch, Mtd, Mcr, Hdr) increased by 19–35 %. Metagenome-assembled genomes (MAG) confirmed complementary metabolic roles, with <em>Microbacter</em> facilitating acidogenesis and <em>Methanoregula/Methanosarcina</em> driving hydrogenotrophic and acetoclastic methanogenesis. Notably, genes associated with intracellular electron transfer in methanogens (Hdr, Frh) were enriched, whereas genes encoding conductive pili or cytochromes were not, suggesting a material-mediated enhancement of electron transfer rather than classical biologically driven DIET. Collectively, this study provides genome-resolved mechanistic evidence that FeOx-based carbon interfaces stabilize anaerobic digestion by coordinating acidification control, syntrophic metabolism, and electron transfer, thereby extending current Fe–C material studies beyond performance enhancement toward mechanistic interpretation.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"229 ","pages":"Article 110110"},"PeriodicalIF":3.7,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146185300","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 : 2026-05-01Epub Date: 2026-01-23DOI: 10.1016/j.bej.2026.110095
Wenbo An , Bin Xu , Junzhen Di , Yifan Liu , Tianzhi Wang
Addressing the environmental toxicity of high-concentration heavy metal pollutants, such as Fe2+, in acid mine drainage (AMD) and the limited adsorption capacity of lignite, this study utilized lignite as a substrate adsorbent. By selecting the environmentally non-toxic Rhodopseudomonas sphaeroides, a novel low-cost modified adsorbent (Rhodopseudomonas sphaeroides-modified lignite, RS-L). Using batch experiments and microscopic characterization methods, the adsorption characteristics and mechanism of RS-L towards Fe2+ were investigated. Results indicated that natural lignite with a mesh size of 60–80 was modified with 12 mL of bacterial solution for 14 days to synthesize RS-L. When the initial Fe2+ concentration was 65 mg/L, pH was 4, and the dosage of RS-L was 1 g, optimal adsorption performance was achieved. The adsorption process followed the pseudo-second-order kinetic model and Langmuir monolayer adsorption, and was a spontaneous (negative ΔG), endothermic (positive ΔH), entropy-increasing (positive ΔS) process. At 45°C, the maximum adsorption capacity of Fe2+ was 13.10 mg/g. The adsorption mechanism primarily involved electrostatic adsorption, the packing effect, chemical precipitation, and complexation reactions. After five adsorption cycles, RS-L maintained an adsorption efficiency of 63.62 % and an adsorption capacity of 8.27 mg/g, demonstrating adaptability to complex ionic environments. This study offers new insights for restoring AMD and provides novel pathways for the resource utilization of lignite.
{"title":"Removal of Fe2 + from acid mine drainage by Rhodopseudomonas sphaeroides-modified lignite: Adsorption characteristics and mechanism","authors":"Wenbo An , Bin Xu , Junzhen Di , Yifan Liu , Tianzhi Wang","doi":"10.1016/j.bej.2026.110095","DOIUrl":"10.1016/j.bej.2026.110095","url":null,"abstract":"<div><div>Addressing the environmental toxicity of high-concentration heavy metal pollutants, such as Fe<sup>2+</sup>, in acid mine drainage (AMD) and the limited adsorption capacity of lignite, this study utilized lignite as a substrate adsorbent. By selecting the environmentally non-toxic <em>Rhodopseudomonas sphaeroides</em>, a novel low-cost modified adsorbent (<em>Rhodopseudomonas sphaeroides</em>-modified lignite, RS-L). Using batch experiments and microscopic characterization methods, the adsorption characteristics and mechanism of RS-L towards Fe<sup>2+</sup> were investigated. Results indicated that natural lignite with a mesh size of 60–80 was modified with 12 mL of bacterial solution for 14 days to synthesize RS-L. When the initial Fe<sup>2+</sup> concentration was 65 mg/L, pH was 4, and the dosage of RS-L was 1 g, optimal adsorption performance was achieved. The adsorption process followed the pseudo-second-order kinetic model and Langmuir monolayer adsorption, and was a spontaneous (negative Δ<em>G</em>), endothermic (positive Δ<em>H</em>), entropy-increasing (positive Δ<em>S</em>) process. At 45°C, the maximum adsorption capacity of Fe<sup>2+</sup> was 13.10 mg/g. The adsorption mechanism primarily involved electrostatic adsorption, the packing effect, chemical precipitation, and complexation reactions. After five adsorption cycles, RS-L maintained an adsorption efficiency of 63.62 % and an adsorption capacity of 8.27 mg/g, demonstrating adaptability to complex ionic environments. This study offers new insights for restoring AMD and provides novel pathways for the resource utilization of lignite.