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Bioinformatics and machine learning tools have made it possible to integrate data across different -omics platforms for novel multiomic insights into diseases. To synergistically process -omics data in an integrative manner, analyte extractions for each -omics type need to be done on the same set of clinical samples. Therefore, we introduce a simultaneous dual extraction method for generating both metabolomic (polar metabolites only) and glycomic (protein-derived N-glycans only) profiles from one sample with good extraction efficiency and reproducibility. As proof of the usefulness of the extraction and joint-omics workflow, we applied it on platelet samples obtained from a cohort study comprising 66 coronary heart disease (CHD) patients and 34 matched healthy community-dwelling controls. The metabolomics and N-glycomics data sets were subjected to block partial least-squares-discriminant analysis (block-PLS-DA) based on sparse generalized canonical correlation analysis (CCA) for identifying relevant mechanistic interactions between metabolites and glycans. This joint-omics investigation revealed intermodulative roles that protein-bound carbohydrates or glycoproteins and amino acids have in metabolic pathways and through intermediate protein dysregulations. It also suggested a protective role of the glyco-redox network in CHD, demonstrating proof-of-principle for a joint-omics analysis in providing new insights into disease mechanisms, as enabled by a simultaneous polar metabolite and protein-derived N-glycan extraction workflow.

Acute myocardial infarction (AMI) is a leading cause of mortality and morbidity worldwide. Diagnostic challenges remain in this highly time-sensitive condition. Using capillary electrophoresis-laser-induced fluorescence, we analyzed the blood plasma N-glycan profile in a cohort study comprising 103 patients with AMI and 69 controls. Subsequently, the data generated was subjected to classification modeling to identify potential AMI biomarkers. An area under the Receiving Operating Characteristic curve (AUCROC) of 0.81 was obtained when discriminating AMI vs. non-MI patients. We postulate that the glycan profile involves a switch from a pro- to an anti-inflammatory state in the AMI pathophysiology. This was supported by significantly decreased levels in galactosylation, alongside increased levels in sialylation, afucosylation and GlcNAc bisection levels in the blood plasma of AMI patients. By substantiating the glycomics analysis with immunoglobulin G (IgG) protein measurements, robustness of the glycan-based classifiers was demonstrated. Changes in AMI-related IgG activities were also confirmed to be associated with alterations at the glycosylation level. Additionally, a glycan-biomarker panel derived from glycan features and current clinical biomarkers performed remarkably (AUCROC = 0.90, sensitivity = 0.579 at 5% false positive rate) when discriminating between patients with ST-segment elevation MI (n = 84) and non-ST-segment elevation MI (n = 19). Moreover, by applying the model trained using glycomics information, AMI and controls can still be discriminated at 1 and 6 months after baseline. Thus, glycomics biomarkers could potentially serve as a valuable complementary test to current diagnostic biomarkers. Additional research on their utility and associated biomechanisms via a large-scale study is recommended.

Acute myocardial infarction (AMI) is a leading cause of mortality and morbidity worldwide. This work aims to investigate the translational potential of a multi-omics study (comprising metabolomics, lipidomics, glycomics, and metallomics) in revealing biomechanistic insights into AMI. Following the N-glycomics and metallomics studies performed by our group previously, untargeted metabolomic and lipidomic profiles were generated and analysed in this work via the use of a simultaneous metabolite/lipid extraction and liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis workflow. The workflow was applied to blood plasma samples from AMI cases (n = 101) and age-matched healthy controls (n = 66). The annotated metabolomic (number of features, n = 27) and lipidomic (n = 48) profiles, along with the glycomic (n = 37) and metallomic (n = 30) profiles of the same set of AMI and healthy samples were integrated and analysed. The integration method used here works by identifying a linear combination of maximally correlated features across the four omics datasets, via utilising both block-partial least squares-discriminant analysis (block-PLS-DA) based on sparse generalised canonical correlation analysis. Based on the multi-omics mapping of biomolecular interconnections, several postulations were derived. These include the potential roles of glycerophospholipids in N-glycan-modulated immunoregulatory effects, as well as the augmentation of the importance of Ca–ATPases in cardiovascular conditions, while also suggesting contributions of phosphatidylethanolamine in their functions. Moreover, it was shown that combining the four omics datasets synergistically enhanced the classifier performance in discriminating between AMI and healthy subjects. Fresh and intriguing insights into AMI, otherwise undetected via single-omics analysis, were revealed in this multi-omics study. Taken together, we provide evidence that a multi-omics strategy may synergistically reinforce and enhance our understanding of diseases.

