Microplastics (MPs) interaction with human cells poses potential health risks yet quantifying this process and its extensiveness remains challenging. In this study, we developed an automated strategy combining darkfield hyperspectral imaging (HSI) with a deep learning pipeline to detect and quantify cell-associated polystyrene (PS) microplastics at the single-cell level. This pipeline includes 3 main steps: the Mask R-CNN segmented individual cells, the least-squares support vector machine (LS-SVM) distinguished microplastic particles from cellular material, and the circular Hough transform (CHT) was used to count the particles detected within segmented cellular regions. This image analysis pipeline demonstrated high performance: Mask R-CNN detected cells with 95% precision, LS-SVM classified particle spectra with 99.7% accuracy, and the CHT detected particles with a precision of 99.6%. Together, these components enabled reliable quantification of particles within cells. Results showed a dose-dependent effect on the number of PS MPs in Caco-2 cells. At lowest concentrations (1 × 103 particles/mL), 21% of cells were detected with PS, while no impact on cell viability was observed. In contrast, at higher concentrations (1 × 108 and 1 × 109 particles/mL), 100% of cells were detected with PS and showed significant reductions in cell viability. Our findings demonstrated that integrating darkfield HSI with deep learning provides a robust quantitative assessment of MPs and cells interaction at single-cell resolution. This approach may be adaptable to other particle types and cell lines, subject to retraining and validation, offering a valuable tool for microplastic toxicology studies and complementing traditional high-throughput assays in evaluating the level of cell association and dose-response relationships.

Barramundi (Lates calcarifer), native to the Indo-Pacific, has rapidly become the fastest-growing aquaculture species worldwide. However, maintaining stable production during the larviculture stage remains challenging, with larval mortality often reaching 100%. As hatchery management practices play a crucial role in regulating water microbiota, understanding microbiome dynamics is essential for improving survival rates. From day post hatch (DPH) 0 to DPH 17 at a commercial barramundi larviculture facility in Vietnam, microbial dynamics and community assembly in rearing tanks were analyzed using flow cytometry (FCM) and 16S rRNA gene amplicon sequencing. The analysis revealed persistent tank-specific differences in microbial communities that fluctuated over time. Stochastic factors played a significant role in microbial assembly, leading to variability between tanks even under similar operational conditions and underscoring the complexity of microbial community dynamics in larviculture systems. Flow cytometry and 16S rRNA sequencing showed moderately correlated diversity metrics and captured comparable overall trends in community composition, reflecting their potential complementarity in monitoring microbial dynamics. This underscores the potential of FCM as a complementary, rapid, and cost-effective approach for monitoring microbial community dynamic in aquaculture.

Unexploded ordnance from World Wars I and II continues to release 2,4,6-trinitrotoluene into marine sediments, yet microbial responses to this chronic contamination remain poorly understood. Here, we characterize the taxonomic and functional potential of sediment microbiomes at the historical submarine wreck UC-30 in the North Sea, combining 16S rRNA amplicon sequencing, shotgun metagenomics, and targeted GC-MS/MS analysis with a parallel aerobic laboratory enrichment. Minewell sediments showed distinct community shifts, with enrichment of Proteobacteria, notably Haliaceae and Rhodobacteraceae, alongside increased representation of oxidoreductases and stress-related enzyme classes, including glutathione S-transferases. Genes associated with TNT transformation, including Old Yellow Enzymes and nitroreductases, were modestly enriched in situ. The laboratory enrichment confirmed TNT removal and presence of N-ethylmaleimide reductase, an Old Yellow Enzyme implicated in TNT transformation. Functional and taxonomic parallels between field and enrichment communities indicate shared adaptive capacities under TNT exposure, positioning contaminated marine microbiomes as reservoirs of bioremediation potential. Long-term TNT exposure at historical shipwrecks can influence sediment microbial communities, according to combined wreck sampling from the North Sea and laboratory incubation tests.

