Aerosolization of micro- and nanoplastics via sea spray: Investigating the role of polymer type, size, and concentration, and potential implications for human exposure.

Micro- and nanoplastics (MNPs) can enter the atmosphere via sea spray aerosols (SSAs), but the effects of plastic characteristics on the aerosolization process are unclear. Furthermore, the importance of the transport of MNPs via these SSAs as a possible new exposure route for human health remains unknown. The aim of this study was two-fold: (1) to examine if a selection of factors affects aerosolization processes of MNPs, and (2) to estimate human exposure to MNPs via aerosols inhalation. A laboratory-based bubble bursting mechanism, simulating the aerosolization process at sea, was used to investigate the influence of MNP as well as seawater characteristics. To determine the potential human exposure to microplastics via inhalation of SSAs, the results of the laboratory experiments were extrapolated to the field based on sea surface microplastic concentrations and the volume of inhaled aerosols. Enrichment seemed to be influenced by MNP size, concentration and polymer type. With higher enrichment for smaller particles and denser polymers. Experiments with different concentrations showed a larger range of variability but nonetheless lower concentrations seemed to result in higher enrichment, presumably due to lower aggregation. In addition to the MNP characteristics, the type of seawater used seemed to influence the aerosolization process. Our human exposure estimate to microplastic via inhalation of sea spray aerosols shows that in comparison with reported inhaled concentrations in urban and indoor environments, this exposure route seems negligible for microplastics. Following the business-as-usual scenario on plastic production, the daily plastic inhalation in coastal areas in 2100 is estimated to increase but remain far below 1 particle per day. This study shows that aerosolization of MNPs is a new plastic transport pathway to be considered, but in terms of human exposure it seems negligible compared to other more important sources of MNPs, based on current reported environmental concentrations.

Cooperative interactions between invader and resident microbial community members weaken the negative diversity-invasion relationship.

The negative diversity-invasion relationship observed in microbial invasion studies is commonly explained by competition between the invader and resident populations. However, whether this relationship is affected by invader-resident cooperative interactions is unknown. Using ecological and mathematical approaches, we examined the survival and functionality of Aminobacter niigataensis MSH1 to mineralize 2,6-dichlorobenzamide (BAM), a groundwater micropollutant affecting drinking water production, in sand microcosms when inoculated together with synthetic assemblies of resident bacteria. The assemblies varied in richness and in strains that interacted pairwise with MSH1, including cooperative and competitive interactions. While overall, the negative diversity-invasion relationship was retained, residents engaging in cooperative interactions with the invader had a positive impact on MSH1 survival and functionality, highlighting the dependency of invasion success on community composition. No correlation existed between community richness and the delay in BAM mineralization by MSH1. The findings suggest that the presence of cooperative residents can alleviate the negative diversity-invasion relationship.

Impact of operational conditions on drinking water biofilm dynamics and coliform invasion potential.

Biofilms within drinking water distribution systems serve as a habitat for drinking water microorganisms. However, biofilms can negatively impact drinking water quality by causing water discoloration and deterioration and can be a reservoir for unwanted microorganisms. In this study, we investigated whether indicator organisms for drinking water quality, such as coliforms, can settle in mature drinking water biofilms. Therefore, a biofilm monitor consisting of glass rings was used to grow and sample drinking water biofilms. Two mature drinking water biofilms were characterized by flow cytometry, ATP measurements, confocal laser scanning microscopy, and 16S rRNA sequencing. Biofilms developed under treated chlorinated surface water supply exhibited lower cell densities in comparison with biofilms resulting from treated groundwater. Overall, the phenotypic as well as the genotypic characteristics were significantly different between both biofilms. In addition, the response of the biofilm microbiome and possible biofilm detachment after minor water quality changes were investigated. Limited changes in pH and free chlorine addition, to simulate operational changes that are relevant for practice, were evaluated. It was shown that both biofilms remained resilient. Finally, mature biofilms were prone to invasion of the coliform, Serratia fonticola. After spiking low concentrations (i.e., ±100 cells/100 mL) of the coliform to the corresponding bulk water samples, the coliforms were able to attach and get established within the mature biofilms. These outcomes emphasize the need for continued research on biofilm detachment and its implications for water contamination in distribution networks. IMPORTANCE: The revelation that even low concentrations of coliforms can infiltrate into mature drinking water biofilms highlights a potential public health concern. Nowadays, the measurement of coliform bacteria is used as an indicator for fecal contamination and to control the effectiveness of disinfection processes and the cleanliness and integrity of distribution systems. In Flanders (Belgium), 533 out of 18,840 measurements exceeded the established norm for the coliform indicator parameter in 2021; however, the source of microbial contamination is mostly unknown. Here, we showed that mature biofilms, are susceptible to invasion of Serratia fonticola. These findings emphasize the importance of understanding and managing biofilms in drinking water distribution systems, not only for their potential to influence water quality, but also for their role in harboring and potentially disseminating pathogens. Further research into biofilm detachment, long-term responses to operational changes, and pathogen persistence within biofilms is crucial to inform strategies for safeguarding drinking water quality.