Persistent organic pollutants (POPs) accumulate in the organisms due to their hydrophobicity and resistance to xenobiotic metabolism. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of most representative POPs. Its pathophysiological effects have been extensively studied on many types of tissues but not on muscles. In this study, female C57BL/6J mouse model was used to analyze the long-term effects of maternal exposure to TCDD during gestation and lactation on the skeletal muscles (soleus, plantaris, and gastrocnemius) of the progeny during adulthood. The effects of re-exposure to TCDD in mice exposed during their development were also characterized. Female C57BL/6J mice were maternally exposed to TCDD or its vehicle (n-nonane in corn oil) and then re-exposed to TCDD or its vehicle at 9 weeks of age. The metabolites in the skeletal muscles were analyzed by gas chromatography–quadrupole time of flight-mass spectrometry (GC–qTOF-MS). Univariate analysis showed significant effects in certain metabolites in the skeletal muscle. It also showed that TCDD exerts a more significant impact on exposure to TCDD at 9 weeks of age than during maternal exposure for the soleus. On the other hand, TCDD exerts a more significant impact on mice maternally exposed to TCDD than at 9 weeks of age for the gastrocnemius. Multivariate analysis showed clear discrimination between the TCDD-exposed mice and the control. This study demonstrates the effects of TCDD observed following maternal exposure; some of them can be reinforced or attenuated by a re-exposure at the adult age, suggesting that the POP which mainly acts through the activation of the AhR leads to metabolic adaptation in the skeletal muscles. The period of exposure was a key factor in our study with TCDD playing a crucial role during the maternal period, as compared to when they were exposed at 9 weeks of age. It was inferred that disruption in amino acid metabolism might lead to a loss in muscle mass which may result in muscular atrophy. Our results also show that the metabolite profiles after perinatal exposure are different in different types of muscles even though they are all classified as skeletal muscles. Therefore, TCDD may affect the organism (specifically different skeletal muscles) in a non-homogenous manner.

Acute myocardial infarction (AMI) is a leading cause of mortality and morbidity worldwide. Using a validated and efficient ICP-MS/MS-based workflow, a total of 30 metallomic features were profiled in a study comprising 101 AMI patients and 66 age-matched healthy controls. The metallomic features include 12 essential elements (Ca, Co, Cu, Fe, K, Mg, Mn, Na, P, S, Se, Zn), 8 non-essential/toxic elements (Al, As, Ba, Cd, Cr, Ni, Rb, Sr, U, V), and 10 clinically relevant element-pair product/ratios (Ca/Mg, Ca×P, Cu/Se, Cu/Zn, Fe/Cu, P/Mg, Na/K, Zn/Se). Preliminary linear regression with feature selection confirmed smoking status as a predominant determinant for the non-essential/toxic elements, and revealed potential routes of action. Univariate assessments with adjustments for covariates revealed insights into the ambivalent relationships of Cu, Fe, and P with AMI, while also confirming cardioprotective associations of Se. Also, beyond their roles as risk factors, Cu and Se may be involved in the response mechanism in AMI onset/intervention, as demonstrated via longitudinal data analysis with 2 additional time-points (1-/6-month follow-up). Finally, based on both univariate tests and multivariate classification modelling, potentially more sensitive markers measured as element-pair ratios were identified (e.g., Cu/Se, Fe/Cu). Overall, metallomics-based biomarkers may have utility for AMI prediction.

