Enhanced Soil Fertility and Carbon Dynamics in Organic Farming Systems: The Role of Arbuscular Mycorrhizal Fungal Abundance.

Arbuscular mycorrhizal fungi (AMF) are critical for soil ecosystem services as they enhance plant growth and soil quality via nutrient cycling and carbon storage. Considering the growing emphasis on sustainable agricultural practices, this study investigated the effects of conventional and organic farming practices on AMF diversity, abundance, and ecological functions in maize, pepper, and potato-cultivated soils. Using next-generation sequencing and quantitative PCR, we assessed AMF diversity and abundance in addition to soil health indicators such as phosphorus content, total nitrogen, and soil organic carbon. Our findings revealed that, while no significant differences in soil physicochemical parameters or AMF diversity were observed across farming systems when all crop data were combined, organic farming significantly enhances AMF abundance and fosters beneficial microbial ecosystems. These ecosystems play vital roles in nutrient cycling and carbon storage, underscoring the importance of organic practices in promoting robust AMF communities that support ecosystem services. This study not only deepens our understanding of AMF's ecological roles but also highlights the potential of organic farming to leverage these benefits for improving sustainability in agricultural practices.

The impact of bioaerosol trajectories on microbial community assembly and physicochemical dynamics in the atmosphere.

This study explored the assembly mechanisms and physicochemical dynamics of microbial communities within atmospheric bioaerosols, focusing on the influence of different aerial trajectories. Over two years, samples near Seoul were classified into 'North', 'Southwest', and 'Others' categories based on their aerial trajectories. Physicochemical analysis of the PM2.5 particles revealed distinct ion compositions for each cluster, reflecting diverse environmental influences. Microbial community analysis revealed that shared dominant bacterial phyla were present in all clusters. However, distinct taxonomic profiles and biomarkers were also evident, such as coastal bacteria in the 'Southwest' cluster correlating with wind speed, and arid soil-originated bacteria in the 'North' cluster correlating with cations. These findings demonstrate that biomarkers in each cluster are representative of the distinct environments associated with their aerial trajectories. Notably, cluster 'Southwest' the highest microbial diversity and a strong alignment with the neutral community model, suggesting a large influence of passive dispersal from marine environments. Contrarily, 'North' and 'Others' were more influenced by niche-dependent factors. This study highlights the complex interplay between environmental factors and microbial dynamics in bioaerosols and provides important insights for environmental monitoring and public health risk assessment.

Assessing long-term ecological impacts of PCE contamination in groundwater using a flow cytometric fingerprint approach.

This study aims to develop and validate a comprehensive method for assessing ecological disturbances in groundwater ecosystems caused by tetrachloroethylene (PCE) contamination, utilizing flow cytometry (FCM) fingerprint approach. We hypothesized that the ecological disturbance resulting from PCE contamination would exhibit 'press disturbance', persisting over extended periods, and inducing notable phenotypic differences in the microbial community compared to undisturbed groundwater. We collected 40 groundwater samples from industrial district with a history of over twenty years of PCE contamination, along with 56 control groundwater from the national surveillance groundwater system. FCM revealed significant alterations in the phenotypic diversity of microbial communities in PCE-contaminated groundwater, particularly during the dry season. The presence of specific dechlorinating bacteria (Dehalococcoides, Dehalogenimonas, and Geobacter) and their syntrophic partners was identified as an indicator of contamination. Phenotypic diversity measures provided clearer and more direct reflections of contamination impact compared to taxonomic diversity measures. This study establishes FCM fingerprinting as a simple, robust, and accurate method for evaluating ecological disturbances, with potential applications in early warning systems and continuous monitoring of groundwater contamination. The findings not only underscore the sensitivity of FCM in detecting phenotypic variations induced by environmental stressors but also highlight its utility in understanding the complex dynamics of microbial communities in contaminated groundwater ecosystems.

A simple protocol for cultivating the bacterivorous soil nematode Caenorhabditis elegans in its natural ecology in the laboratory.

