When ARTP Meets Microfluidics: Recent Advances in Next-Generation High-Throughput Microbial Selection Systems

Introduction

In today’s era of rapid advancements in biomanufacturing and synthetic biology, microorganisms serve as highly efficient ‘cellular factories’, and their performance directly determines the yield and economic viability of the target products—be they pharmaceuticals, chemicals, fuels or food additives. Traditional microbial breeding methods, such as chemical mutagenesis and radiation mutagenesis, are often hampered by bottlenecks such as uncontrollable mutations, inefficient screening and limited throughput. How to rapidly and efficiently obtain high-performance industrial strains has long been a central challenge in this field.

In recent years, the convergence of two cutting-edge technologies has offered a breakthrough solution to this challenge: Atmospheric and room-temperature plasma (ARTP) mutagenesis technology, with its highly efficient and safe physical mutagenic properties, provides a powerful engine for generating genetic diversity; whilst droplet-based microfluidics (DBMF) technology, with its ultra-high throughput, miniaturisation and precise manipulation capabilities, equips the screening of vast numbers of mutants with a ‘microscope’ and a ‘robotic arm’. On 30 December 2025, a review article titled ‘Advances in high-throughput mutation breeding systems integrating atmospheric and room-temperature plasma (ARTP) with droplet-based microfluidics’, published in the journal *Microbial Cell Factories*, systematically outlined the principles, advantages and broad application prospects of this integrated system across multiple fields, sketching out a new blueprint for the intelligent future of microbial breeding.

I. Bottlenecks in Traditional Breeding and the New Paradigm of Integrated Systems

Microbial breeding plays a cornerstone role in fields such as fermentation engineering, pharmaceuticals and environmental protection. Traditional methods, such as chemical mutagenesis, are constrained by the specificity and concentration of reagents, making mutations difficult to predict; radiation mutagenesis is often accompanied by high mortality rates, which impair cell viability; whilst transgenic technology, although capable of selectively enhancing traits, faces challenges regarding regulatory compliance and public acceptance. These limitations have created an urgent need for advanced mutagenesis and screening systems.

Against this backdrop, the integration of ARTP with droplet microfluidics has emerged. ARTP technology can rapidly induce a wide range of mutations in microorganisms at ambient temperature and pressure. Its operating principle is as follows: when an inert gas flows between two exposed metal electrodes, under the influence of an applied radio-frequency electric field, electrons gain energy and undergo elastic and inelastic collisions with neutral gas molecules, causing the gas to ionise and form a plasma. The active particles within this plasma can effectively interact with the genetic material of cells, causing damage to the DNA structure. This, in turn, utilises the cells’ own highly error-tolerant repair mechanisms to generate a large number of mutation sites, ultimately yielding a high-capacity gene mutation library.

Figure 1 ǀ Schematic diagram of an atmospheric and room-temperature plasma (ARTP) generation system. This figure illustrates the structure and operating principle of the ARTP generation system, which primarily comprises a plasma generator, a gas flow system and a power supply unit.

Droplet microfluidics represents an advanced branch of microfluidics. Its core lies in the precise manipulation of two immiscible fluid phases to generate, in situ within micro-scale channels, thousands to millions of discrete microdroplets, each of which functions as an independent, isolated micro-reactor. Leveraging the synergistic advantages of miniaturisation, parallelisation and isolation, this technology provides a highly promising platform for high-throughput screening in microbial engineering.

Figure 2 ǀ Droplet Operating System (A: Droplet generation; B: Generation of droplets with multiple concentration gradients; C: Droplet splitting and fusion; D: Droplet sorting)

A. The droplet generation process demonstrates how droplets are generated using specific equipment and techniques. B. The generation of droplets with multiple concentration gradients demonstrates how droplets of different concentrations can be generated within the same system to meet the requirements of subsequent experiments. C. The process of droplet splitting and fusion illustrates how the physical properties of droplets can be controlled to achieve splitting and fusion, thereby altering their volume and composition. D. Droplet sorting demonstrates how specific methods can be used to classify and sort droplets with different characteristics, facilitating subsequent analysis and experiments.

II. Core Operating Principles of the ARTP-DBMF Integrated System

This integrated system establishes a robust closed-loop platform that seamlessly bridges random mutagenesis and high-resolution screening through three functionally coupled stages, thereby accelerating the ‘mutagenesis–screening–learning’ cycle by several orders of magnitude.