Recombinant protein biopharmaceuticals comprise a significant portion of the current drug development landscape. The glycosylation profile of these proteins is a key quality parameter as it can affect their safety, efficacy, and stability. However, glycan analysis is challenging because of the complexity of their structures. To overcome this challenge in achieving accurate glycan identification, cross-identification of N-Glycans by CE-LIF method using two capillary coatings and three labeling dyes was developed in this work. This work explored whether complementary separation capabilities can be achieved using homemade polyvinyl alcohol (PVA) coating and commercial Guarant™ (Guarant) coating in the analysis of N-glycans. Similar separation profiles were observed using the two capillary coatings, and hence the N-glycan GU databases generated by these coatings were comparable and complementary. The performance of cross-validation by labeling with three fluorescent dyes indicated that low covariance of APTS and Turquoise™ labeling can be obtained, and hence these two labeling mechanisms provided better accuracy for the identification of glycans. Superior reproducibility with RSDs less than 1% for all target glycan standards was achieved by the internal standards (IS) method using maltodextrin ladders as additives in the separation buffer. The developed CE-LIF analysis method was applied to the identification of N-glycans in IgG samples.

The flame retardant triphenyl phosphate (TPhP) has been widely detected in surface waters. Yet, little information is known regarding its impact on microalgae. We investigated the uptake and toxicity of TPhP on two freshwater microalgae Chlorella vulgaris (CV) and Scenedesmus obliquus (SO) after exposure to 10 μg/l–10 mg/l for 5 days. The presence of microalgae significantly enhanced TPhP degradation, with the final concentrations dropped to 5.5–35.1% of the original concentrations. Most of the medium TPhP were sorbed and transformed by microalgae in just one day. Growth of CV was inhibited in a concentration-dependent manner, whereas growth of SO were only inhibited significantly at 10 mg/l TPhP exposure. Mass spectrometry-based untargeted metabolomics revealed concentration- and species-dependent metabolic responses. Exposure to TPhP in CV resulted in enhanced respiration (increase of fumarate and malate) and osmoregulation (increase of sucrose and myo-inositol), synthesis of membrane lipids (accumulation of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), decrease of lysoglycerolipids, fatty acids, and glyceryl-glucoside). Exposure to TPhP in SO resulted in enhanced osmoregulation (increase of valine, proline, and raffinose) and lipolysis (decrease of MGDG, accumulation of fatty acids, lysophospholipids, and glycerol phosphate). Although chlorophyll a and b contents did not change significantly, decrease of chlorophyll derivatives was observed in both CV and SO at high exposure concentrations. Further bioassays confirmed that CV exhibited enhanced membrane integrity and decreased cellular reactive oxygen species (ROS) possibly as a defense strategy, whereas SO showed disruption of membrane integrity and induction of ROS at 10 mg/l exposure. This study demonstrated the potential of microalgae to remove TPhP in water, and offered new insights for the risk assessment of TPhP on freshwater microalgae using metabolomics.

Profiles of combat injuries worldwide have shown that penetrating trauma is one of the most common injuries sustained during battle. This is usually accompanied by severe bleeding or hemorrhage. If the soldier does not bleed to death, he may eventually succumb to complications arising from trauma hemorrhagic shock (THS). THS occurs when there is a deficiency of oxygen reaching the organs due to excessive blood loss. It can trigger massive metabolic derangements and an overwhelming inflammatory response, which can subsequently lead to the failure of organs and possibly death. A better understanding of the acute metabolic changes occurring after THS can help in the development of interventional strategies, as well as lead to the identification of potential biomarkers for rapid diagnosis of hemorrhagic shock and organ failure. In this preliminary study, a metabolomic approach using the complementary platforms of nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography coupled with mass spectrometry (LC-MS) was used to determine the metabolic changes occurring in a porcine model of combat trauma injury comprising of penetrating trauma to a limb with hemorrhagic shock. Several metabolites associated with the acute-phase reaction, inflammation, energy depletion, oxidative stress, and possible renal dysfunction were identified to be significantly changed after a thirty-minute shock period.

With possible food crises looming in the near future, urban farming, including small-scale community and home gardens for home consumption, presents a promising option to improve food security in cities. These small-scale farms and gardens often use planter boxes and raised beds filled with lightweight soil or potting mixes. While previous studies on biochar focused on its application on large-scale contiguous farmlands, this study aimed to evaluate the suitability of biochar as a partial soil substitute to produce a durable and lightweight soil-biochar mix for small-scale urban farms. The effects of biochar on the chemical properties of the soil-biochar mix, crop yield and, particularly, crop nutrients and metabolic content were assessed. A germination test using pak choi seeds (Brassica rapa L. cultivar group Pak choi, Green-Petioled Form) showed that the biochar contained phytostimulants. Through a nursery pot experiment over four growth cycles, biochar treatments performed better than pure soil at retaining water-soluble NO3- and K+ ions, but were worse at retaining PO43- ions. Nonetheless, despite its positive effect on soil NO3- retention, biochar application did not improve crop yield significantly when the application rate varied from 0% to 60% (v/v). Untargeted metabolomic analyses showed that biochar application may increase the production of carbohydrates and certain flavonoids and glucosinolates. The results of this study showed that biochar can potentially be used to improve pak choi nutritional values and applied in large quantity to obtain a lightweight soil mix for urban farming.