The complex interplay between an animal and its surrounding environment requires constant attentive observation in natural settings. Moreover, how ecological interactions are affected by an animal's genes is difficult to ascertain outside the laboratory. Genetic studies with the bacterivorous nematode Caenorhabditis elegans have elucidated numerous relationships between genes and functions, such as physiology, behaviors, and lifespan. However, these studies use standard laboratory culture that does not reflect C. elegans true ecology. C. elegans is found growing in nature and reproduced in large numbers in soils enriched with rotting fruit or vegetation, a source of abundant and diverse microbes that nourish the thriving populations of nematodes. We developed a simple mesocosm we call soil-fruit-natural-habitat that simulates the natural ecology of C. elegans in the laboratory. Apples were placed on autoclaved potted soils, and after a soil microbial solution was added, the mesocosm was subjected to day-night, temperature, and humidity cycling inside a growth chamber. After a period of apple-rotting, C elegans were added, and the growing worm population was observed. We determined optimal conditions for the growth of C. elegans and then performed an ecological succession experiment observing worm populations every few days. Our data showed that the mesocosm allows abundant growth and reproduction of C. elegans that resembles populations of the nematode found in rotting fruit in nature. Overall, our study presents a simple protocol that allows the cultivation of C. elegans in a natural habitat in the laboratory for a broad group of scientists to study various aspects of animal and microbial ecology.

Case report: Intrathecal baclofen therapy improved gait pattern in a stroke patient with spastic dystonia.

Background: Intrathecal baclofen (ITB) therapy, a viable alternative for unsuitable candidates of conventional spasticity medications, is a preferred method of administration over the oral route. Owing to its enhanced bioavailability, ITB ensures a more effective delivery at the target site. Objective: There is a lack of conclusive evidence regarding the use of ITB treatment in managing ambulatory patients with spastic dystonia. Before ITB pump implantation, patients commonly undergo an ITB bolus injection trial to rule out potential adverse reactions and verify the therapeutic effects on hypertonic issues. In this report, we highlight a case of spastic dystonia, particularly focusing on an ambulatory patient who demonstrated significant improvement in both the modified Ashworth scale (MAS) score and gait pattern following the ITB injection trial. Case report: This case report outlines the medical history of a 67-year-old male diagnosed with left-side hemiplegia and spastic dystonia, resulting from his second episode of intracranial hemorrhage in the right thalamus. An ITB injection trial was initiated because the patient was not suitable for continued botulinum toxin injections and oral medications. This was due to the persistent occurrence of spastic dystonia in both the upper and lower extremities. The patient underwent a four-day ITB injection trial with progressively increasing doses, resulting in improved MAS scores and gait parameters, including cadence, step length, step time, stride length, and stride time were increased. Particularly, kinematic gait analysis demonstrates a substantial improvement of increased knee flexion in the swing phase in stiff knee gait pattern. These findings indicated a gradual reduction in spasticity-related symptoms, signifying the positive effect of the ITB injection trial. The patient eventually received an ITB pump implantation. Conclusion: In this post-stroke patient with spastic dystonia, ITB therapy has demonstrated effective and substantial management of spasticity, along with improvement in gait patterns.

Determinants of microbial colonization on microplastics through wastewater treatment processes: The role of polymer type and sequential treatment.

This study examines the microbial colonization characteristics of microplastics (MPs) in wastewater treatment plants (WWTPs), focusing on polymer types (High-Density Polyethylene (HDPE) and Polyethylene Terephthalate (PET)) and various stages of wastewater treatments. Through individual and sequential deployment approaches, the research aimed to identify the determinants of bacterial colonization on MPs, whether they were introduced at each stage of treatment individually or in sequence from primary to tertiary stages. The study revealed that the stage of wastewater treatment profoundly influenced bacterial colonization on the polymer types MPs, with bacterial attachment being largely niche-specific. HDPE showed increased sensitivity to wastewater composition, leading to selective biofilm formation. For instance, in HDPE, Firmicutes accounted for 25.1 ± 0.04 % during primary treatment, while Alphaproteobacteria increased significantly in the tertiary treatment to 19.8 ± 0.1 %. Conversely, PET exhibited a stochastic pattern of bacterial colonization due to differences in surface hydrophilicity. Additionally, in sequential deployments, a notable shift towards stochastic bacterial attachment on MPs, particularly with HDPE was observed. The Shannon diversity values for MP biofilms were consistently higher than those for wastewater across all stages, with PET showing an increase in diversity in sequential deployments (Shannon diversity: 5.01 ± 0.03 for tertiary stage). These findings highlight the critical role of MPs as carriers of diverse bacteria, emphasizing the necessity for strategies to mitigate their impact in WWTPs. This study presents a significant advancement in our understanding of the interactions between MPs and microbial populations in WWTP environments.