The clinical application of probiotics for oral health is increasing, yet their colonization dynamics remain poorly understood. This study investigated whether administration timing, strain origin, and host-specific factors influence oral persistence.Two Limosilactobacillus reuteri strains (oral vs. non-oral isolate) were evaluated in two in vivo studies. In the first, participants consumed probiotics either during the day or before bedtime; in the second, daily administration continued for 28 days, followed by a 7-day washout. Probiotic abundance was qPCR quantified and oral microbiomes were sequenced. Additionally, a retrospective analysis of periodontitis patients receiving probiotics was performed.Bedtime administration significantly prolonged probiotic detectability compared to daytime intake. Both strains were largely transient, disappearing within days post-administration, though the oral isolate showed enhanced adhesion. Notably, a subset of participants retained the probiotic for up to a week, suggesting interindividual variability and potential biomarkers of colonization. Periodontitis patients who had higher concentrations of probiotics also displayed more improvement in pocket probing depth of deep pockets.These findings highlight the need to consider timing, strain selection, and host factors in probiotic-based oral health interventions.

The growing demand for sustainable protein sources is driving the food industry to explore alternative production systems. To this end, we explored microbial protein (MP) production from synthetic cheese whey permeate (CWP) using kefir, a synergistic culture of yeasts and bacteria, under nearly-extreme conditions relevant to the food industry (acidic pH, high salinity, upper mesophilic temperatures), and different operational modes (continuous, fed-batch). The composition and yield of MP were influenced by the cultivation conditions, with bacteria being substantially more impacted than yeasts. The most promising conditions for continuous aerobic, protein-rich MP production were mildly acidic pH, low salt content and mesophilic temperature, achieving final biomass concentrations up to similar to 12 g L-1 with a protein content up to 51%. The community composition was highly dependent on the applied conditions, with higher NaCl concentrations leading to greater bacterial diversity, while the yeast community was more stable with Trichosporon and Meyerozyma dominating in almost all cases. Fed-batch cultivation resulted in higher biomass concentrations compared to continuous mode, but yielded MP with lower protein content and slightly reduced nutritional quality, potentially due to the different culture dynamics as a result of the operational strategy. The MP produced had a high essential amino acid (AA) content, with 100 g of microbial biomass produced with 0% NaCl in continuous and fed-batch mode covering at least 60 and 86% of the human daily requirements in essential AA, respectively. These findings show the potential for tailored MP production from CWP by selectively increasing bacterial or yeast abundance and impacting their biomass properties to achieve the desired macromolecular composition. Since synthetic cheese whey permeate was used, the results should be considered an idealized baseline, as real wastewater matrices may contain complex organics and trace inhibitors that could affect microbial community dynamics and process performance.

BACKGROUND: Drinking water systems (DWS) are often an overlooked source of microbial contamination of drinking water in broiler and piglet production. Persistent biofilms within water lines can act as reservoirs of contamination, reintroducing microorganisms into the flowing water and potentially compromising animal health. This study investigates the microbial composition of biofilms in the DWS of broiler houses and pig nursery units, their impact on drinking water quality, and the influence of the source water on both water quality and biofilm communities. RESULTS: The bacterial load of DWS biofilm swabs, collected at the end of production cycles before cleaning and disinfection was evaluated, and the dominant bacterial taxa were identified. Furthermore, 16S gene metabarcoding was applied to the biofilm samples. No significant differences in microbial load were observed between the two sectors, with a median total aerobic count of 3.6 log CFU/cm2. Enterococci, a faecal indicator, were detected in 80% of all samples. Moreover, Escherichia coli was found more frequently in broiler houses (47%) than in pig nursery units (27%). The two dominant identified genera were Staphylococcus and Pseudomonas. The Staphylococcus saprophyticus species was the most frequently identified isolate, accounting for 10.6% of all isolates across both broiler houses and pig nursery units. In broiler houses, the next most frequently identified species were Pseudomonas aeruginosa (5.6%) and Stenotrophomonas maltophilia (5.3%). In contrast, in pig nursery units, Pseudomonas fluorescens (6.3%) and Psychrobacter faecalis/pulmonis (5.5%) were most frequently identified. Research showed that the drinking water microbial community not only depended on the source water but was also influenced by biofilms in DWS, as similar bacterial taxa were found in both the drinking water at the drinking nipples and in biofilms on water-contact surfaces. CONCLUSIONS: The presence of faecal indicator bacteria and potential animal pathogens underscores the risks associated with the biofilms. These biofilms can contaminate drinking water to animals, underscoring the need for targeted strategies to monitor and mitigate biofilm formation.