Oral Biofilm Composition, Dissemination to Keratinocytes, and Inflammatory Attenuation Depend on Probiotic and Synbiotic Strain Specificity.

Several inflammatory diseases are characterized by a disruption in the equilibrium between the host and its microbiome. Due to the increase in resistance, the use of antibiotics for the widespread, nonspecific killing of microorganisms is at risk. Pro-microbial approaches focused on stimulating or introducing beneficial species antagonistic toward pathobionts may be a viable alternative for restoring the host-microbiome equilibrium. Unfortunately, not all potential probiotic or synbiotic species and even subspecies (to strain level) are equally effective for the designated pathology, leading to conflicting accounts of their efficacy. To assess the extent of these species- and strain-specific effects, 13 probiotic candidates were evaluated for their probiotic and synbiotic potential with glycerol on in vitro oral biofilms, dissemination from biofilms to keratinocytes, and anti-inflammatory activity. Species- and strain-specific effects and efficacies were observed in how they functioned as probiotics or synbiotics by influencing oral pathobionts and commensals within biofilms and affected the dissemination of pathobionts to keratinocytes, ranging from ineffective strains to strains that reduced pathobionts by 3 + log. In addition, a minority of the candidates exhibited the ability to mitigate the inflammatory response of LPS-stimulated monocytes. For a comprehensive assessment of probiotic therapy for oral health, a judicious selection of fully characterized probiotic strains that are specifically tailored to the designated pathology is required. This approach aims to challenge the prevailing perception of probiotics, shifting the focus away from "form over function." Rather than using unproven, hypothetical probiotic strains from known genera or species, one should choose strains that are actually functional in resolving the desired pathology before labelling them probiotics.

The immunostimulatory activity of sea spray aerosols: bacteria and endotoxins activate TLR4, TLR2/6, NF-κB and IRF in human cells.

Frequent exposure to sea spray aerosols (SSA) containing marine microorganisms and bioactive compounds may influence human health. However, little is known about potential immunostimulation by SSA exposure. This study focuses on the effects of marine bacteria and endotoxins in SSA on several receptors and transcription factors known to play a key role in the human innate immune system. SSA samples were collected in the field (Ostend, Belgium) or generated in the lab using a marine aerosol reference tank (MART). Samples were characterized by their sodium contents, total bacterial counts, and endotoxin concentrations. Human reporter cells were exposed to SSA to investigate the activation of toll-like receptor 4 (TLR4) in HEK-Blue hTLR4 cells and TLR2/6 in HEK-Blue hTLR2/6 cells, as well as the activation of nuclear factor kappa B (NF-κB) and interferon regulatory factors (IRF) in THP1-Dual monocytes. These responses were then correlated to the total bacterial counts and endotoxin concentrations to explore dose-effect relationships. Field SSA contained from 3.0 × 103 to 6.0 × 105 bacteria/m3 air (averaging 2.0 ± 1.9 × 105 bacteria/m3 air) and an endotoxin concentration ranging from 7 to 1217 EU/m3 air (averaging 389 ± 434 EU/m3 air). In contrast, MART SSA exhibited elevated levels of total bacterial count (from 2.0 × 105 to 2.4 × 106, averaging 7.3 ± 5.5 × 105 cells/m3 air) and endotoxin concentration from 536 to 2191 (averaging 1310 ± 513 EU/m3 air). SSA samples differentially activated TLR4, TLR2/6, NF-κB and IRF. These immune responses correlated dose-dependently with the total bacterial counts, endotoxin levels, or both. This study sheds light on the immunostimulatory potential of SSA and its underlying mechanisms, highlighting the need for further research to deepen our understanding of the health implications of SSA exposure.