Environmental consequences of transformation products from an antibiotic mixture and their mitigation in a wastewater microbiome using an HCl-modified adsorbent.

This study enhanced our understanding of antibiotic mixtures' occurrence, transformation, toxicity, and ecological risks. The role of acid-modified biochar (BC) in treating antibiotic residues was explored, shedding light on how BC influences the fate, mobility, and environmental impact of antibiotics and transformation products (TPs) in an activated sludge (AS) microbiome. A mixture of oxytetracycline and sulfamethoxazole was found to synergistically (or additively) inhibit cell growth of AS and disrupt the microbiome structure, species richness/diversity, and function. The formation of TPs with potentially higher toxicity and persistence than the original compounds was identified, explaining the microbiome disruption. Agricultural waste-derived BC was optimized for contaminant adsorption, leading to a reduction in toxicity when added to AS by sequestering TPs on its surface. This work highlighted adsorbents as a practical engineering strategy for mitigating liquid-phase contaminants' toxicological consequences, proactively controlling the fate and effects of antibiotics and TPs.

Microbial interaction-induced siderophore dynamics lead to phenotypic differentiation of Staphylococcus aureus.

This study investigated the impact of microbial interactions on siderophore dynamics and phenotypic differentiation of Staphylococcus aureus under iron-deficient conditions. Optimization of media demonstrated that the glycerol alanine salts medium was best suited for analyzing the dynamics of siderophore production because of its stable production of diverse siderophore types. The effects of pH and iron concentration on siderophore yield revealed a maximum yield at neutral pH and low iron concentration (10 µg). Microbial interaction studies have highlighted variations in siderophore production when different strains (Staphylococcus epidermidis, Pseudomonas aeruginosa, and Escherichia coli) are co-cultured with S. aureus. Co-culture of S. aureus with P. aeruginosa eliminated siderophore production in S. aureus, while co-culture of S. aureus with E. coli and S. epidermidis produced one or two siderophores, respectively. Raman spectroscopy revealed that microbial interactions and siderophore dynamics play a crucial role in directing the phenotypic differentiation of S. aureus, especially under iron-deficient conditions. Our results suggest that microbial interactions profoundly influence siderophore dynamics and phenotypic differentiation and that the study of these interactions could provide valuable insights for understanding microbial survival strategies in iron-limited environments.

Phenotypic shifts induced by environmental pre-stressors modify antibiotic resistance in Staphylococcus aureus.

Introduction: Escalating prevalence of antibiotic resistance in Staphylococcus aureus has necessitated urgent exploration into the fundamental mechanisms underlying antibiotic resistance emergence, particularly in relation to its interaction with environmental stressors. This study aimed to investigate the effects of environmental stressors prior to antibiotic exposure on the antibiotic resistance of S. aureus. Methods: We used Raman spectroscopy and flow cytometry to measure prior stress-induced phenotypic alterations of S. aureus, and identified the association between phenotypic shifts and the antibiotic resistance. Results: The results revealed a multifaceted relationship between stressors and the development of antibiotic resistance. The stressors effectuate distinct phenotypic diversifications and subsequently amplify these phenotypic alterations following antibiotic treatments, contingent upon the specific mode of action; these phenotypic shifts in turn promote the development of antibiotic resistance in S. aureus. This study's findings demonstrated that the presence of pre-stress conditions triggered an augmentation of resistance to vancomycin (VAN), while concurrently attenuating resistance to norfloxacin. Marked shifts in Raman peaks associated with lipids and nucleic acids demonstrated correlations with elevated survival rates following VAN treatment. Conclusion: Consequently, these observations indicate that pre-stress conditions "prime" bacterial cells for differential responses to antibiotics and bear significant implications for formulating clinical therapeutic strategies.

Single-cell phenotypes revealed as a key biomarker in bacterial-fungal interactions: a case study of Staphylococcus and Malassezia.

IMPORTANCE: Evaluating bacterial-fungal interactions is important for understanding ecological functions in a natural habitat. Many studies have defined bacterial-fungal interactions according to changes in growth rates when co-cultivated. However, the current literature lacks detailed studies on phenotypic changes in single cells associated with transcriptomic profiles to understand the bacterial-fungal interactions. In our study, we measured the single-cell phenotypes of bacteria co-cultivated with fungi using Raman spectroscopy with its transcriptomic profiles and determined the consequence of these interactions in detail. This rapid and reliable phenotyping approach has the potential to provide new insights regarding bacterial-fungal interactions.