Meat analogues have gained traction as they provide a more sustainable and ethical alternative to traditional meat, with microbial biomass being a promising feedstock due to its nutritional value and potentially environmentally-friendly production. However, it remains unclear whether the microbial cultivation conditions influence its processability and applicability for conversion into meat analogues. This study explored the effects of microbial cultivation parameters, such as salt (NaCl) levels in the medium and cultivation temperature, on the quality of the biomass from the Gram-negative bacterium Paracoccus zeaxanthinifaciens for hybrid meat analogue production (1:1 w/w microbial biomass to wheat gluten). Cultivation in bioreactors under five different conditions revealed substantial impacts on the macromolecular composition, physicochemical, thermal and rheological properties, and the ability to create a texturized meat analogue. Overall, the cultivation at temperatures below the optimal levels for growth yielded the most promising results. Specifically, biomass cultivated at low temperature yielded the highest total amino acid content and strongest fibers in a texturized meat analogue, whereas cultivation in high salt levels increased the lipid content. Microbial biomass grown under control and low-temperature conditions formed strong fibers suitable for texturized meat analogues, but cultivation at high salt and high temperature compromised texturization via shear cell, with the latter yielding unstructured paste. This study highlights for the first time the importance of optimizing cultivation conditions to enhance the quality and functionality of microbial biomass as a feedstock for meat analogue production.

Context The North Sea hosts numerous historic shipwrecks. Two World War II vessels with ferrous hull were investigated, namely, a German Sperrbrecher 141 and Allied HMS Basilisk. Aims Their disturbance of the coastal seabed was determined. More specifically, the microbial composition, diversity and introduction of iron, aluminium, sulfur and zinc was measured in marine sediment samples. Methods Elemental analysis was conducted by microwaved-assisted HF destruction prior to inductively coupled plasma–optical emission spectroscopy (ICP-OES). Microbial characterisation involved the use of flow-cytometry, ATP spectroscopic assay and 16S rRNA amplicon sequencing. Key results The wrecks cause enrichment in sulfur and largely varying iron concentrations (4.6–153 mg kg−1). Aluminium, which was less abundant, correlated to some extent with the bacterial cell density. Marine bacteria detected include Verrucomicrobiaceae, Woeseia and Flavobacteriaceae, next to sulfur and iron cyclers, similar to Desulfocapsaceae, Sulfurovaceae, Desulfuromonas and B2M28 that are active in dissimilatory iron reduction. Close to the wrecks, the alpha diversity in the seabed was significantly decreased. Conclusions Hence, the submerged wrecks still alter the coastal sediment geochemistry and microbiology. Implications Marine ecosystems near sunken war ships show an adaptive response that microbially cycle iron and sulfur, enriched in those environments. Results show special relevance to recent off-shore structures in coastal areas.

The microbial and biochemical landscape of clinically normal-appearing skin in individuals with acne remains uncharacterized. Here, we performed longitudinal multi-omics profiling of facial skin from 10 moderate acne patients and 10 healthy controls, integrating 16S rRNA gene sequencing, shotgun metagenomics, and untargeted metabolomics across lesional and non-lesional sites. Compositional tensor factorization revealed that non-lesional acne skin occupies a distinct intermediate state between healthy and lesional skin in both the microbiome and the metabolome. Machine learning models distinguished healthy from non-lesional acne skin with 70% accuracy, demonstrating that molecular dysbiosis occurs in skin without visible lesions. Non-lesional sites exhibited reduced microbial diversity, strain-level shifts in Corynebacterium and Lawsonella correlating with disease severity, and metabolic alterations, including elevated lipids and perturbed amino acid and dipeptide profiles. Microbe–metabolite co-occurrence network analyses revealed that healthy skin is enriched for protective metabolites such as urocanic acid, while acne-associated skin shows distinct co-occurrence patterns. These findings establish acne as a field effect disorder, with molecular alterations extending beyond visible lesions across the entire facial skin ecosystem. This molecular signature of pre-lesional skin provides potential biomarkers for early intervention and suggests that effective acne treatment may require holistic approaches targeting the broader skin environment rather than individual lesions.

