Biochemical Engineering Research Articles

An In Vitro Hybrid Biocatalytic System Enabled by a Combination of Surface-Displayed, Purified, and Cell-Free Expressed Enzymes.

Enzymatic cascades have become a green and sustainable approach for the synthesis of valuable chemicals and pharmaceuticals. Using sequential enzymes to construct a multienzyme complex is an effective way to enhance the overall performance of biosynthetic routes. Here we report the design of an efficient in vitro hybrid biocatalytic system by assembling three enzymes that can convert styrene to (S)-1-phenyl-1,2-ethanediol. Specifically, we prepared the three enzymes in different ways, which were cell surface-displayed, purified, and cell-free expressed. To assemble them, we fused two orthogonal peptide-protein pairs (i.e., SpyTag/SpyCatcher and SnoopTag/SnoopCatcher) to the three enzymes, allowing their spatial organization by covalent assembly. By doing this, we constructed a multienzyme complex, which could enhance the production of (S)-1-phenyl-1,2-ethanediol by 3 times compared to the free-floating enzyme system without assembly. After optimization of the reaction system, the final product yield reached 234.6 μM with a substrate conversion rate of 46.9% (based on 0.5 mM styrene). Taken together, our strategy integrates the merits of advanced biochemical engineering techniques, including cellular surface display, spatial enzyme organization, and cell-free expression, which offers a new solution for chemical biosynthesis by enzymatic cascade biotransformation. We, therefore, anticipate that our approach will hold great potential for designing and constructing highly efficient systems to synthesize chemicals of agricultural, industrial, and pharmaceutical significance.

Waste-to-Energy Generation: Complex World Project Analysis

Sustainable development and the circular economy mandate efficacious management of waste. The annually increasing volumes of municipal solid waste pose a formidable global challenge. Waste-to-energy conversion, utilizing thermochemical or biochemical technologies, presents a viable solution for mitigating waste disposal concerns. This study conducts a thorough analysis of extant projects to evaluate the economic viability and environmental benefits across various technologies. Employing a self-compiled, unique database, our examination spans enterprises operational from 1980 to 2022, including 37 of the most representative facilities across Europe, North America, and East and Southeast Asia. Economic efficiency is gauged through the levelized cost of electricity generated by these installations, while environmental impacts are assessed based on the statistics on prevented greenhouse gas emissions. The methodology encompasses correlation and techno–economic analyses and expert evaluation. Contrary to conventional wisdom, our findings challenge the ubiquity of scale effects among technologies and the presumed decline in electricity generation costs with newer technologies. However, they corroborate the enhanced environmental benefits of recent technological advancements. The insights derived from this research are poised to inform strategic municipal solid waste management planning in Russia and beyond, offering a foundation for the design of new facilities. The scientific novelty of this work lies in its holistic approach to analyzing the ecological and economic efficiencies of all extant technologies.

Honeycomb-Inspired Surface-Enhanced Raman Scattering Microarray for Large-Area Automated Testing of Urease in Saliva Samples.

Surface-enhanced Raman scattering (SERS) technology, as an important analytical tool, has been widely applied in the field of chemical and biomedical sensing. Automated testing is often combined with biochemical analysis technologies to shorten the detection time and minimize human error. The present SERS substrates for sample detection are time-consuming and subject to high human error, which are not conducive to the combination of SERS and automated testing. Here, a novel honeycomb-inspired SERS microarray is designed for large-area automated testing of urease in saliva samples to shorten the detection time and minimize human error. The honeycomb-inspired SERS microarray is decorated with hexagonal microwells and a homogeneous distribution of silver nanostars. Compared with the other four common SERS substrates, the optimal honeycomb-inspired SERS microarray exhibits the best SERS performance. The RSD of 100 SERS spectra continuously collected from saliva samples is 6.56%, and the time of one detection is reduced from 5 min to 10 s. There is a noteworthy linear relationship with a R2 of 0.982 between SERS intensity and urease concentration, indicating the quantitative detection capability of the urease activity in saliva samples. The honeycomb-inspired SERS microarray, combined with automated testing, provides a new way in which SERS technology can be widely used in biomedical applications.

Spatial neurolipidomics-MALDI mass spectrometry imaging of lipids in brain pathologies.