Unlocking the mechanism of action: a cost-effective flow cytometry approach for accelerating antimicrobial drug development.

Antimicrobial resistance is one of the greatest challenges to global health. While the development of new antimicrobials can combat resistance, low profitability reduces the number of new compounds brought to market. Elucidating the mechanism of action is crucial for developing new antimicrobials. This can become expensive as there are no universally applicable pipelines. Phenotypic heterogeneity of microbial populations resulting from antimicrobial treatment can be captured through flow cytometric fingerprinting. Since antimicrobials are classified into limited groups, the mechanism of action of known compounds can be used for predictive modeling. We demonstrate a cost-effective flow cytometry approach for determining the mechanism of action of new compounds. Cultures of Actinomyces viscosus and Fusobacterium nucleatum were treated with different antimicrobials and measured by flow cytometry. A Gaussian mixture mask was applied over the data to construct phenotypic fingerprints. Fingerprints were used to assess statistical differences between mechanism of action groups and to train random forest classifiers. Classifiers were then used to predict the mechanism of action of cephalothin. Statistical differences were found among the different mechanisms of action groups. Pairwise comparison showed statistical differences for 35 out of 45 pairs for A. viscosus and for 32 out of 45 pairs for F. nucleatum after 3.5 h of treatment. The best-performing random forest classifier yielded a Matthews correlation coefficient of 0.92 and the mechanism of action of cephalothin could be successfully predicted. These findings suggest that flow cytometry can be a cheap and fast alternative for determining the mechanism of action of new antimicrobials.IMPORTANCEIn the context of the emerging threat of antimicrobial resistance, the development of novel antimicrobials is a commonly employed strategy to combat resistance. Elucidating the mechanism of action of novel compounds is crucial in this development but can become expensive, as no universally applicable pipelines currently exist. We present a novel flow cytometry-based approach capable of determining the mechanism of action swiftly and cost-effectively. The workflow aims to accelerate drug discovery and could help facilitate a more targeted approach for antimicrobial treatment of patients.

Detection of antibiotic residues in groundwater with a validated multiresidue UHPLC-MS/MS quantification method.

The occurrence of antibiotic residues in the environment has received considerable attention because of their potential to select for bacterial resistance. The overuse of antibiotics in human medicine and animal production results in antibiotic residues entering the aquatic environment, but concentrations are currently not well determined. This study investigates the occurrence of antibiotics in groundwater in areas strongly related to agriculture and the antibiotic treatment of animals. A multiresidue method was validated according to EU Regulation 2021/808, to allow (semi-)quantitative analysis of 78 antibiotics from 10 different classes: ß-lactams, sulfonamides, tetracyclines, lincosamides, amphenicols, (fluoro)quinolones, macrolides, pleuromutilins, ansamycins and diaminopyrimidines using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). This method was used to test different storage conditions of these water samples during a stability study over a period of 2 weeks. Sulfonamides, lincosamides and pleuromutilins were the most stable. Degradation was most pronounced for ß-lactam antibiotics, macrolides and ansamycins. To maintain stability, storage of samples at -18 °C is preferred. With the validated method, antibiotic residues were detected in groundwater, sampled from regions associated with intensive livestock farming in Flanders (Belgium). Out of 50 samples, 14% contained at least one residue. Concentrations were low, ranging from < LOD to 0.03 µg/L. Chloramphenicol, oxolinic acid, tetracycline and sulfonamides (sulfadiazine, sulfadoxine, sulfamethazine and sulfisoxazole) were detected. This study presents a new method for the quantification of antibiotic residues, which was applied to investigate the presence of antibiotic residues in groundwater in Flanders.

Bacterially enhanced plant-growing media for controlled environment agriculture.