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"229 ","pages":"Article 110095"},"PeriodicalIF":3.7,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146075984","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 : 2026-05-01Epub Date: 2026-01-30DOI: 10.1016/j.bej.2026.110101
Minjun Zhao , Shuaijie Jin , Wenzhuo Li , Yuke Xu , Qin Zhang
Urea has been documented as an excellent promoter for improving sewage sludge (SS) fermentation, considering its effectiveness and economic feasibility, yet its effects on the fates of antibiotic resistance genes (ARGs) during this process are still unknown. Herein, the responses of ARGs distribution to urea exposure were studied, and the results revealed that urea exacerbated ARGs propagation, as evidenced by an increase of 66.8 % total abundance. Mechanistic exploration demonstrated that the presence of urea and free ammonia (FA) stripped the extracellular polymeric substances (EPS) and increased the cell membrane permeability, contributing to ARGs and mobile genetic elements (MGEs) release and consequently improved their horizontal transfer. Also, urea exhibited “screening effects” to enrich some harboring ARGs carriers (e.g., Bacteroidetes_norank, Tissierella and Firmicutes_norank). Further analysis found that the generated FA induced oxidative stress (e.g., katE and SOD1) and activated the SOS response (e.g., recA, recO, and recR), promoting ARGs formation, which could be further improved by unhydrolyzed urea through upregulating the metabolic functions (e.g., TCA cycle) associated with energy production. The structural equation model suggested that the upregulation of key metabolic pathways was the predominant contributor to the ARGs propagation. Collectively, this work explored the effects and underlying mechanisms of urea on ARGs' fates during SS fermentation, highlighting the potential environmental risks of urea-based treatment on resource recovery from SS.
{"title":"Urea-based treatment promotes the propagation of antibiotic resistance genes during sludge fermentation: Insights into its multifaceted roles in structure disruption, microbial community reshaping, and metabolic regulation","authors":"Minjun Zhao , Shuaijie Jin , Wenzhuo Li , Yuke Xu , Qin Zhang","doi":"10.1016/j.bej.2026.110101","DOIUrl":"10.1016/j.bej.2026.110101","url":null,"abstract":"<div><div>Urea has been documented as an excellent promoter for improving sewage sludge (SS) fermentation, considering its effectiveness and economic feasibility, yet its effects on the fates of antibiotic resistance genes (ARGs) during this process are still unknown. Herein, the responses of ARGs distribution to urea exposure were studied, and the results revealed that urea exacerbated ARGs propagation, as evidenced by an increase of 66.8 % total abundance. Mechanistic exploration demonstrated that the presence of urea and free ammonia (FA) stripped the extracellular polymeric substances (EPS) and increased the cell membrane permeability, contributing to ARGs and mobile genetic elements (MGEs) release and consequently improved their horizontal transfer. Also, urea exhibited “screening effects” to enrich some harboring ARGs carriers (<em>e.g., Bacteroidetes_norank, Tissierella</em> and <em>Firmicutes_norank</em>). Further analysis found that the generated FA induced oxidative stress (<em>e.g., katE and SOD1</em>) and activated the SOS response (<em>e.g., recA</em>, <em>recO</em>, and <em>recR</em>), promoting ARGs formation, which could be further improved by unhydrolyzed urea through upregulating the metabolic functions (<em>e.g.,</em> TCA cycle) associated with energy production. The structural equation model suggested that the upregulation of key metabolic pathways was the predominant contributor to the ARGs propagation. Collectively, this work explored the effects and underlying mechanisms of urea on ARGs' fates during SS fermentation, highlighting the potential environmental risks of urea-based treatment on resource recovery from SS.