Prediction of groundwater quality index to assess suitability for drinking purpose using averaged neural network and geospatial analysis.

Groundwater quality management is pivotal for ensuring public health and ecological resilience. However, the conventional water quality indices often face challenges related to parameter selection, geographic coverage, and scalability. The integration of machine learning and spatial analysis represents a promising methodological shift, allowing for high accuracy and adaptive management strategies. The Safe Groundwater Project in Unsupplied Areas (2017-2020) employed a comprehensive Groundwater Quality Index (GQI) to evaluate potable groundwater quality across South Korea, utilizing a large dataset comprising 28 water quality parameters and 3552 wells. This study revealed that over 50 % of the evaluated wells (Total 8326 wells) were inappropriate as sources of drinking water, indicating a pressing need for policy revision. The averaged neural network model achieved a high predictive accuracy of approximately 95 % for GQI grades, outperforming other classification models. The introduction of 2D spatial analysis in conjunction with machine learning algorithms notably increased the predictive accuracy for unevenly distributed groundwater samples. Moreover, this combined approach enabled the intuitive visualization of groundwater vulnerability across various regions, which can inform targeted interventions for effective resource allocation and management. This research represents a methodologically robust, interdisciplinary approach that holds significant implications for a framework for future groundwater quality management and vulnerability assessment.

Continuous operation of fungal wheel reactor based on solid-state fermentation for the removal of pharmaceutical and personal care products.

Wood-rotting fungi and their enzymatic systems represent promising biocatalysts for the removal of pharmaceuticals and personal care products (PPCPs) from wastewater. We designed a fungal wheel reactor (FWR) based on solid-state fermentation (SSF) of Trametes versicolor and a lignocellulosic substrate, which was used as an immobilization carrier for fungal biomass and the sole initial nutrient source for producing fungal oxidative enzymes. Three pharmaceutical and personal care products, acetaminophen, bisphenol A and carbamazepine, were spiked into the synthetic wastewater and the treatment was carried out under non-sterile conditions. Acetaminophen was completely removed from the FWR until laccase was observed. The acetaminophen removal efficiency was retrieved by replacing the fungal wheel with fresh SSF products. Bisphenol A and carbamazepine were removed via enzymatic activity and adsorption. When the fungal wheel was replaced, acetaminophen began to be completely removed, even after laccase depletion. The microbial community analysis indicated that the continuous removal of acetaminophen was mainly due to the high proportion of T. versicolor. The relative abundance of the co-occurring microbial community might be responsible for the divergence in acetaminophen removal between two of fungal wheel-replaced reactors. Overall, FWRs are promising tools for the removal of PPCPs by highly reactive enzymatic mechanisms as well as adsorption on the carrier surface. By replacing SSF and settled microbial communities, FWRs may continuously contribute to bioremediation over a long-term period.

Bacterial Resuscitation from Starvation-Induced Dormancy Results in Phenotypic Diversity Coupled with Translational Activity Depending on Carbon Substrate Availability.

Dormancy is a survival strategy of stressed bacteria inhabiting a various environment. Frequent dormant-active transitions owing to environmental changes play an important role in functional redundancy. However, a proper understanding of the phenotypic changes in bacteria during these transitions remains to be clarified. In this study, orthogonal approaches, such as electron microscopy, flow cytometry, and Raman spectroscopy, which can evaluate phenotypic heterogeneity at the single-cell level, were used to observe morphological and molecular phenotypic changes in resuscitated cells, and RNA sequencing (RNASeq) was used to determine the genetic characteristics associated with phenotypes. Within 12 h of the resuscitation process, morphological (cell size and shape) and physiological (growth and viability) characteristics as well as molecular phenotypes (cellular components) were found to be recovered to the extent that they were similar to those in active cells. The recovery rate and detailed phenotypic properties of the resuscitated cells differed significantly depending on the type or concentration of carbon sources. RNASeq analysis revealed that genes related to translation were significantly upregulated under all resuscitation conditions. The simpler the carbon source (e.g., glucose), the higher the expression of genes involved in cellular repair, and the more complex the carbon source (e.g., beef extract), the higher the expression of genes associated with increased energy production associated with cellular aerobic respiration. This study of phenotypic plasticity of resuscitated cells provides fundamental insight into understanding the adaptive fine-tuning of the microbiome in response to environmental changes and the functional redundancy resulting from phenotype heterogeneity.