Microbe-plant interactions in the root zone not only shape crop performance in soil but also in hydroponic cultivation systems. The biological and physicochemical properties of the plant-growing medium determine the root-associated microbial community and influence bacterial inoculation effectiveness, which affects plant growth. This study investigated the combined impact of plant-growing media composition and bacterial community inoculation on the root-associated bacterial community of hydroponically grown lettuce (Lactuca sativa L.). Ten plant-growing media were composed of varying raw materials, including black peat, white peat, coir pith, wood fibre, composted bark, green waste compost, perlite and sand. In addition, five different bacterial community inocula (BCI S1-5) were collected from the roots of lettuce obtained at different farms. After inoculation and cultivation inside a vertical farm, lettuce root-associated bacterial community structures, diversity and compositions were determined by evaluating 16S rRNA gene sequences. The study revealed distinct bacterial community structures among experimental replicates, highlighting the influence of raw material variations on root-associated bacterial communities, even at the batch level. However, bacterial community inoculation allowed modulation of the root-associated bacterial communities independently from the plant-growing medium composition. Bacterial diversity was identified as a key determinant of plant growth performance with green waste compost introducing Bacilli and Actinobacteria, and bacterial community inoculum S3 introducing Pseudomonas, which positively correlated with plant growth. These findings challenge the prevailing notion of hydroponic cultivation systems as sterile environments and highlight the significance of proper plant-growing media raw material selection and bacterial community inoculation in shaping root-associated microbiomes that provide stability through microbial diversity. This study supports the concept of creating bacterially enhanced plant-growing media to promote plant growth in controlled environment agriculture.

Addition of (bio)surfactants in the biofiltration of hydrophobic volatile organic compounds in air.

The removal of volatile organic compounds (VOCs) in air is of utmost importance to safeguard both environmental quality and human well-being. However, the low aqueous solubility of hydrophobic VOCs results in poor removal in waste gas biofilters (BFs). In this study, we evaluated the addition of (bio)surfactants in three BFs (BF1 and BF2 mixture of compost and wood chips (C + WC), and BF3 filled with expanded perlite) to enhance the removal of cyclohexane and hexane from a polluted gas stream. Experiments were carried out to select two (bio)surfactants (i.e., Tween 80 and saponin) out of five (sodium dodecyl sulfate (SDS), Tween 80, surfactin, rhamnolipid and saponin) from a physical-chemical (i.e., decreasing VOC gas-liquid partitioning) and biological (i.e., the ability of the microbial consortium to grow on the (bio)surfactants) point of view. The results show that adding Tween 80 at 1 critical micelle concentration (CMC) had a slight positive effect on the removal of both VOCs, in BF1 (e.g., 7.0 ± 0.6 g cyclohexane m-3 h-1, 85 ± 2% at 163 s; compared to 6.7 ± 0.4 g cyclohexane m-3 h-1, 76 ± 2% at 163 s and 0 CMC) and BF2 (e.g., 4.3 ± 0.4 g hexane m-3 h-1, 27 ± 2% at 82 s; compared to 3.1 ± 0.7 g hexane m-3 h-1, 16 ± 4% at 82 s and 0 CMC), but a negative effect in BF3 at either 1, 3 and 9 CMC (e.g., 2.4 ± 0.4 g hexane m-3 h-1, 30 ± 4% at 163 s and 1 CMC; compared to 4.6 ± 1.0 g hexane m-3 h-1, 43 ± 8% at 163 s and 0 CMC). In contrast, the performance of all BFs improved with the addition of saponin, particularly at 3 CMC. Notably, in BF3, the elimination capacity (EC) and removal efficiency (RE) doubled for both VOCs (i.e., 9.1 ± 0.6 g cyclohexane m-3 h-1, 49 ± 3%; 4.3 ± 0.3 g hexane m-3 h-1, 25 ± 3%) compared to no biosurfactant addition (i.e., 4.5 ± 0.4 g cyclohexane m-3 h-1, 23 ± 3%; hexane 2.2 ± 0.5 g m-3 h-1, 10 ± 2%) at 82 s. Moreover, the addition of the (bio)surfactants led to a shift in the microbial consortia, with a different response in BF1-BF2 compared to BF3. This study evaluates for the first time the use of saponin in BFs, it demonstrates that cyclohexane and hexane RE can be improved by (bio)surfactant addition, and it provides recommendations for future studies in this field.