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"229 ","pages":"Article 110101"},"PeriodicalIF":3.7,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146075983","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 : 2026-05-01Epub Date: 2026-02-09DOI: 10.1016/j.bej.2026.110114
Fuyao Guan , Min Wu , Fei Tang , Xin Xu , Haoju Wang , Qihang Chen , Wei Sun , Huiru Cui , Jiahui Jiang , Hongguang Yang , Ping Yu , Min Chen
The inner membrane transporter GadC in Escherichia coli can facilitate glutamate uptake and γ-aminobutyric acid (GABA) efflux. Based on a previously engineered glutamate decarboxylase mutant GadBD304G/F433L (with a broadened pH adaptability and an enhanced activity) expressed in E. coli BL21(DE3)/pETDuet-1-gadBD304G/F433L, this study cloned gadC into the plasmid pETDuet-1-gadBD304G/F433L to construct recombinant strain E. coli BL21(DE3)/pETDuet-1-gadBD304G/F433L-gadC. Whole-cell biocatalysis yielded 5.1 g/L GABA with the single-gene strain and 2.3 g/L GABA with the double-gene (gadBD304G/F433L-gadC) strain. Subsequent optimization of the biocatalytic conditions (60 °C, pH 3.2, 15 g/L cell biomass, 0.75 mM L-monosodium glutamate, 0.2 mM PLP) via single-factor and orthogonal experiments significantly increased GABA production to 54.84 g/L. Finally, implementing a whole-cell biocatalyst recycling strategy for 5 batches enabled the efficient synthesis of 382.59 g/L GABA from sodium glutamate. This study contributes to a possible industrial production of GABA by engineered strain E. coli BL21(DE3)/pETDuet-1-gadBD304G/F433L.
大肠杆菌的内膜转运蛋白GadC可促进谷氨酸的摄取和γ-氨基丁酸（GABA）的外排。本研究以大肠杆菌BL21(DE3)/pETDuet-1-gadBD304G/F433L中表达的谷氨酸脱羧酶突变体GadBD304G/F433L为基础，将gadC克隆到质粒pETDuet-1-gadBD304G/F433L中，构建重组菌株BL21(DE3)/pETDuet-1-gadBD304G/F433L-gadC。单基因菌株全细胞生物催化产GABA为5.1 g/L，双基因菌株（gadBD304G/F433L-gadC）产GABA为2.3 g/L。随后通过单因素和正交实验优化生物催化条件（60°C, pH 3.2, 15 g/L细胞生物量，0.75 mM L-味精，0.2 mM PLP）， GABA产量显著提高至54.84 g/L。最后，采用5批全细胞生物催化剂循环策略，使谷氨酸钠高效合成382.59 g/L GABA。本研究为利用工程菌株大肠杆菌BL21(DE3)/pETDuet-1-gadBD304G/F433L工业化生产GABA提供了可能。
{"title":"Efficient biosynthesis of γ-aminobutyric acid by a GadBD304G/F433L mutant-based whole-cell biocatalyst","authors":"Fuyao Guan , Min Wu , Fei Tang , Xin Xu , Haoju Wang , Qihang Chen , Wei Sun , Huiru Cui , Jiahui Jiang , Hongguang Yang , Ping Yu , Min Chen","doi":"10.1016/j.bej.2026.110114","DOIUrl":"10.1016/j.bej.2026.110114","url":null,"abstract":"<div><div>The inner membrane transporter GadC in <em>Escherichia coli</em> can facilitate glutamate uptake and γ-aminobutyric acid (GABA) efflux. Based on a previously engineered glutamate decarboxylase mutant GadB<sup>D304G/F433L</sup> (with a broadened pH adaptability and an enhanced activity) expressed in <em>E. coli</em> BL21(DE3)/pETDuet-1-<em>gadB</em><sup>D304G/F433L</sup>, this study cloned <em>gadC</em> into the plasmid pETDuet-1-<em>gadB</em><sup>D304G/F433L</sup> to construct recombinant strain <em>E. coli</em> BL21(DE3)/pETDuet-1-<em>gadB</em><sup>D304G/F433L</sup>-<em>gadC</em>. Whole-cell biocatalysis yielded 5.1 g/L GABA with the single-gene strain and 2.3 g/L GABA with the double-gene (<em>gadB</em><sup>D304G/F433L</sup>-<em>gadC</em>) strain. Subsequent optimization of the biocatalytic conditions (60 °C, pH 3.2, 15 g/L cell biomass, 0.75 mM <span>L</span>-monosodium glutamate, 0.2 mM PLP) via single-factor and orthogonal experiments significantly increased GABA production to 54.84 g/L. Finally, implementing a whole-cell biocatalyst recycling strategy for 5 batches enabled the efficient synthesis of 382.59 g/L GABA from sodium glutamate. This study contributes to a possible industrial production of GABA by engineered strain <em>E. coli</em> BL21(DE3)/pETDuet-1-<em>gadB</em><sup>D304G/F433L</sup>.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"229 ","pages":"Article 110114"},"PeriodicalIF":3.7,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146185298","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 : 2026-05-01Epub Date: 2026-02-03DOI: 10.1016/j.bej.2026.110107