Phenotypic convergence of bacterial adaption to sub-lethal antibiotic treatment.

Microorganisms can adapt quickly to changes in their environment, leading to various phenotypes. The dynamic for phenotypic plasticity caused by environmental variations has not yet been fully investigated. In this study, we analyzed the time-series of phenotypic changes in Staphylococcus cells during adaptive process to antibiotics stresses using flow cytometry and Raman spectroscopy. The nine antibiotics with four different mode of actions were treated in bacterial cells at a sub-lethal concentration to give adaptable stress. Although the growth rate initially varied depending on the type of antibiotic, most samples reached the maximum growth comparable to the control through the short-term adaptation after 24 h. The phenotypic diversity, which showed remarkable changes depending on antibiotic treatment, converged identical to the control over time. In addition, the phenotype with cellular biomolecules converted into a bacterial cell that enhance tolerance to antibiotic stress with increases in cytochrome and lipid. Our findings demonstrated that the convergence into the phenotypes that enhance antibiotic tolerance in a short period when treated with sub-lethal concentrations, and highlight the feasibility of phenotypic approaches in the advanced antibiotic treatment.

Transcriptional Response and Plant Growth Promoting Activity of Pseudomonas fluorescens DR397 under Drought Stress Conditions.

Drought is one of the most vulnerable factors that affect crop productivity. Little is known about plant-associated microbiomes and their functional roles in assisting plant growth under drought. We investigated the genetic and transcriptomic characteristics of opportunistic beneficial microorganisms that selectively alleviate stress through plant-bacteria interactions under drought. Pseudomonas fluorescens DR397 was isolated from the drought-prone rhizospheric soil of soybean and showed high metabolic activity at -1.25 Mpa. The genome of DR397 possesses several genes related to the synthesis of compatible solutes (choline and glycine-betaine), exopolysaccharides (alginate and cellulose), and secretion systems (type II, III, IV, and VI), as well as genes related to plant growth promotion (indole-3-acetic acid, transketolase, and thiamine phosphate synthesis). The expression of these genes was significantly upregulated (8- to 263-fold change) only under drought conditions with plant root exudate treatment, whereas subtle transcriptomic changes were observed under solely root exudate treatment. When DR397 was placed on both legume cultivars (Pisum sativum and Phaseolus vulgaris), growth was hardly affected under well-watered conditions, but the shoot and root growths were increased by up from 62.0% to 149.1% compared with the control group under drought conditions. These results provide fundamental insight on the plant-bacterial interactions that alleviate plant stress as an important ecological strategy for improving drought tolerance. IMPORTANCE Drought is a serious abiotic stress on plants as wells as the microbes that coexist with plants, which significantly lowers their fitness. The plant-bacterial interaction is an important strategy to enhance their fitness under drought. However, many knowledge gaps still exist in our understanding of transcriptomic features of bacteria interacting with plant under drought. Here, by investigating the transcriptomic profiles and pot cultivation with legume, we show that the interactions of Pseudomonas fluorescens DR397 with plants change with drought. We, therefore, provide a fundamental evidence of a hidden hero in the soil that promote plant fitness from external stress.

Shifts in benthic bacterial communities associated with farming stages and a microbiological proxy for assessing sulfidic sediment conditions at fish farms.

To assess the aquaculture-induced sediment conditions associated with sulfur cycles, shifts in bacterial communities across farming stages were investigated. The sulfate reduction rate (SRR), and concentrations of acid volatile sulfide (AVS) and H2S were significantly higher at the mid- and post-farming stages than at the early stage, indicating that the aquaculture effects persist even after harvest. Incomplete organic carbon-oxidizing sulfate-reducing bacteria (IO-SRB) affiliated with Desulfobulbaceae, and gammaproteobacterial sulfur oxidizing bacteria (SOB) (Thiohalobacter, Thioprofundum, and Thiohalomonas) were dominant during the early stage, whereas fermenting bacteria (Bacteroidetes and Firmicutes) and complete oxidizing SRB (CO-SRB) belonging to Desulfobacteraceae, and epsilonproteobacterial SOB (Sulfurovum) dominated during the mid- and post-stages. The shift in SRB and SOB communities well reflected the anoxic and sulfidic conditions of farm sediment. Especially, the Sulfurovum-like SOB correlated highly and positively with H2S, AVS, and SRR, suggesting that they could be relevant microbiological proxies to assess sulfidic conditions in farm sediment.