The nutritional composition and cell size of microbial biomass for food applications are defined by the growth conditions.

BACKGROUND: It is increasingly recognized that conventional food production systems are not able to meet the globally increasing protein needs, resulting in overexploitation and depletion of resources, and environmental degradation. In this context, microbial biomass has emerged as a promising sustainable protein alternative. Nevertheless, often no consideration is given on the fact that the cultivation conditions affect the composition of microbial cells, and hence their quality and nutritional value. Apart from the properties and nutritional quality of the produced microbial food (ingredient), this can also impact its sustainability. To qualitatively assess these aspects, here, we investigated the link between substrate availability, growth rate, cell composition and size of Cupriavidus necator and Komagataella phaffii. RESULTS: Biomass with decreased nucleic acid and increased protein content was produced at low growth rates. Conversely, high rates resulted in larger cells, which could enable more efficient biomass harvesting. The proteome allocation varied across the different growth rates, with more ribosomal proteins at higher rates, which could potentially affect the techno-functional properties of the biomass. Considering the distinct amino acid profiles established for the different cellular components, variations in their abundance impacts the product quality leading to higher cysteine and phenylalanine content at low growth rates. Therefore, we hint that costly external amino acid supplementations that are often required to meet the nutritional needs could be avoided by carefully applying conditions that enable targeted growth rates. CONCLUSION: In summary, we demonstrate tradeoffs between nutritional quality and production rate, and we discuss the microbial biomass properties that vary according to the growth conditions.

Effect of inoculum type, packing material and operational conditions on the biofiltration of a mixture of hydrophobic volatile organic compounds in air.

The emission of volatile organic compounds (VOCs) into the atmosphere causes negative environmental and health effects. Biofiltration is known to be an efficient and cost-effective treatment technology for the removal of VOCs in waste gas streams. However, little is known on the removal of VOC mixtures and the effect of operational conditions, particularly for hydrophobic VOCs, and on the microbial populations governing the biofiltration process. In this study, we evaluated the effect of inoculum type (acclimated activated sludge (A-AS) versus Rhodococcus erythropolis) and packing material (mixture of compost and wood chips (C + WC) versus expanded perlite) on the removal of a mixture of hydrophobic VOCs (toluene, cyclohexane and hexane) in three biofilters (BFs), i.e., BF1: C + WC and R. erythropolis; BF2: C + WC and A-AS; and BF3: expanded perlite and R. erythropolis. The BFs were operated for 374 days at varying inlet loads (ILs) and empty bed residence times (EBRTs). The results showed that the VOCs were removed in the following order: toluene > cyclohexane > hexane, which corresponds to their air-water partitioning coefficient and thus bioavailability of each VOC. Toluene is the most hydrophilic VOC, while hexane is the most hydrophobic. BF2 outperformed BF1 and BF3 in each operational phase, with average maximum elimination capacities (ECmax) of 21 ± 3 g toluene m-3 h-1 (removal efficiency (RE): 100 %; EBRT: 82 s), 11 ± 2 g cyclohexane m-3 h-1 (RE: 86 ± 6 %; EBRT: 163 s) and 6.2 ± 0.9 g hexane m-3 h-1 (RE: 96 ± 4 %; EBRT: 245 s). Microbial analysis showed that despite having different inocula, the genera Rhodococcus, Mycobacterium and/or Pseudonocardia dominated in all BFs but at different relative abundances. This study provides new insights into the removal of difficult-to-degrade VOC mixtures with limited research to date on biofiltration.

Opportunities in optical and electrical single-cell technologies to study microbial ecosystems.

New techniques are revolutionizing single-cell research, allowing us to study microbes at unprecedented scales and in unparalleled depth. This review highlights the state-of-the-art technologies in single-cell analysis in microbial ecology applications, with particular attention to both optical tools, i.e., specialized use of flow cytometry and Raman spectroscopy and emerging electrical techniques. The objectives of this review include showcasing the diversity of single-cell optical approaches for studying microbiological phenomena, highlighting successful applications in understanding microbial systems, discussing emerging techniques, and encouraging the combination of established and novel approaches to address research questions. The review aims to answer key questions such as how single-cell approaches have advanced our understanding of individual and interacting cells, how they have been used to study uncultured microbes, which new analysis tools will become widespread, and how they contribute to our knowledge of ecological interactions.