Keying Xu , Xinru Liu , Juan Zhu , Fenghe Li , Xiaoyan Liu , Jie Wang
The polymerase/nickase cycling amplification is a classic method for isothermal nucleic acid amplification, praised for its efficiency. However, its sensitivity and specificity are lacking for trace miRNA analysis. Herein, we have developed a novel modular polymerase/nickase cycling amplification technology (M-PNP) based on traditional polymerase/nickase probes (PNP), enhancing detection efficiency, sensitivity, and specificity. Specifically, M-PNP is formed by introducing a hairpin structure at the 5’ end of PNP and adding an auxiliary probe at the 3’ end. The hairpin structure acts like a lock, inhibiting non-specific nucleic acid amplification. In the presence of miRNA, the hairpin structure recognizes the miRNA and undergoes a conformational change, transforming into a new micro-hairpin structure. Through the action of polymerase and nickase, this transformation activates the polymerase/nickase cycling amplification reaction and promotes miRNA cycling, thereby enhancing detection sensitivity. The introduction of the auxiliary probe enables immediate activation of fluorescence signals during the target-triggered polymerase/nickase cycling amplification, significantly increasing detection speed and reducing time consumption. We used miR-21 from the urine of clinical kidney injury patients as the detection target and employed M-PNP for practical testing. The results showed that this platform can accurately distinguish kidney injury patients. This optimization and improvement injects new vitality and innovative ideas into the design of traditional polymerase/nickase cycling amplification probes.
{"title":"Hairpin-locked modular polymerase/nickase cycle amplification enables ultrasensitive urinary miRNA detection for acute kidney injury diagnosis","authors":"Keying Xu , Xinru Liu , Juan Zhu , Fenghe Li , Xiaoyan Liu , Jie Wang","doi":"10.1016/j.bej.2026.110107","DOIUrl":"10.1016/j.bej.2026.110107","url":null,"abstract":"<div><div>The polymerase/nickase cycling amplification is a classic method for isothermal nucleic acid amplification, praised for its efficiency. However, its sensitivity and specificity are lacking for trace miRNA analysis. Herein, we have developed a novel modular polymerase/nickase cycling amplification technology (M-PNP) based on traditional polymerase/nickase probes (PNP), enhancing detection efficiency, sensitivity, and specificity. Specifically, M-PNP is formed by introducing a hairpin structure at the 5’ end of PNP and adding an auxiliary probe at the 3’ end. The hairpin structure acts like a lock, inhibiting non-specific nucleic acid amplification. In the presence of miRNA, the hairpin structure recognizes the miRNA and undergoes a conformational change, transforming into a new micro-hairpin structure. Through the action of polymerase and nickase, this transformation activates the polymerase/nickase cycling amplification reaction and promotes miRNA cycling, thereby enhancing detection sensitivity. The introduction of the auxiliary probe enables immediate activation of fluorescence signals during the target-triggered polymerase/nickase cycling amplification, significantly increasing detection speed and reducing time consumption. We used miR-21 from the urine of clinical kidney injury patients as the detection target and employed M-PNP for practical testing. The results showed that this platform can accurately distinguish kidney injury patients. This optimization and improvement injects new vitality and innovative ideas into the design of traditional polymerase/nickase cycling amplification probes.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"229 ","pages":"Article 110107"},"PeriodicalIF":3.7,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146185326","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 : 2026-05-01Epub Date: 2026-02-07DOI: 10.1016/j.bej.2026.110113
Lei Cao , Yunfei Shao , Yang Zhou , Liang Zhao , Yan Zhou , Qian Ye , Wen-Song Tan