Given the complexity of nervous tissues, understanding neurochemical pathophysiology puts high demands on bioanalytical techniques with respect to specificity and sensitivity. Mass spectrometry imaging (MSI) has evolved to become an important, biochemical imaging technology for spatial biology in biological and translational research. The technique facilitates comprehensive, sensitive elucidation of the spatial distribution patterns of drugs, lipids, peptides, and small proteins in situ. Matrix-assisted laser desorption ionization (MALDI)-based MSI is the dominating modality due to its broad applicability and fair compromise of selectivity, sensitivity price, throughput, and ease of use. This is particularly relevant for the analysis of spatial lipid patterns, where no other comparable spatial profiling tools are available. Understanding spatial lipid biology in nervous tissue is therefore a key and emerging application area of MSI research. The aim of this review is to give a concise guide through the MSI workflow for lipid imaging in central nervous system (CNS) tissues and essential parameters to consider while developing and optimizing MSI assays. Further, this review provides a broad overview of key developments and applications of MALDI MSI-based spatial neurolipidomics to map lipid dynamics in neuronal structures, ultimately contributing to a better understanding of neurodegenerative disease pathology.

Numerical modeling of magnetohydrodynamic non‐Newtonian flow in a cross‐slot

AbstractThis study numerically investigates the characteristics of a non‐Newtonian magnetohydrodynamic flow in a cross‐slot. Numerical simulations are performed using power law, Bird–Carreau and Casson non‐Newtonian fluid models. The flow characteristics and shear viscosity behavior in the flow region are analyzed for different values of magnetic field. Additionally, the dynamic behavior of the bifurcated flow in the cross‐slot is studied in detail, and the computational results are compared with existing models. It is shown that the fluid velocity is reduced in both the inlet and outlet channels of the cross‐slot due to the magnetic field, regardless of the viscosity model. Moreover, it is found that the fluid viscosity increases along the centerline of the inlet channels and decreases in the outlet channels of the cross‐slot for all non‐Newtonian fluid models tested here. Furthermore, this study shows that the flow properties of the non‐Newtonian fluids can be controlled by changing the magnetic field strength. The results of this study will be useful for analyzing the flow behavior of blood in a microfluidic cross‐slot and other rheological fluids used in biochemical engineering and industrial processes, where higher mass transfer and mixing efficiency can be achieved by imposing an external magnetic field.Highlights Simulation of non‐Newtonian magnetohydrodynamic flow in a cross‐slot is conducted. Analysis of the flow and shear viscosity behavior for different Hartman number is performed. Flow properties were successfully controlled with the imposed external magnetic field.

Advances Treatment Technologies in Chemical Industry Wastewater

The main treatment methods of chemical wastewater were reviewed. Physical, chemical, and biochemical treatment technologies effective for removing those pollutants were presented with their applicability, effectiveness and advantages. The problems facing the wastewater treatment in the petroleum chemical industry and coal chemical industry were introduced. In the end, the new progress and trend of the processes were overviewed.

Evaluation of ultimate biochemical carbon separation technology based on ultra-short sludge retention time activated sludge system

The ultra-short sludge retention time (SRT) activated sludge (USSAS) process (SRT≤4 d) can achieve efficient carbon separation and has great potential for achieving energy savings and resource recovery. Following the principles of system stabilization, organics separation, organics recovery and organics resourcing, in the present study, the effects of different SRTs (0.5, 1, 2, 3 and 4 d) and hydraulic retention times (HRTs, 1, 1.5, 2, 3 and 4 h) on the stability, adsorption capacity and resource recovery potential of the USSAS system were investigated, and biochemical carbon separation mechanisms were summarized. The results showed that the organic component content of the USSAS system was greater than that of conventional activated sludge (CAS) system, but the sludge settling performance of the sludge was poor, and the boundary conditions for stable operation were SRT= 2 d and HRT= 2 h. Once the boundary conditions were breached, the system showed potential risks of bulking. The USSAS system exhibited high organic matter separation capacity and good adsorption performance, while the carbon recovery potential showed that the carbon was bio-captured more than it was mineralized and that microbial degradation of organic matter was limited. The results of resource potential studies showed that the polyhydroxyalkanoate (PHA) content and sludge calorific value in the USSAS system were greater than those in the CAS, indicating that the USSAS system offers high resource utilization potential.