Tri-n-butyl phosphate (TBP) is widely used in various industrial processes and has been detected in all environmental matrices. So far, little work has been done regarding the metabolism of TBP on terrestrial invertebrates. We investigated the metabolism of TBP in the earthworm, Perionyx excavatus, after acute exposure to TBP for one and two days in filter paper contact test, as well as after chronic exposure for 28 days in soil experiment. Biotransformation products were identified by using liquid chromatography coupled with quadrupole time-of-flight mass spectrometry, and by exploiting the information dependent acquisition in tandem mass spectrometry. TBP exhibited low accumulation in earthworm-soil ecosystem at 10 mg/kg and 50 mg/kg. The presence of earthworms significantly enhanced TBP degradation at 50 mg/kg in soil. Dibutyl phosphate and hydroxylated TBP were the major phase I metabolites. Three novel phase II metabolites were identified: ethanol dibutyl phosphate and its sulfate conjugate, and the phosphate conjugate of hydroxylated TBP. Hydroxylation and further phosphorylation dominated metabolism in chronic exposure. An extensive metabolic pathway of TBP in earthworm was proposed. This is the first report of TBP metabolism in terrestrial invertebrates and highlights the necessity to identify metabolites of contaminants when evaluating their bioaccumulation and toxicity.

Lichens can be used as cost-effective biomonitors of elements from road traffic in the urban environment. However, the nature of comprehensive interactions between hazardous heavy metals from road traffic and lichen metabolites remains unclear. In this study, elemental and metabolic profiles of the lichen Dirinaria picta after exposure to road traffic for 3 months in 9 sites of Singapore city were investigated. Concentrations of 34 elements reveal strong correlations among each other and a general increase with traffic exposure level. A variety of metabolites, i.e. sugar alcohols, sugars, amino acids, low-molecular mass organic acids, secondary metabolites and nucleosides, are identified through a multi-platform approach combining liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF/MS), gas chromatography quadrupole mass spectrometry (GC-Q/MS) and 1H nuclear magnetic resonance (1H NMR). The total amount of the heavy metals Fe, Zn, Cu, Cd, Pb and Co can be significantly correlated with 21 metabolites positively and 9 metabolites negatively. These correlations reveal a heavy metal detoxification mechanism through ROS scavenging, osmoregulation and chelation with N atoms and sulfhydryl groups, as well as depletion of arabitol and inhibited synthesis of certain secondary metabolites such as quinones and steroids due to heavy metal stress. This study provides new insights into the metabolic toxicity and detoxification mechanisms in lichens under heavy metal exposure.

To compare aquatic organisms’ responses to the toxicity of copper oxide (CuO) nanoparticles (NPs) with those of CuO microparticles (MPs) and copper (Cu) ions, a global metabolomics approach was employed to investigate the changes of both polar and nonpolar metabolites in microalga Chlorella vulgaris after 5-day exposure to CuO NPs and MPs (1 and 10 mg/L), as well as the corresponding dissolved Cu ions (0.08 and 0.8 mg/L). Unchanged growth, slight reactive oxygen species production, and significant membrane damage (at 10 mg/L CuO particles) in C. vulgaris were demonstrated. A total of 75 differentiated metabolites were identified. Most metabolic pathways perturbed after CuO NPs exposure were shared by those after CuO MPs and Cu ions exposure, including accumulation of chlorophyll intermediates (max. 2.4–5.2 fold), membrane lipids remodeling for membrane protection (decrease of phosphatidylethanolamines (min. 0.6 fold) and phosphatidylcholines (min. 0.2–0.7 fold), as well as increase of phosphatidic acids (max. 1.5–2.9 fold), phosphatidylglycerols (max. 2.2–2.3 fold), monogalactosyldiacylglycerols (max. 1.2–1.4 fold), digalactosylmonoacylglycerols (max. 1.9–3.8 fold), diacylglycerols (max. 1.4 fold), lysophospholipids (max. 1.8–3.0 fold), and fatty acids (max. 3.0–6.2 fold)), perturbation of glutathione metabolism induced by oxidative stress, and accumulation of osmoregulants (max. 1.3–2.6 fold) to counteract osmotic stress. The only difference between metabolic responses to particles and those to ions was the accumulation of fatty acids oxidation products: particles caused higher fold changes (particles/ions ratio 1.9–3.0) at 1 mg/L and lower fold changes (particles/ions ratio 0.4–0.7) at 10 mg/L compared with ions. Compared with microparticles, there was no nanoparticle-specific pathway perturbed. These results confirm the predominant role of dissolved Cu ions on the toxicity of CuO NPs and MPs, and also reveal particle-specific toxicity from a metabolomics perspective.