Advances in Chinese hamster ovary (CHO) cell line development have improved monoclonal antibody (mAb) production through efficient selection systems. These systems avoid the purification complexities and biosafety concerns of antibiotic-based approaches while improving cellular metabolism. The glutamine synthetase (GS) system reduces ammonia accumulation, whereas the tyrosine biosynthesis–based triple-marker system eliminates alkaline tyrosine feeding. Despite these distinct advantages, conventional selection strategies — particularly those relying on a single marker like GS — often lack control over the light-to-heavy chain (LC/HC) ratio, which limits mAb yield. To address this, we developed a dual‑pressure selection platform through the combined disruption of the tyrosine biosynthesis pathway (triple knockout) and the GS gene in CHO cells. Compared to single‑pressure systems in high‑density fed‑batch and semi‑perfusion cultures, the dual‑pressure cells showed consistently higher cell-specific productivity. Under nutrient‑restricted semi‑perfusion, cell‑specific productivity increased 1.59‑fold and 5.04‑fold relative to the tyrosine and GS systems, respectively, yielding higher final titers despite lower growth. Mechanistic studies indicated that the dual‑pressure system redirects central carbon and amino acid metabolism, improving glucose utilization and lowering lactate and ammonia accumulation. It also enhances LC/HC ratio control, thereby overcoming a major limitation of GS‑only selection. This integrated strategy not only simplifies high‑density bioprocessing but also advances our understanding of metabolic selection and offers a scalable platform for gene co-expression and productivity enhancement in CHO cell engineering.
{"title":"Enhancing CHO cell productivity through a dual-pressure selection system based on tyrosine and glutamine biosynthetic pathways","authors":"Lei Cao , Yunfei Shao , Yang Zhou , Liang Zhao , Yan Zhou , Qian Ye , Wen-Song Tan","doi":"10.1016/j.bej.2026.110113","DOIUrl":"10.1016/j.bej.2026.110113","url":null,"abstract":"<div><div>Advances in Chinese hamster ovary (CHO) cell line development have improved monoclonal antibody (mAb) production through efficient selection systems. These systems avoid the purification complexities and biosafety concerns of antibiotic-based approaches while improving cellular metabolism. The glutamine synthetase (GS) system reduces ammonia accumulation, whereas the tyrosine biosynthesis–based triple-marker system eliminates alkaline tyrosine feeding. Despite these distinct advantages, conventional selection strategies — particularly those relying on a single marker like GS — often lack control over the light-to-heavy chain (LC/HC) ratio, which limits mAb yield. To address this, we developed a dual‑pressure selection platform through the combined disruption of the tyrosine biosynthesis pathway (triple knockout) and the <em>GS</em> gene in CHO cells. Compared to single‑pressure systems in high‑density fed‑batch and semi‑perfusion cultures, the dual‑pressure cells showed consistently higher cell-specific productivity. Under nutrient‑restricted semi‑perfusion, cell‑specific productivity increased 1.59‑fold and 5.04‑fold relative to the tyrosine and GS systems, respectively, yielding higher final titers despite lower growth. Mechanistic studies indicated that the dual‑pressure system redirects central carbon and amino acid metabolism, improving glucose utilization and lowering lactate and ammonia accumulation. It also enhances LC/HC ratio control, thereby overcoming a major limitation of GS‑only selection. This integrated strategy not only simplifies high‑density bioprocessing but also advances our understanding of metabolic selection and offers a scalable platform for gene co-expression and productivity enhancement in CHO cell engineering.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":"229 ","pages":"Article 110113"},"PeriodicalIF":3.7,"publicationDate":"2026-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146185299","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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