Stage 1: ARTP Mutagenesis. ARTP exposes microbial cells to active plasma species at atmospheric pressure, inducing a broad range of genomic mutations through a non-contact and gentle treatment. This process generates a genetically diverse mutant library whilst maintaining high cell survival rates, providing the necessary genetic foundation for downstream microdroplet encapsulation.

Stage Two: Droplet Encapsulation and Cultivation. Microfluidic droplet technology disperses and encapsulates the ARTP-induced mutants into picolitre-scale droplets. Each droplet functions as an independent cultivation and reaction unit, completely eliminating the effects of intercellular growth rate differences and interspecies competition, whilst allowing for the rapid accumulation of metabolites, thereby activating concentration-dependent physiological pathways such as quorum sensing. The microfluidic device can generate highly uniform droplets at a rate exceeding 20,000 per second, enabling high-throughput analysis.

Stage Three: High-throughput screening. During cultivation, target phenotypes within the droplets are dynamically monitored via on-chip fluorescent probes or absorbance sensors. Subsequently, an active sorting mechanism isolates high-performance mutants at a rate exceeding 20,000 droplets per hour—a process that is 10⁴ to 10⁵ times faster than microplate-based detection methods.

The synergy of the entire system lies in the fact that ARTP provides a vast mutation breadth, whilst droplet microfluidics enables ultra-high-throughput single-cell phenotypic analysis within a controlled microenvironment. This closed-loop process, spanning from mutation induction to precise screening, significantly accelerates the strain improvement cycle.

Figure 3 ǀ The integrated ARTP-DBMF system. This figure illustrates the structure and functionality of the integrated ARTP-DBMF system. The system combines artificial intelligence technology with biomedical research, with the aim of improving experimental efficiency and the accuracy of data analysis. Through real-time monitoring and data processing, the system is able to optimise experimental conditions and facilitate the analysis and processing of biological samples.

III. Seven Key Advantages of the Integrated System

Compared with traditional methods, the ARTP-DBMF integrated system offers a range of synergistic advantages:

. High mutagenic diversity whilst maintaining viability: ARTP mutagenesis technology can induce effective genomic mutations in large numbers of microorganisms or cells within a short period of time, without the need for harsh chemical reagents or causing thermal damage, thereby preserving the physiological integrity of the microbial cells. This enables almost all mutants to undergo immediate downstream encapsulation and growth within the droplets.

. Precise screening and high-throughput detection: In droplet microfluidics, each ARTP-derived mutant is isolated within an oil-in-water droplet, eliminating inter-cell interference and enabling precise measurement of enzyme activity, metabolic output or stress resistance. Compared with flow cytometry, the droplet method allows for the simultaneous assessment of enzyme expression, stability, stereoselectivity and substrate specificity within a fully customisable microenvironment.

. Diversity and Adaptability: The system generates a high degree of genetic diversity during the mutagenesis process. The diverse phenotypes resulting from random mutations provide researchers with a rich pool of material from which to select, enabling them to optimise microbial performance more flexibly according to research objectives and practical requirements.

. Adaptive and Evolvable Feedback Loop: The combination of ARTP-induced genetic variation and real-time phenotypic analysis enabled by droplet microfluidics forms an adaptive cycle: the optimal mutants identified in each round inform subsequent mutagenesis parameters, thereby enabling directed evolution with minimal human intervention.

. Extremely low reagent consumption and operational costs: By coupling the rapid, uniform mutagenesis of ARTP with the parallel droplet generation enabled by droplet microfluidics, the overall screening speed is 10⁴ to 10⁵ times faster than traditional microplate assays. At the same time, reagent consumption can be reduced by a factor of 10⁶ to 10⁷, making large-scale mutant screening economically viable.

. Environmentally friendly and safe: Unlike traditional chemical mutagens, ARTP mutagenesis employs a physical method, thereby avoiding potential environmental pollution and health risks to operators. The closed nature of the microfluidic system further minimises aerosol exposure to biological materials, providing additional safety assurance for operators.

. Simplified Operational Workflow: The integration of ARTP with droplet microfluidic culture technology simplifies the mutation breeding process and reduces experimental complexity. Researchers can utilise automated equipment and data analysis software to achieve efficient screening and cultivation of mutants, thereby further enhancing laboratory productivity and the reliability of results.