Raman-Deuterium Isotope Probing and Metagenomics Reveal the Drought Tolerance of the Soil Microbiome and Its Promotion of Plant Growth.

Drought has become a major agricultural threat leading crop yield loss. Although a few species of rhizobacteria have the ability to promote plant growth under drought, the drought tolerance of the soil microbiome and its relationship with the promotion of plant growth under drought are scarcely studied. This study aimed to develop a novel approach for assessing drought tolerance in agricultural land by quantitatively measuring microbial phenotypes using stable isotopes and Raman spectroscopy. Raman spectroscopy with deuterium isotope probing was used to identify the Raman signatures of drought effects from drought-tolerant bacteria. Counting drought-tolerant cells by applying these phenotypic properties to agricultural samples revealed that 0% to 52.2% of all measured single cells had drought-tolerant properties, depending on the soil sample. The proportions of drought-tolerant cells in each soil type showed similar tendencies to the numbers of revived pea plants cultivated under drought. The phenotype of the soil microbiome and plant behavior under drought conditions therefore appeared to be highly related. Studying metagenomics suggested that there was a reliable link between the phenotype and genotype of the soil microbiome that could explain mechanisms that promote plant growth in drought. In particular, the proportion of drought-tolerant cells was highly correlated with genes encoding phytohormone production, including tryptophan synthase and isopentenyl-diphosphate delta-isomerase; these enzymes are known to alleviate drought stress. Raman spectroscopy with deuterium isotope probing shows high potential as an alternative technology for quantitatively assessing drought tolerance through phenotypic analysis of the soil microbiome. IMPORTANCE Soil microbiome has played a critical role in the plant survival during drought. However, the drought tolerance of soil microbiome and its ability to promote plant growth under drought is still scarcely studied. In this study, we identified the Raman signature (i.e., phenotype) of drought effects from drought-tolerant bacteria in agricultural soil samples using Raman-deuterium isotope probing (Raman-DIP). Moreover, the number of drought-tolerant cells measured by Raman-DIP was highly related to the survival rate of plant cultivation under drought and the abundance of genes encoding phytohormone production alleviating drought stress in plant. These results suggest Raman-DIP is a promising technology for measuring drought tolerance of soil microbiome. This result give us important insight into further studies of a reliable link between phenotype and genotype of soil microbiome for future plant-bacteria interaction research.

Template Engineering of Metal-to-Insulator Transitions in Epitaxial Bilayer Nickelate Thin Films.

Understanding metal-to-insulator phase transitions in solids has been a keystone not only for discovering novel physical phenomena in condensed matter physics but also for achieving scientific breakthroughs in materials science. In this work, we demonstrate that the transport properties (i.e., resistivity and transition temperature) in the metal-to-insulator transitions of perovskite nickelates are tunable via the epitaxial heterojunctions of LaNiO3 and NdNiO3 thin films. A mismatch in the oxygen coordination environment and interfacial octahedral coupling at the oxide heterointerface allows us to realize an exotic phase that is unattainable in the parent compound. With oxygen vacancy formation for strain accommodation, the topmost LaNiO3 layer in LaNiO3/NdNiO3 bilayer thin films is structurally engineered and it electrically undergoes a metal-to-insulator transition that does not appear in metallic LaNiO3. Modification of the NdNiO3 template layer thickness provides an additional knob for tailoring the tilting angles of corner-connected NiO6 octahedra and the linked transport characteristics further. Our approaches can be harnessed to tune physical properties in complex oxides and to realize exotic physical phenomena through oxide thin-film heterostructuring.

Flow cytometric monitoring of the bacterial phenotypic diversity in aquatic ecosystems.

Flow cytometry is a promising tool used to identify the phenotypic features of bacterial communities in aquatic ecosystems by measuring the physical and chemical properties of cells based on their light scattering behavior and fluorescence. Compared to molecular or culture-based approaches, flow cytometry is suitable for the online monitoring of microbial water quality because of its relatively simple sample preparation process, rapid analysis time, and high-resolution phenotypic data. Advanced statistical techniques (e.g., denoising and binning) can be utilized to successfully calculate phenotypic diversity by processing the scatter data obtained from flow cytometry. These phenotypic diversities were well correlated with taxonomic-based diversity computed using next-generation 16S RNA gene sequencing. The protocol provided in this paper should be a useful guide for a fast and reliable flow cytometric monitoring of bacterial phenotypic diversity in aquatic ecosystems.