Fluoride mouthwashes are commonly used for prevention of dental caries. The most common forms of fluoride are stannous fluoride (SnF2), amine fluoride (AmF) and sodium fluoride (NaF), each with differing activities against oral bacteria. Since the microbiome has been linked to interindividual differences in responses to treatment, the phenotype of the microbiome may provide information on potential treatment responses. Flow cytometry successfully tracks microbial phenotypic heterogeneity and may prove useful for capturing interindividual treatment responses. We compared the effect of fluoride-containing mouthwashes, Elmex Anti Caries (AmF/NaF), Listerine Anti Caries (NaF) and Meridol (AmF/SnF2) on the salivary microbiome in vitro. We determined the effect on the microbial community structure using 16 S rRNA gene amplicon sequencing, assessed the acidogenic potential by measuring organic acids after incubation, and constructed flow cytometric fingerprints of the microbial community to evaluate if it could be used to capture the response to treatment and to assess if it has potential for precision medicine. We observed mouthwash- and donor-dependent changes in the salivary microbiome composition and acidogenic potential. Meridol affected more bacterial taxa and showed the strongest reduction in organic acids, followed by Elmex. The response to treatment could be captured in the flow cytometric fingerprint of the initial intact salivary microbiota and a random forest classifier predicting the optimal mouthwash for each donor was successfully trained (MCC = 0.95). Treatment with fluoride mouthwashes induces a shift in the salivary microbiome in vitro and alter its potential to produce organic acids, contributing to the prevention of dental caries. Moreover, flow cytometry shows promise for tailoring treatments to patients.

Small intestinal microbial overgrowth (SIMO) results from a breakdown in the delicate equilibrium between luminal environment, gut motility, and microbial ecology. Despite extensive research, these factors have largely been investigated as separate entities, with limited integrative insights into their interplay. This review is the first comprehensive synthesis of physicochemical, mechanical, and microbial parameters shaping SIMO pathogenesis. By reviewing both clinical and experimental data, we reveal how alterations in pH, transit time, digestive secretion dynamics, bile acid composition and impaired intestinal absorption collectively reshape microbial load, diversity, and metabolic output, establishing a self-perpetuating loop of dysfunction. We further discuss the limitations of current diagnostic tools and the transformative potential of emerging approaches, from sampling capsules enabling molecular analyses, to in vitro models simulating human small intestinal ecosystem. This integrative perspective shifts the paradigm from a microbe-centered to an ecosystem-based understanding of SIMO, outlining key challenges and opportunities for personalized diagnostics, mechanistic research, and microbiota-targeted next-generation therapeutics including pre-, pro-, postbiotics and faecal transplantation.

Enhancing our understanding of the role of microbial life strategies and their trade-offs in the functioning of microbial communities is essential for improving the management of microbial communities. In aquaculture microbiomes, management aimed at increasing the dominance of K-strategists has experimentally been shown to influence cultivation performance. To understand the mechanisms behind such observations, we need to improve our understanding of the typical properties and behaviour of r- and K-strategists. Several studies have advanced our understanding of theoretical trade-offs that may shape these life strategies, but our understanding of which trade-offs are relevant under natural conditions is still limited. In this study, we investigated the in situ growth strategies of bacterial taxa in rearing water microbiomes of whiteleg shrimp (Litopenaeus vannamei) larviculture by reconstructing 67 high quality metagenome assembled genomes (MAGs), which covered between 31 and 85% of the sampled communities. We found evidence for niche separation between r- and K-biased strategists residing in these communities, with r-biased strategists typically encoding more and more versatile transport and metabolism pathways, and having a higher fitness for exploitation of spatially structured nutrient hotspots. We further increased the knowledge regarding the influence of r- and K-biased strategistson aquaculture cultivation performance by showing that the in situ growth activity of r-biased strategists could be linked better with cultivation performance than the relative abundance of r- and K-biased strategists.

The bioconversion of CO2‑derived methanol into higher‑value chemicals offers an attractive route for hybrid catalytic-biotechnological carbon capture and utilization (CCU). Clostridium luticellarii is one of the few acetogens able to produce isobutyric acid. However, operational and metabolic factors driving its production are poorly understood. This work investigates how CO2 availability shapes the product spectrum of C. luticellarii during methylotrophic growth and assesses whether CO2 supply can be used as a process lever to promote isobutyric acid formation. Batch experiments with varying initial bicarbonate concentrations revealed that conditions leading to CO2 limitation (i.e., DIC depletion at ≤ 30 mM NaHCO3) redirected carbon and electron fluxes away from acetic acid toward butyric and isobutyric acids, with the latter accounting for up to 41% of total products. This metabolic switch was not observed when CO2 was in excess (>45 mM). High acetic acid supplementation (100 mM) triggered isobutyric acid production even while CO2 was still available, indicating a combined regulation of dissolved inorganic carbon (DIC) and acetic acid availability. Net acetic acid consumption took place in all isobutyric acid-producing experiments. These observations were reproduced in 3-L bioreactors and further exploited through a fed‑batch strategy in which an initial acetic‑acid‑accumulating phase was followed by CO2‑limited feeding. This approach achieved complete conversion of methanol and CO2 and yielded an isobutyric acid titer of 2.70 ± 0.04 g·L-1. Controlling CO2 availability is a viable operational tool to steer C. luticellarii metabolism toward isobutyric acid production, in interaction with electron acceptor availability.