Tris(1,3-dichloro-2-propyl)phosphate (TDCPP) is one of the most widely used organophosphate ester flame retardants. The presence of TDCPP in surface waters and aquatic organisms have been reported worldwide, yet the ecological risk of TDCPP on microalgae is rarely studied. We investigated the biotransformation of TDCPP and its toxicity on the microalga Scenedesmus obliquus using an untargeted metabolomics approach. Exposure to TDCPP resulted in a dose-response decrease of micoalgal biomass. In the presence of microalgae, TDCPP concentration in the media decreased by 25.3-40.6% after 5 days. TDCPP metabolites were identified in the media including hydrolysis and hydroxyl-substituted dechlorination products. A dose-response separation of metabolic profiles of microalgae was observed, with effect seen at the lowest concentration of 10 µg/L tested, which is slightly higher than environmentally relevant concentrations. Differentiated metabolites identified include 52 lipids and 6 polar metabolites. Analysis of altered lipid pathways suggests that microalgal cells reinforce thylakoid membranes (function to protect photosynthesis) by compromising the integrity of plasma membrane (function to protect cellular substances) and extraplastidial cellular membranes. Changes in the polar metabolites might indicate osmotic stress and improved NO signaling after TDCPP exposure. Consistent with perturbation of membrane lipids, further experiment confirmed that exposure to 10 mg/L TDCPP resulted in significant (p 0.01) plasma membrane damage. This study indicates biotransformation and the membrane damage toxicity mechanism of TDCPP on S. obliquus, demonstrating the usefulness of metabolomics for the toxicity mechanism elucidation of emerging pollutants.

Celery (Apium graveolens L. var dulce) is a widely cultivated vegetable which is popularly consumed due to its nutrient content and contains bioactive metabolites with positive effects on human physiology. In this study, 1H NMR spectroscopy coupled with multivariate statistical analyses was used to distinguish celery stem and leaf samples from different geographical origins. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were employed to investigate the differences between celery extracts from three geographical origins: Australia, Taiwan and China. Sugars, amino acids and organic acids were found to contribute significantly to the differentiation between origins, with mannitol identified as an important discriminating metabolite. It was demonstrated that NMR-based metabolomics is an effective approach for establishing reliable metabolomic fingerprints and profiles, enabling the identification of metabolite biomarkers for the possible discrimination of geographical origin.

To determine the bactericidal mechanism of electrolyzed water and mild heat against Escherichia coli O157:H7, we performed ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-qTOF-MS/MS) coupled with multivariate analysis to profile the intracellular metabolites of E. coli O157:H7 in response to electrolyzed water (EW) and mild heat treatment. The results indicated that EW (4 mg/L free available chlorine) combined with heat treatment at 50 °C resulted in 2.31 log CFU/mL reduction of E. coli O157:H7 and the reactive oxygen species fluorescence intensity of EW at 50 °C was ten times higher than that of the control group. Data demonstrated that treatment with EW and heat caused significant perturbation of metabolic pathways that were functionally related with amino acid metabolism, nucleotides synthesis, and lipid biosynthesis. EW at 50 °C resulted in major alterations to pathways involved in acyl carrier protein metabolism, anhydromuropeptides recycling, biosynthesis of CDP-diacylglycerol, trehalose and lipid IVa. Transcriptome analysis revealed that heat and EW affected the transcription levels of some genes in opposite ways. The expression of rpoS, oxyR, soxR, gadA, gadB, sucA, and sucB in the 50 °C group was downregulated, but upregulated in EW exposed cells. The expression of most genes was reduced in response to the combined treatment, with 0.024- and 0.286-fold downregulation of udk (encoding uridine kinase) and gadA (encoding glutamate decarboxylase alpha), respectively, being observed in cells treated with EW at 50 °C. These results provided further evidence of the metabolomic and transcriptomic response of E. coli O157:H7 to oxidation and heat stress.