IV. Successful Applications in Biosynthesis and Fermentation Engineering

This system has yielded significant results in enhancing the synthetic capacity of microbial products; specific applications can be summarised as follows:

1. Selection and breeding of high-yield enzyme and metabolite strains:

· Erythritol: By integrating ARTP mutagenesis with the automated microbial adaptive evolution instrument (EVOL cell), a growth-coupled, biosensor-guided adaptive evolution platform was established to enhance erythritol synthesis in Yarrowia lipolytica. The engineered strain G31 achieved an erythritol titer of 220.5 g/L in a 5 L bioreactor.

· Chitosanase: Using ARTP mutagenesis on Bacillus cereus, a high-yield chitosanase-producing strain was screened, with enzyme activity increased by a factor of 3.66.

· Coenzyme Q10: ARTP mutagenesis was performed on Rhodobacter sphaeroides, yielding the mutant R.S 17, which exhibited an 80.37 per cent increase in product yield. In batch fermentation with feed supplementation, the coenzyme Q10 concentration reached 236.7 mg/L.

· Chitin deacetylase: By combining ARTP mutagenesis with a microbial microdroplet culture system, the Rhodococcus maris strain B4 was selected, with a maximum CDA yield 3.15 times that of the parent strain.

2. Development of high-yield amino acid-producing strains:

·L-histidine: Wild-type *Bacillus glutamicus* was mutagenised using ARTP, and following automated high-throughput microbial microdroplet culture and plate screening, the resistant mutant Cg-F4 was obtained, with an L-histidine yield of (0.561 ± 0.016) g/L.

· L-glutamine: Through ARTP mutagenesis and high-throughput screening, a strain of *Glutamic acid bacillus* was obtained with an L-glutamine yield of 25.7 ± 2.7 g/L.

· Microalgal amino acids: Directed breeding of Auxenochlorella pyrenoidosa was carried out using ARTP mutagenesis combined with a high-throughput microbial microdroplet culture system, resulting in the high-yield amino acid mutant strain MMC-8, with a total amino acid content of 44.35 per cent of dry weight.

3. Construction of high-yield antibiotic and vitamin-producing strains:

· Macrolide antibiotics: By combining ARTP mutagenesis with a microbial microdroplet culture system, high-throughput screening and adaptive evolution were carried out on Bacillus siamensis, resulting in the mutant strain IMD4036, whose macrolide yield is 3.0 times that of the parental strain.

· Vitamin B12: ARTP mutagenesis was used to enhance vitamin B12 biosynthesis in Ensifer adhaerens; the vitamin B12 titre of the optimal mutant, BCA-24, increased significantly from 65.64 mg/L to 104.54 mg/L.

4. Enhancement of fermentation engineering strains:

· Tolerance of Saccharomyces cerevisiae: By integrating ARTP mutagenesis with a microbial microdroplet culture system, the tolerance of Saccharomyces cerevisiae to inhibitors during lignocellulosic ethanol fermentation was improved, resulting in the mutant strain M8, which exhibits strong stress resistance.

Yeast single-cell protein: Utilising ARTP mutagenesis combined with microfluidic fluorescence-activated droplet sorting technology based on green fluorescent protein (GFP) and red fluorescent protein (RFP), mutagenic breeding was conducted on Saccharomyces cerevisiae, yielding mutant strains HF5 and UD11, whose protein content increased by 7.40 per cent and 10.92 per cent, respectively.

V. Innovative Applications in the Bio-feed Industry

This system has demonstrated considerable potential in the screening and cultivation of high-quality feed microbial strains. Its main areas of application include:

1. Engineering of probiotic strains:

· Clostridium butyricum: Through ARTP mutagenesis, Clostridium butyricum was engineered to yield mutant strains with enhanced antimicrobial activity and improved tolerance to high temperatures and bile salts.

· Bacillus coagulans: By combining ARTP mutagenesis with adaptive evolution, acid- and bile salt-tolerant mutant strains of Bacillus coagulans were developed. When applied to solid-state fermentation of soya bean meal, these mutant strains improved nutritional quality, increasing the content of acid-soluble protein, crude protein and total amino acids by 18.2 per cent, 12.7 per cent and 9.8 per cent, respectively.

· Lactobacillus plantarum: Through ARTP mutagenesis, Lactobacillus plantarum mutant strains with enhanced antimicrobial activity were obtained; their growth rate increased by 19.4 per cent and their antimicrobial rate by 37.5 per cent.