Rainwater backflow caused by system cross-connection into drinking water distribution systems (DWDSs), resulting from pipe breaks, leaks, manual installation errors, or misconnections associated with third-pipe systems for rainwater and greywater reuse, can disrupt biostability of the DWDS by introducing microorganisms and nutrients. Using a pilot-scale DWDS with a mature biofilm, this study simulated a conservative worst-case backflow scenario by supplying a 1 : 1 mixture of rainwater and tap water for two consecutive weeks. This was done in recirculation mode, followed by recovery through replacement with uncontaminated tap water without chemical disinfection or hydraulic flushing. This experiment was conducted twice under identical conditions. Microbial abundance, activity, and community composition in bulk water and the biofilm were assessed using culture-based methods, total organic carbon (TOC), ATP measurements, online flow cytometry, 16S rRNA gene amplicon sequencing, and a continuous Orb in-line probe that measures fluorescence-based bio-load. Rainwater backflow had an immediate impact with a one log increase in total cell concentrations, a two-fold increase in TOC values, and peaks in ATP concentrations of 75.71 ng L-1 compared to 3.50 ng L-1 (experiment I) and 0.913 ng L-1 (experiment II) for regular tap water, alongside pronounced shifts in bulk water community composition, demonstrating the duration and scale of the backflow event. Rainwater-associated taxa were temporarily detected in the bulk and biofilm, even after 100% tap water was supplied, but remained at low relative abundances. The culture-based methods (CCA, PCN, ADA) demonstrated that regulatory limits were exceeded during and after the backflow, reflecting the delay in compliance after a backflow event. These results highlight the resilience of biostable DWDSs to short-term rainwater backflow and demonstrate the value of high-frequency and high-resolution microbial monitoring for rapid detection and management of contamination events.

Organic micropollutants (OMPs) compose a group of emerging contaminants that occur in environmental waters at trace concentrations (μg/L to ng/L) with suspected adverse effects on ecosystems and human health. Conventional wastewater treatment plants (WWTPs) are not designed to eliminate the more recalcitrant OMPs. As such, WWTP-effluents are a major source of OMPs in the aquatic environment, and sustainable advanced treatment options are required. One option concerns the biodegradation of OMPs, but several studies show that it might be constrained by the residual low energy content in effluent waters. Since molecular hydrogen (H2) has been identified as a universally available energy source utilised by various bacteria in oligotrophic environments, supporting mixotrophic growth, we examined the hypothesis that H2 can enhance OMP-removal and concomitant ecotoxicity from domestic wastewater effluent. To this end, a lab-scale biological trickling filter supplemented with H2 (1.5% in the aeration with ambient air) was operated in continuous mode for treating a field-collected domestic WWTP-effluent. In total, 51 OMPs were detected in the WWTP-effluent, of which azithromycin and clarithromycin contributed to 76.5% of the total ecotoxic effects, as demonstrated in a cyanobacterial growth inhibition assay. The H2-supplemented trickling filter enhanced the removal of azithromycin and clarithromycin by (69.5 ± 1.5)% and (46.0% ±2.9)%, respectively, and increased the reduction of total OMP-associated ecotoxic effects by 56.5%, compared to the non-H2-supplemented control. Moreover, H2 supplementation improved PO43- and NO3- removal by a factor of 2.3 and 1.5, respectively. These findings demonstrate that H2 supplementation may support microbial processes involved in OMP-removal from domestic wastewater in a microbial treatment system, thereby reducing concomitant ecotoxicity.