Triphenyl phosphate (TPHP) is one of the most highly utilized organophosphorus flame retardants, and has been frequently detected in various environmental matrices, including soil. So far, limited information is known regarding the potential toxicity of TPHP to the earthworm-soil ecosystem. We investigated the metabolism of TPHP and the perturbation of the endogenous metabolome in the earthworm, Perionyx excavatus, using gas chromatography mass spectrometry (GC-MS) and liquid chromatography quadrupole time-of-flight (LC-QTOF)-based untargeted metabolomics approach after acute exposure to TPHP for one and two days through a filter paper contact test, as well as after chronic exposure for 28 days in a soil microcosm experiment. TPHP showed low bioaccumulation potential in the earthworm-soil ecosystem at concentrations of 10 mg/kg and 50 mg/kg. Identified phase I metabolites include diphenyl phosphate, mono-hydroxylated and di-hydroxylated TPHP. Two groups of phase II metabolites, thiol conjugates (including mercaptolactic acid, cysteine, cysteinylglycine, and mercaptoethanol conjugates) and glucoside conjugates (including glucoside, glucoside-phosphate, and C14H19O10P conjugates), were putatively identified. Only acute TPHP exposure caused significant perturbations of the endogenous metabolome in earthworms, featuring fluctuations in amino acids, glucose, inosine and phospholipids. These results reveal novel phase II metabolism and toxicity of TPHP in P. excavatus.

Determining and linking the structural heterogeneity of recombinant antibodies to function is critical in the biopharmaceutical industry. We introduce a new microfluidic capillary electrophoresis—mass spectrometry (μCE-MS) approach to characterize intact monoclonal antibody (mAb) and simultaneously quantifying distinct variants. Our MS analysis of intact NIST mAb (RM8671) shows 18 variants identified amongst proteolytic and glycolytic modifications with a range of relative abundances between 0.1% and 100%. In order to verify our quantitative MS results, we used an established system based on capillary electrophoresis—with laser induced fluorescence (CE-LIF) for profiling the N-glycans. All major glycans were identified and further substantiated by exoglycosidase digestion. Interestingly, the µCE-MS analysis of intact NIST mAb consistently yielded higher amounts of G2FG2F-Hex glycoform (~3.4%), whereas the CE-LIF analysis indicates that only 1.4% of G2F-Gal is present. Therefore, the additional hexose adduct observed in µCE-MS may have been the glycation product of the mAb. Further analysis of deglycosylated mAb with µCE-MS made it possible to reveal the glycation with 10.5% of one hexose product and 4.9% of two hexose product in the intact deglycosylated antibody. An integrated solution using two orthogonal and complementary techniques for characterizing antibody glycosylation is provided here.

In this study, (1)H nuclear magnetic resonance (NMR)-based metabolomics approach was used to characterize the metabolic response of the earthworm Perionyx excavatus in continuous vermifiltration for two months under hydraulic loading rates of 1m(3)m(-2)d(-1) (VF1) and 1.5m(3)m(-2)d(-1) (VF1.5). Both VF1 and VF1.5 showed higher removal of chemical oxygen demand and total nitrogen than the biofilter without earthworms. Principal component analysis of the NMR spectra of earthworm metabolites showed significant separations between those not subjected to wastewater filtration (control) and VF1 or VF1.5. Temporal variations of earthworm biomass, and the identified metabolites that are significantly different between control, VF1 and VF1.5 revealed that worms underwent increasing metabolic activity within 20days in VF1 and 14days in VF1.5, then decreasing metabolic activity. The use of NMR-based metabolomics in monitoring earthworm metabolism was demonstrated to be a novel approach in studying engineered vermifiltration systems.