Spatiotemporal monitoring of a periodontal multispecies biofilm model: demonstration of prebiotic treatment responses.

Biofilms are complex polymicrobial communities which are often associated with human infections such as the oral disease periodontitis. Studying these complex communities under controlled conditions requires in vitro biofilm model systems that mimic the natural environment as close as possible. This study established a multispecies periodontal model in the drip flow biofilm reactor in order to mimic the continuous flow of nutrients at the air-liquid interface in the oral cavity. The design is engineered to enable real-time characterization. A community of five bacteria, Streptococcus gordonii-GFPmut3*, Streptococcus oralis-GFPmut3*, Streptococcus sanguinis-pVMCherry, Fusobacterium nucleatum, and Porphyromonas gingivalis-SNAP26 is visualized using two distinct fluorescent proteins and the SNAP-tag. The biofilm in the reactor develops into a heterogeneous, spatially uniform, dense, and metabolically active biofilm with relative cell abundances similar to those in a healthy individual. Metabolic activity, structural features, and bacterial composition of the biofilm remain stable from 3 to 6 days. As a proof of concept for our periodontal model, the 3 days developed biofilm is exposed to a prebiotic treatment with L-arginine. Multifaceted effects of L-arginine on the oral biofilm were validated by this model setup. L-arginine showed to inhibit growth and incorporation of the pathogenic species and to reduce biofilm thickness and volume. Additionally, L-arginine is metabolized by Streptococcus gordonii-GFPmut3* and Streptococcus sanguinis-pVMCherry, producing high levels of ornithine and ammonium in the biofilm. In conclusion, our drip flow reactor setup is promising in studying spatiotemporal behavior of a multispecies periodontal community.ImportancePeriodontitis is a multifactorial chronic inflammatory disease in the oral cavity associated with the accumulation of microorganisms in a biofilm. Not the presence of the biofilm as such, but changes in the microbiota (i.e., dysbiosis) drive the development of periodontitis, resulting in the destruction of tooth-supporting tissues. In this respect, novel treatment approaches focus on maintaining the health-associated homeostasis of the resident oral microbiota. To get insight in dynamic biofilm responses, our research presents the establishment of a periodontal biofilm model including Streptococcus gordonii, Streptococcus oralis, Streptococcus sanguinis, Fusobacterium nucleatum, and Porphyromonas gingivalis. The added value of the model setup is the combination of simulating continuously changing natural mouth conditions with spatiotemporal biofilm profiling using non-destructive characterization tools. These applications are limited for periodontal biofilm research and would contribute in understanding treatment mechanisms, short- or long-term exposure effects, the adaptation potential of the biofilm and thus treatment strategies.

Cellular and bioenergetic effects of polystyrene microplastic in function of cell type, differentiation status and post-exposure time.

The ubiquity of microplastics (MPs) in food sources and personal care products increasingly raises concerns on human health. However, little is known about the duration of the effects of MPs and whether effects depend on cellular differentiation status. Herein, cellular and bioenergetic effects of MPs in different exposure scenarios on four types of human cell lines derived from lung (A549 and BEAS-2B), colon (Caco-2) and liver (HepG2) were investigated. These cell lines are models for the major exposure routes in the body (inhalation, ingestion and physiological transport through the liver by the portal vein). To this aim, different scenarios were implemented by exposing undifferentiated and differentiated cells to single dosing of 2-µm polystyrene (PS) (102-105 particles/mL) for 48 h and 12 days. The undifferentiated Caco-2 cells with short exposure (48 h) showed the highest uptake rate of PS yet without significant cellular and mitochondrial responses. The biological effects, with the exception of ROS production, were not influenced by differentiation states of A549 and Caco-2 cells although differentiated cells showed much weaker ability to internalize PS. However, PS had significantly long-term impacts on cellular and mitochondrial functions even after the initial exposure period. In particular, Caco-2 cells that were post-exposed for 12 days after single PS dosing suffered higher oxidative stress and exhibited mitochondrial dysfunction than that for short exposure. Correspondingly, we observed that PS particles still remained in cell membrane and even in nuclei with high retention rate by 14-d post exposure during which metabolism and exchange of internalization and release occurred in cells. This indicates PS could induce chronic stress and even harmful effects on human cells after single intake that persists for a long time. This study paves the way for assessing the influence of PS on human health at low particle concentrations and with multiple exposure scenarios.