Microbially induced calcium carbonate precipitation is a widespread natural phenomenon with numerous technical applications. Recent advances have shown that bacterial calcium carbonates (BCC) form nonclassically via amorphous calcium carbonate (ACC) precursors in the presence of organics, but the role of organics in the formation and nanostructural features of BCCs is not fully understood. Here we show that two bacterial strains produce BCCs with diverse textural and structural features at the macroscale but similar at the micro and nanoscale. We show that bacterial organics guide precipitation of calcite, stabilizing ACC to produce nanogranular crystals and these organics are then trapped within the crystal, rather than being released as previously suggested. These organics are N-rich and create regions of low Z-contrast aligned perpendicular to the c-axis of the bacterial calcite crystal, yielding a "Swiss-cheese-like" mesostructure. Moreover, it is these occluded organics that lead to the distinctive biosignatures observed in BCC. Finally, we also observe crystalline 2D films, possibly proteins, templating the oriented crystallization of bacterial calcite. These ultrastructural features help to disclose how microbial CaCO3 biomineralization takes place leading to improved technical applications and may provide fingerprints for their identification in nature.

Microbial phenotypes vary at the single-cell level, shaping key community traits like resilience and adaptability. Yet, current methods either lack resolution (e.g., culturing, sequencing), are too costly, or technically complex, limiting widespread use. To address this gap, we introduce and validate a workflow called DE-SWIRL (Double Emulsion–Sorting Workflow with sImple, Rapid emuLsification), an accessible, low-cost workflow enabling ultrahigh-throughput (~10 7 microcultures/experiment) screening of individual microbial cells. DE-SWIRL integrates a published droplet-templated emulsification protocol producing uniform double emulsions from monodisperse single emulsions with Fluorescence-Activated Cell Sorting (FACS). We validated that double emulsions of 6 and 24 pL can be reliably formed, with ~45% droplet survival. To address persistent large-particle contaminants, we validated a gating strategy and show it enables accurate screening and sorting. When starting from a monodisperse single emulsion population, oil layer variability is higher for droplet-templated emulsification than for on-chip microfluidics, but maintains a comparably uniform core emulsion while offering substantial time savings. We demonstrate DE-SWIRL's utility by isolating viable strains from a synthetic community with up to 99% sorting purity and isolating droplet cocultures from a mixed community. This workflow provides a fast, accessible, and affordable workflow for screening entire microbiomes at a single-cell level using a fluorescent assay of interest.

Micronutrient malnutrition affects over 3 billion people worldwide. This study evaluated biofortified wheat breads with iodine, selenium, and zinc, applied individually or in combination, and examined mineral retention, bioaccessibility, and intestinal epithelial responses. Mineral concentrations in wheat and their changes after baking unfermented flatbread and fermented sourdough were quantified, while intestinal bioaccessibility and epithelial effects were assessed using in-vitro digestion and cell culture models. Biofortification significantly increased mineral concentrations in wheat; however, retention during baking varied by mineral and product. Flour composition and baking method influenced iodine and zinc levels, whereas selenium retention was primarily affected by wheat cultivar and fermentation. In biofortified Bezostaja-1, Se bioaccessibility reached 68% in flatbread, while iodine and zinc reached 49% and 12% in sourdough. Selenium enhanced mitochondrial activity in intestinal cells in both bread types, and zinc-enriched sourdough increased epithelial integrity by 15% and reduced cellular permeability by 30%.

Oxygen depletion and sulphide formation, resulting from the accumulation of organic waste, are common challenges in shrimp ponds that could result in complete harvest failure. The stage at which these circumstances occur during the shrimp growth period remains elusive, yet, knowledge of the timing of oxygen depletion and sulphide formation is essential to enable remediating actions. Here, we used an experimental shrimp pond model at different stages in the shrimp growth period to determine when oxygen depletion and sulphide production occur. Microscale depth measurements of oxygen and H2S were determined using microelectrodes to visualize their profiles at different depths of the water-sediment interface and the sediment. We evaluated the potential of different molybdate concentrations at different stages to determine the optimal conditions to suppress H2S formation. Oxygen depletion and sulphide production took place in the middle of the shrimp growth cycle in the simulated model of waste accumulation. The addition of molybdate was only effective in the early stages of the onset of oxygen depletion and H2S formation, and residual molybdate was required to ensure a continuous suppression of sulphide production. However, oxygen depletion could not be prevented and reintroduction of oxygen did not occur when molybdate was added. In conclusion, molybdate appeared to be an effective strategy to suppress H2S formation at the onset of its production in a shrimp pond bottom model.