Application of (bio) chemical engineering concepts and tools to model genetic regulatory circuits, and some essential central carbon metabolism pathways in living cells. Part 4. Applications in the design of some Genetically Modified Micro-Organisms (GMOs)

In the first part of this work, the general Chemical and Biochemical Engineering (CBE) concepts and rules are briefly reviewed, together with the rules of the control theory of Nonlinear Systems (NSCT), all in the context of (i) deriving deterministic Modular Structured Kinetic Models (MSDKM) to describe the dynamics of metabolic processes in living cells, and (ii) of Hybrid Structured Modular Dynamic Models (HSMDM) (with continuous variables, linking the cell-nano-scale MSDKM state variables to the macro-scale state variables of the bioreactor dynamic model). Thus, in the HSMDM model, both prediction quality and its validity range are improved. By contrast, the current (classical/default) approach in bioengineering practice for solving design, optimization, and control problems based on the math models of industrial biological reactors is to use unstructured Monod (for cell culture reactor) or simple Michaelis-Menten (if only enzymatic reactions are retained) global kinetic models by ignoring detailed representations of metabolic cellular processes. By contrast, as reviewed, and exemplified in the second part of this work, an accurate and realistic math modelling of the dynamic individual GERMs (gene expression regulatory module), or genetic regulatory circuits (GRC), and cell-scale CCM (central carbon metabolism) key-modules can be done by only using the novel holistic ’Whole-Cell Of Variable-Volume’ (WCVV) modelling framework, under isotonic/homeostatic conditions/constraints introduced and promoted by the author. An example was given in the same Part 2 for the case study of a dynamic model for the oscillating glycolysis coupled with the Tryptophan (TRP) oscillating synthesis in the E. coli cells.

Genome-scale identification and comparative analysis of transcription factors in thermophilic cyanobacteria

BackgroundThe transcription factors (TFs) in thermophilic cyanobacteria might represent a uniquely evolved gene repertoire in light of the strong selective pressure caused by hostile habitats. Understanding the molecular composition of the TF genes in thermophilic cyanobacteria will facilitate further studies regarding verifying their exact biochemical functions and genetic engineering. However, limited information is available on the TFs of thermophilic cyanobacteria. Herein, a thorough investigation and comparative analysis were performed to gain insights into the molecular composition of the TFs in 22 thermophilic cyanobacteria.ResultsThe results suggested a fascinating diversity of the TFs among these thermophiles. The abundance and type of TF genes were diversified in these genomes. The identified TFs are speculated to play various roles in biological regulations. Further comparative and evolutionary genomic analyses revealed that HGT may be associated with the genomic plasticity of TF genes in Thermostichus and Thermosynechococcus strains. Comparative analyses also indicated different pattern of TF composition between thermophiles and corresponding mesophilic reference cyanobacteria. Moreover, the identified unique TFs of thermophiles are putatively involved in various biological regulations, mainly as responses to ambient changes, may facilitating the thermophiles to survive in hot springs.ConclusionThe findings herein shed light on the TFs of thermophilic cyanobacteria and fundamental knowledge for further research regarding thermophilic cyanobacteria with a broad potential for transcription regulations in responses to environmental fluctuations.

Prediction of Cotton Shell from Sodecoton Behavior in Thermal Conversion and Theoretical Energy Potential

This work focuses on the evaluation of the energy potential of cotton shells from SODECOTON and to predict suitable thermal conversion conditions. To achieve this goal, structural and proximate analyses were performed, while ultimate analysis, flue gas and higher heating value were calculated from models. This study reveals that cotton shells highly contain cellulose, volatile matter, lignin and carbon, which make it suitable for bioethanol, bio-oil/syngas and biochar production via biochemical and thermochemical technologies as well as fuel briquette. Heating value from cotton shell ranging from 17 MJ/kg to 19 MJ/kg, is set in the interval to be used as fuel. From the results, the value of flue gas is within acceptable limits and the pollutant emissions are less than the values of regulated threshold set. The energy potential released is around 1.30 GJ per year, 1.58 × 107 Nm3/year, 1.62 × 104 m3/year, 15.21 × E6 L and 26 × E6 L for annual combustion energy, syngas, methane, bioethanol and bio oil, respectively. Amongst them combustion via fuel briquette is suitable for less developing country such as Cameroon thus, it could supply 40% of cooking energy consumption in Garoua center town and can improve environmental sustainability in company.