Full-electric microfluidic platform to capture, analyze and selectively release single cells.

Current single-cell technologies require large and expensive equipment, limiting their use to specialized labs. In this paper, we present for the first time a microfluidic device which demonstrates a combined method for full-electric cell capturing, analyzing, and selectively releasing with single-cell resolution. All functionalities are experimentally demonstrated on Saccharomyces cerevisiae. Our microfluidic platform consists of traps centered around a pair of individually accessible coplanar electrodes, positioned under a microfluidic channel. Using this device, we validate our novel Two-Voltage method for trapping single cells by positive dielectrophoresis (pDEP). Cells are attracted to the trap when a high voltage (VH) is applied. A low voltage (VL) holds the already trapped cell in place without attracting additional cells, allowing full control over the number of trapped cells. After trapping, the cells are analyzed by broadband electrochemical impedance spectroscopy. These measurements allow the detection of single cells and the extraction of cell parameters. Additionally, these measurements show a strong correlation between average phase change and cell size, enabling the use of our system for size measurements in biological applications. Finally, our device allows selectively releasing trapped cells by turning off the pDEP signal in their trap. The experimental results show the techniques potential as a full-electric single-cell analysis tool with potential for miniaturization and automation which opens new avenues towards small-scale, high throughput single-cell analysis and sorting lab-on-CMOS devices.

Predicting the performance of chain elongating microbiomes through flow cytometric fingerprinting.

As part of the circular bio-economy paradigm shift, waste management and valorisation practices have moved away from sanitation and towards the production of added-value compounds. Recently, the development of mixed culture bioprocess for the conversion of waste(water) to platform chemicals, such as medium chain carboxylic acids, has attracted significant interest. Often, the microbiology of these novel bioprocesses is less diverse and more prone to disturbances, which can lead to process failure. This issue can be tackled by implementing an advanced monitoring strategy based on the microbiology of the process. In this study, flow cytometry was used to monitor the microbiology of lactic acid chain elongation for the production of caproic acid, and assess its performance both qualitatively and quantitatively. Two continuous stirred tank reactors for chain elongation were monitored flow cytometrically for over 336 days. Through community typing, four specific community types could be identified and correlated to both a specific functionality and genotypic diversity. Additionally, the machine-learning algorithms trained in this study demonstrated the ability to predict production rates of, amongst others, caproic acid with high accuracy in the present (R² > 0.87) and intermediate accuracy in the near future (R² > 0.63). The identification of specific community types and the development of predictive algorithms form the basis of advanced bioprocess monitoring based on flow cytometry, and have the potential to improve bioprocess control and optimization, leading to better product quality and yields.

Probiotics for oral health: do they deliver what they promise?

Probiotics have demonstrated oral health benefits by influencing the microbiome and the host. Although promising, their current use is potentially constrained by several restrictions. One such limiting factor lies in the prevailing preparation of a probiotic product. To commercialize the probiotic, a shelf stable product is achieved by temporarily inactivating the live probiotic through drying or freeze drying. Even though a lyophilized probiotic can be kept dormant for an extended period of time, their viability can be severely compromised, making their designation as probiotics questionable. Additionally, does the application of an inactive probiotic directly into the oral cavity make sense? While the dormancy may allow for survival on its way towards the gut, does it affect their capacity for oral colonisation? To evaluate this, 21 probiotic product for oral health were analysed for the number of viable (probiotic), culturable (CFU) and dead (postbiotic) cells, to verify whether the commercial products indeed contain what they proclaim. After isolating and uniformly lyophilizing three common probiotic species in a simple yet effective lyoprotective medium, the adhesion to saliva covered hydroxyapatite discs of lyophilized probiotics was compared to fresh or reactivated lyophilized probiotics. Unfortunately, many of the examined products failed to contain the claimed amounts of viable cells, but also the strains used were inadequately characterized and lacked clinical evidence for that unknown strain, questioning their label of a 'probiotic'. Additionally, lyophilized probiotics demonstrated low adhesive capacity compared to their counterparts, prompting the question of why fresh or reactivated probiotics are not currently used.