2. Production of antibiotic alternatives:

· Salinomycin: Through ARTP and ribosome engineering, a breeding process for Streptomyces griseus was established. The mutant strain Tet30Chl25 achieved a salinomycin yield of 34,712 mg/L, more than 2.0 times that of the parent strain S12.

VI. Extended Applications in Edible Fungus Breeding and Environmental Remediation

The scope of application of this system has extended beyond traditional microorganisms to more complex biological systems and environmental fields.

1. Breeding for Increased Yield of Bioactive Compounds in Edible Fungi:

· Ganoderma triterpenes: Using ARTP mutagenesis combined with a microbial microdroplet culture system for directed breeding of the Ganoderma strain G0023, mutant strains YB05 and YB18 were successfully screened; their triterpene yields increased significantly by 32.10 per cent and 15.72 per cent, respectively.

· Phellinus linteus flavonoids and polysaccharides: Through ARTP mutagenesis of its protoplasts, a high-yield flavonoid mutant strain, A67, was obtained, with an 88.24 per cent increase in intracellular flavonoid yield. Similarly, ARTP-mutagenised Hericium erinaceus strains exhibited increases in fermented mycelial polysaccharide content of 23.25 per cent and 47.45 per cent, respectively.

2. Targeted selection of strains for environmental remediation:

This system also performs exceptionally well in engineering microorganisms to enhance their environmental remediation capabilities:

· Hydrocarbon degradation: Combining UV and plasma mutagenesis with a high-throughput screening method based on a multi-functional microplate reader and dual-wavelength UV spectroscopy, highly efficient total hydrocarbon-degrading bacteria were identified, achieving a degradation rate of up to 85.1 per cent—a 48 per cent improvement over the wild-type strain.

· Heavy metal tolerance and adsorption: Through ARTP mutagenesis, a Bacillus velezensis mutant strain with significantly enhanced chromium resistance was obtained; its minimum inhibitory concentration (MIC) for chromium increased from 80 mg/L in the parent strain to 400 mg/L. In another study, the Pseudomonas fluorescens mutant strain T4-2, obtained using ARTP technology, exhibited significantly increased extracellular polymer production and flocculation activity, as well as high adsorption capacity for chromium and cadmium.

· Biological control: Utilising a high-throughput screening system combining ARTP mutagenesis with a droplet-encapsulated microfluidic cell sorter, the Bacillus safensis mutant strain R1-15 was optimised and selected from a soil microbial community; this strain exhibited a 42.82 per cent increase in the diameter of its antibacterial zone and a 62 per cent enhancement in antimicrobial activity.

VII. Future Prospects and Challenges

Although the high-throughput mutagenic breeding system integrating ARTP with droplet microfluidics has demonstrated immense potential, research in this field remains at an early stage. Most existing studies still utilise either ARTP or droplet microfluidics technology in isolation, with only a few having achieved iterative coupling within a closed-loop evolutionary framework. Furthermore, challenges persist regarding system scalability, long-term genetic stability, process parameter robustness and industrial implementation.

Nevertheless, this integrated strategy offers unique advantages in accelerating random mutagenesis, enabling screening with single-cell precision, and enhancing evolutionary efficiency. With continuous advancements in related technologies, the system is expected to find applications in a wider range of fields, thereby increasing its practical value. Future developments may focus on deeper integration of automation and intelligence, such as utilising machine learning models to analyse high-throughput screening data and retrospectively predict optimal ARTP mutagenesis parameters, thereby establishing a fully autonomous, self-optimising intelligent breeding cycle.

Conclusion

The integration of ARTP with droplet microfluidics marks the dawn of a new ‘ultra-high-throughput’ era in microbial breeding. This represents not merely a technical combination, but a revolution in the research paradigm—integrating the two previously separate key steps of ‘generating diversity’ and ‘screening for functionality’ into a single, highly efficient, precise and controllable continuous-flow platform. From enhancing the synthesis of enzymes and chemicals to developing novel feed additives and environmental remediation agents, this system is demonstrating its powerful driving force across multiple dimensions of biomanufacturing.

As our understanding of this system deepens and our engineering capabilities improve, we can look forward to designing even smarter and more efficient ‘next-generation cell factories’ in the future, providing a core driving force for a sustainable bioeconomy. This breeding revolution, driven jointly by physical stimulation and microfluidic control, is quietly reshaping our blueprint for modifying and utilising microorganisms.