Activation of LXRs alleviates neuropathic pain-induced cognitive dysfunction by modulation of microglia polarization and synaptic plasticity via PI3K/AKT pathway.

Cognitive dysfunction is a common comorbidity in patients with chronic pain. Activation of Liver X receptors (LXRs) plays a potential role in improving cognitive disorders in central nervous diseases. In this study, we investigated the role of LXRs in cognitive deficits induced by neuropathic pain. We established the spared nerve injury (SNI) model to investigate pain-induced memory dysfunction. Pharmacological activation of LXRs with T0901317 or inhibition with GSK2033 was applied. PI3K inhibitor LY294002 was administered to explore the underlying mechanism of LXRs. Changes in neuroinflammation, microglia polarization, and synaptic plasticity were assessed using biochemical technologies. We found that SNI-induced cognitive impairment was associated with reduced LXRβ expression, increased M1-phenotype microglia, decreased synaptic proteins, and inhibition of PI3K/AKT signaling pathway in the hippocampus. Activation of LXRs using T0901317 effectively alleviated SNI-induced cognitive impairment. Additionally, T0901317 promoted the polarization of microglia from M1 to M2, reduced pro-inflammatory cytokines, and upregulated synaptic proteins in the hippocampus. However, administration of GSK2033 or LY294002 abolished these protective effects of T0901317 in SNI mice. LXRs activation alleviates neuropathic pain-induced cognitive impairment by modulating microglia polarization, neuroinflammation, and synaptic plasticity, at least partly via activation of PI3K/AKT signaling in the hippocampus. LXRs may be promising targets for addressing pain-related cognitive deficits.

A neuro-fuzzy modular system for modeling nonlinear systems

The real world is nonlinear and in the control application field, this aspect needs to be resolved to build models so we need to refer to nonlinear system modeling techniques. Neuro-fuzzy systems and modular neural networks (NNs) are among the best modeling approaches for nonlinear systems. The combined features of both approaches provide better models. Thus, we propose in this paper a neuro-fuzzy modular architecture for modeling nonlinear systems. The modular architecture consists of dividing a nonlinear problem into several simpler subproblems. We assigned to each subproblem an NN. Each NN provides individual solutions that will be combined to provide a general solution to the original problem. In this respect, the decomposition of the original problem is based on a fuzzy decision mechanism. This mechanism consists of a set of fuzzy rules for processing nonlinear problems using two different strategies. The first involves training only the network weights, and the second adds the fuzzy set parameters to the training step. A comparative study of both strategies reveals the competence of the second strategy in providing better accuracy and simplicity. Using the neuro-fuzzy combination among the modular NNs reduces the complexity of the original problem and achieves much better performance. The proposed architecture is evaluated by two second-order nonlinear systems, a numerical system and a real system called “the chemical reactor,” which is used to carry out a chemical reaction not only in chemical and biochemical engineering, but also in the petrochemical industry. For both systems, the proposed approach provides better performance in terms of the learning time, learning error, and number of neurons.

Advances in Distributed Fiber Optic Biochemical Sensing Technology

Advances in Distributed Fiber Optic Biochemical Sensing Technology

Application of Molecular Biology Technologies in Environmental restoration

With the development of molecular biology, various new technologies have been developed and applied to the monitoring and management of the ecological environment. This includes the biodiversity of the oceans, soils, and plant and microbial ecosystems. This paper introduces the types of molecular biology techniques and discusses their applications in bioremediation and monitoring of contaminated environments. Conventional techniques include genetic engineering, DNA sequence analysis, electrophoresis separation and purification, nucleic acid probe detection, and polymerase chain reaction. Denaturing gradient gel electrophoresis (denaturing gradient gel electrophoresis) is a new electrophoresis technique. It is a gel system that separates DNA fragments according to their melting properties. Gene microarray technology (also known as gene chip) began to be widely used in the 1890s with biological monitoring technology. There are thousands of nucleic acid probes, which detect nucleic acids in living organisms using fluorescence or electricity, in gene microarray technology. These technologies can be used to regulate the diversity of microbial communities, solve the virus infection of plants, and explore the contaminant degradation genes and functional strains. The development and progress of modern molecular biology technology and biochemical technology have promoted the research and detection of ecological environments. These methods would become very effective tools for the study of environmental ecology.