A New Colorimetric Test for Accurate Determination of Plastic Biodegradation.

As plastic waste is accumulating in both controlled waste management settings and natural settings, much research is devoted to search for solutions, also in the field of biodegradation. However, determining the biodegradability of plastics in natural environments remains a big challenge due to the often very low biodegradation rates. Many standardised test methods for biodegradation in natural environments exist. These are often based on mineralisation rates in controlled conditions and are thus indirect measurements of biodegradation. It is of interest for both researchers and companies to have tests that are more rapid, easier, and more reliable to screen different ecosystems and/or niches for their plastic biodegradation potential. In this study, the goal is to validate a colorimetric test, based on carbon nanodots, to screen biodegradation of different types of plastics in natural environments. After introducing carbon nanodots into the matrix of the target plastic, a fluorescent signal is released upon plastic biodegradation. The in-house-made carbon nanodots were first confirmed regarding their biocompatibility and chemical and photostability. Subsequently, the effectivity of the developed method was evaluated positively by an enzymatic degradation test with polycaprolactone with Candida antarctica lipase B. Finally, validation experiments were performed with enriched microorganisms and real environmental samples (freshwater and seawater), of which the results were compared with parallel, frequently used biodegradation measures such as O2 and CO2, dissolved organic carbon, growth and pH, to assess the reliability of the test. Our results indicate that this colorimetric test is a good alternative to other methods, but a combination of different methods gives the most information. In conclusion, this colorimetric test is a good fit to screen, in high throughput, the depolymerisation of plastics in natural environments and under different conditions in the lab.

Mode of killing determines the necrotrophic response of oral bacteria.

Background: Bacteria respond to changes in their environment, such as nutrient depletion and antimicrobials exposure. Antimicrobials result not only in bacterial death, but also have a hand in determining species abundances and ecology of the oral biofilms. Proximity of dead bacterial cells to living ones is an important environmental change or stress factor. Dead bacteria represent high concentrations of nutrients, such as proteins, lipids, sugars, and nucleic acids. Living bacteria can use these biomasses as a nutrients source, which is termed necrotrophy. Aim: This study investigates the effect of exposing living oral bacteria (planktonic and biofilms) to their dead siblings after being killed by heat or hydrogen peroxide. Results: Tested bacterial species showed different responses towards the dead cells, depending on the mode of killing, the nutritional value of the culture media, and the the dead cells density. The multispecies oral biofilms showed different responses towards the supplementation of dead cells during biofilm development, while matured biofilms were more resilient. Conclusion: This study indicates that dead bacteria resulting from antiseptics use may imbalance the nutrient availability in the oral cavity, resulting in overgrowth of opportunistic species, and hence ecological changes in oral communities, or introducing new bacterial phenotypes.

Enhanced removal of warfare agent tri-nitro-toluene by a Methylophaga-dominated microbiome.

Historical exposure of the marine environment to 2,4,6-trinitrotoluene (TNT) happened due to the dumping of left-over munitions. Despite significant research on TNT decontamination, the potential of marine microbiome for TNT degradation remains only little explored. In this study, TNT degradation experiments were conducted with sediment located near the World War I munition dumpsite - Paardenmarkt in the Belgian part of North Sea. A slow removal was observed using TNT as sole source of C and N, which could be enhanced by adding methanol. Degradation was reflected in nitro-reduced metabolites and microbial growth. 16S Illumina sequencing analysis revealed several enriched genera that used TNT as a sole source of C and N - Colwellia, Thalossospira, and Methylophaga. Addition of methanol resulted in increased abundance of Methylophaga, which corresponded to the rapid removal of TNT. Methanol enhanced the degradation by providing additional energy and establishing syntrophic association between methanol-utilizing and TNT-utilizing bacteria.