Revealing the origins of vortex cavitation in a Venturi tube by high speed X-ray imaging

Hydrodynamic cavitation is useful in many processing applications, for example, in chemical reactors, water treatment and biochemical engineering. An important type of hydrodynamic cavitation that occurs in a Venturi tube is vortex cavitation known to cause luminescence whose intensity is closely related to the size and number of cavitation events. However, the mechanistic origins of bubbles constituting vortex cavitation remains unclear, although it has been concluded that the pressure fields generated by the cavitation collapse strongly depends on the bubble geometry. The common view is that vortex cavitation consists of numerous small spherical bubbles. In the present paper, aspects of vortex cavitation arising in a Venturi tube were visualized using high-speed X-ray imaging at SPring-8 and European XFEL. It was discovered that vortex cavitation in a Venturi tube consisted of angulated rather than spherical bubbles. The tangential velocity of the surface of vortex cavitation was assessed considering the Rankine vortex model.

Enzyme‐Polymer‐Conjugate‐Based Pickering Emulsions for Cell‐Free Expression and Cascade Biotransformation

AbstractIn this study, we addressed the limitations of conventional enzyme‐polymer‐conjugate‐based Pickering emulsions for interfacial biocatalysis, which traditionally suffer from nonspecific and uncontrollable conjugation positions that can impede catalytic performance. By introducing a non‐canonical amino acid (ncAA) at a specific site on target enzymes, we enabled precise polymer‐enzyme conjugation. These engineered conjugates then acted as biocatalytically active emulsifiers to stabilize Pickering emulsions, while encapsulating a cell‐free protein synthesis (CFPS) system in the aqueous phase for targeted enzyme expression. The resulting cascade reaction system leveraged enzymes expressed in the aqueous phase and on the emulsion interface for optimized chemical biosynthesis. The use of the cell‐free system eliminated the need for intact whole cells or purified enzymes, representing a significant advancement in biocatalysis. Remarkably, the integration of Pickering emulsion, precise enzyme‐polymer conjugation, and CFPS resulted in a fivefold enhancement in catalytic performance as compared to traditional single‐phase reactions. Therefore, our approach harnesses the combined strengths of advanced biochemical engineering techniques, offering an efficient and practical solution for the synthesis of value‐added chemicals in various biocatalysis and biotransformation applications.

Enzyme-Polymer-Conjugate-Based Pickering Emulsions for Cell-Free Expression and Cascade Biotransformation.

In this study, we addressed the limitations of conventional enzyme-polymer-conjugate-based Pickering emulsions for interfacial biocatalysis, which traditionally suffer from nonspecific and uncontrollable conjugation positions that can impede catalytic performance. By introducing a non-canonical amino acid (ncAA) at a specific site on target enzymes, we enabled precise polymer-enzyme conjugation. These engineered conjugates then acted as biocatalytically active emulsifiers to stabilize Pickering emulsions, while encapsulating a cell-free protein synthesis (CFPS) system in the aqueous phase for targeted enzyme expression. The resulting cascade reaction system leveraged enzymes expressed in the aqueous phase and on the emulsion interface for optimized chemical biosynthesis. The use of the cell-free system eliminated the need for intact whole cells or purified enzymes, representing a significant advancement in biocatalysis. Remarkably, the integration of Pickering emulsion, precise enzyme-polymer conjugation, and CFPS resulted in a fivefold enhancement in catalytic performance as compared to traditional single-phase reactions. Therefore, our approach harnesses the combined strengths of advanced biochemical engineering techniques, offering an efficient and practical solution for the synthesis of value-added chemicals in various biocatalysis and biotransformation applications.

Preface: 4th International Symposium on the Frontiers of Biotechnology and Bioengineering (FBB 2023)

This proceeding includes original and peer-reviewed research papers from the 4th International Symposium on the Frontiers of Biotechnology and Bioengineering (FBB 2023: http://www.fbbconf.org/), in Hong Kong, China during October 14-15, 2023.
 For thousands of years, humankind has used biotechnology in agriculture, food production, and medicine. Biotechnology is the research and development in the laboratory using bioinformatics for exploration, extraction, exploitation and production from any living organisms and any source of biomass by means of biochemical engineering. Bioengineering is the application of the principles of engineering and natural sciences to tissues, cells and molecules. FBB aims to bring together leading academic scientists, researchers and research scholars to exchange and share their experiences and research results on all aspects of Biotechnology and Bioengineering. It also provides a platform for academic scientists, researchers and research scholars, doctors, doctoral students and related professionals to present and discuss the most recent innovations, trends, and concerns as well as practical challenges encountered and solutions adopted in the fields of Biotechnology and Bioengineering. All authors and participants are kindly encouraged to contribute to and help shape the conference through submissions of their research abstracts, papers and posters.
 FBB 2023 received 57 submissions, and more than 20 of them had been accepted by our reviewers in FBB 2023. By submitting a paper to FBB 2023, the authors agreed to the review process and understood that papers undergo a peer-review process. Manuscripts were reviewed by appropriately qualified experts in the field selected by the Conference Committee, who gave detailed comments and-if the submission was accepted the authors would submit a revised version that took into account this feedback. All papers were reviewed using a double-blind review process: authors declared their names and affiliations in the manuscript for the reviewers to see, but reviewers did not know each other’s identities, nor did the authors receive information about who had reviewed their manuscript. The Committees of FBB 2023 invested great efforts in reviewing the papers submitted to the conference and organizing the sessions to enable the participants to gain maximum benefit.
 Hopefully, all participants and other interested readers will benefit scientifically from the proceedings and also find it stimulating in the process.
 With our warmest regards,
 F. C. Cheuk, Lv Yang
 Conference Organizing Chair

Genome-wide prediction and transcriptome analysis of sugar transporters in four ascomycete fungi

The import of plant-derived small sugars by sugar transporters (STs) has received increasing interest due to its important biological role and great industrial potential. STs are important targets of genetic engineering to improve fungal plant biomass conversion. Comparatively analysis of the genome-wide prevalence and transcriptomics of STs was performed in four filamentous fungi: Aspergillus niger, Aspergillus nidulans, Penicillium subrubescens and Trichoderma reesei. Using phylogenetic analysis and literature mining, their predicted STs were divided into ten subfamilies with putative sugar specificities assigned. In addition, transcriptome analysis revealed complex expression profiles among different STs subfamilies and fungal species, indicating a sophisticated transcriptome regulation and functional diversity of fungal STs. Several STs showed strong co-expression with other genes involved in sugar utilization, encoding CAZymes and sugar catabolic enzymes. This study provides new insights into the diversity of STs at the genomic/transcriptomic level, facilitating their biochemical characterization and metabolic engineering.

Creating Designer Engineered Extracellular Vesicles for Diverse Ligand Display, Target Recognition, and Controlled Protein Loading and Delivery.

Efficient and targeted delivery of therapeutic agents remains a bottleneck in modern medicine. Here, biochemical engineering approaches to advance the repurposing of extracellular vesicles (EVs) as drug delivery vehicles are explored. Targeting ligands such as the sugar GalNAc are displayed on the surface of EVs using a HaloTag-fused to a protein anchor that is enriched on engineered EVs. These EVs are successfully targeted to human primary hepatocytes. In addition, the authors are able to decorate EVs with an antibody that recognizes a GLP1 cell surface receptor by using an Fc and Fab region binding moiety fused to an anchor protein, and they show that this improves EV targeting to cells that overexpress the receptor. The authors also use two different protein-engineering approaches to improve the loading of Cre recombinase into the EV lumen and demonstrate that functional Cre protein is delivered into cells in the presence of chloroquine, an endosomal escape enhancer. Lastly, engineered EVs are well tolerated upon intravenous injection into mice without detectable signs of liver toxicity. Collectively, the data show that EVs can be engineered to improve cargo loading and specific cell targeting, which will aid their transformation into tailored drug delivery vehicles.