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At least 19 records

Simulating the metabolic pathway dynamics of an organism

Disclosed herein are systems and methods for determining metabolic pathway dynamics using time series multiomics data. In one example, after receiving time series multiomics data comprising time-series metabolomics data associated a metabolic pathway and time-series proteomics data associated with the metabolic pathway, derivatives of the time series multiomics data can be determined. A machine learning model, representing a metabolic pathway dynamics model, can be trained using the time series multiomics data and the derivatives of the time series multiomics data, wherein the metabolic pathway dynamics model relates the time-series metabolomics data and time-series proteomics data to the derivatives of the time series multiomics data. The method can include simulating a virtual strain of the organism using the metabolic pathway dynamics model.

Costello, Zachary↗

On the levels of enzymatic substrate specificity: Implications for the early evolution of metabolic pathways

The most frequently invoked explanation for the origin of metabolic pathways is the retrograde evolution hypothesis. In contrast, according to the so-called 'patchwork' theory, metabolism evolved by the recruitment of relatively inefficient small enzymes of broad specificity that could react with a wide range of chemically related substrates. In this paper it is argued that both sequence comparisons and experimental results on enzyme substrate specificity support the patchwork assembly theory. The available evidence supports previous suggestions that gene duplication events followed by a gradual neoDarwinian accumulation of mutations and other minute genetic changes lead to the narrowing and modification of enzyme function in at least some primordial metabolic pathways.

Lazcano, A.↗

Effects of 5-ion 6-beam sequential irradiation in the presence and absence of hindlimb or control hindlimb unloading on behavioral performances and plasma metabolic pathways of Fischer 344 rats

Introduction Effects and interactions between different spaceflight stressors are expected to be experienced by crew on missions when exposed to microgravity and galactic cosmic rays (GCRs). One of the limitations of previous studies on simulated weightlessness using hindlimb unloading (HU) is that a control HU condition was not included. Methods We characterized the behavioral performance of male Fischer rats 2 months after sham or total body irradiation with a simplified 5-ion 6-mixed-beam exposure representative of GCRs in the absence or presence of HU. Six months later, the plasma, hippocampus, and cortex were processed to determine whether the behavioral effects were associated with long-term alterations in the metabolic pathways. Results In the open field without and with objects, interactions were observed for radiation × HU. In the plasma of animals that were not under the HU or control HU condition, the riboflavin metabolic pathway was affected most for sham irradiation vs. 0.75 Gy exposure. Analysis of the effects of control HU on plasma in the sham-irradiated animals showed that the alanine, aspartate, glutamate, riboflavin, and glutamine metabolisms as well as arginine biosynthesis were affected. The effects of control HU on the hippocampus in the sham-irradiated animals showed that the phenylalanine, tyrosine, and tryptophan pathway was affected the most. Analysis of effects of 0.75 Gy irradiation on the cortex of control HU animals showed that the glutamine and glutamate metabolic pathway was affected similar to the hippocampus, while the riboflavin pathway was affected in animals that were not under the control HU condition. The effects of control HU on the cortex in sham-irradiated animals showed that the riboflavin metabolic pathway was affected. Animals receiving 0.75 Gy of irradiation showed impaired glutamine and glutamate metabolic pathway, whereas animals receiving 1.5 Gy of irradiation showed impaired riboflavin metabolic pathways. A total of 21 plasma metabolites were correlated with the behavioral measures, indicating that plasma and brain biomarkers associated with behavioral performance are dependent on the environmental conditions experienced. Discussion Phenylalanine, tyrosine, and tryptophan metabolism as well as phenylalanine and tryptophan as plasma metabolites are biomarkers that can be considered for spaceflight as they were revealed in both Fischer and WAG/Rij rats exposed to simGCRsim and/or HU.

Physiology↗

FatPlants: a comprehensive information system for lipid-related genes and metabolic pathways in plants

Abstract FatPlants, an open-access, web-based database, consolidates data, annotations, analysis results, and visualizations of lipid-related genes, proteins, and metabolic pathways in plants. Serving as a minable resource, FatPlants offers a user-friendly interface for facilitating studies into the regulation of plant lipid metabolism and supporting breeding efforts aimed at increasing crop oil content. This web resource, developed using data derived from our own research, curated from public resources, and gleaned from academic literature, comprises information on known fatty-acid-related proteins, genes, and pathways in multiple plants, with an emphasis on Glycine max, Arabidopsis thaliana, and Camelina sativa. Furthermore, the platform includes machine-learning based methods and navigation tools designed to aid in characterizing metabolic pathways and protein interactions. Comprehensive gene and protein information cards, a Basic Local Alignment Search Tool search function, similar structure search capacities from AphaFold, and ChatGPT-based query for protein information are additional features. Database URL: https://www.fatplants.net/

59 BASIC BIOLOGICAL SCIENCES↗

On the origin of metabolic pathways

The heterotrophic theory of the origin of life is the only proposal available with experimental support. This comes from the ease of prebiotic synthesis under strongly reducing conditions. The prebiotic synthesis of organic compounds by reduction of CO(2) to monomers used by the first organisms would also be considered an heterotrophic origin. Autotrophy means that the first organisms biosynthesized their cell constituents as well as assembling them. Prebiotic synthetic pathways are all different from the biosynthetic pathways of the last common ancestor (LCA). The steps leading to the origin of the metabolic pathways are closer to prebiotic chemistry than to those in the LCA. There may have been different biosynthetic routes between the prebiotic and the LCAs that played an early role in metabolism but have disappeared from extant organisms. The semienzymatic theory of the origin of metabolism proposed here is similar to the Horowitz hypothesis but includes the use of compounds leaking from preexisting pathways as well as prebiotic compounds from the environment.

NASA Discipline Exobiology↗

Effects of photon irradiation in the presence and absence of hindlimb unloading on the behavioral performance and metabolic pathways in the plasma of Fischer rats

The space environment astronauts experience during space missions consists of multiple environmental challenges, including microgravity. In this study, we assessed the behavioral and cognitive performances of male Fisher rats 2 months after sham irradiation or total body irradiation with photons in the absence or presence of simulated microgravity. We analyzed the plasma collected 9 months after sham irradiation or total body irradiation for distinct alterations in metabolic pathways and to determine whether changes to metabolic measures were associated with specific behavioral and cognitive measures. A total of 344 male Fischer rats were irradiated with photons (6 MeV; 3, 8, or 10 Gy) in the absence or presence of simulated weightlessness achieved using hindlimb unloading (HU). To identify potential plasma biomarkers of photon radiation exposure or the HU condition for behavioral or cognitive performance, we performed regression analyses. The behavioral effects of HU on activity levels in an open field, measures of anxiety in an elevated plus maze, and anhedonia in the M&M consumption test were more pronounced than those of photon irradiation. Phenylalanine, tyrosine, and tryptophan metabolism, and phenylalanine metabolism and biosynthesis showed very strong pathway changes, following photon irradiation and HU in animals irradiated with 3 Gy. Here, 29 out of 101 plasma metabolites were associated with 1 out of 13 behavioral measures. In the absence of HU, 22 metabolites were related to behavioral and cognitive measures. In HU animals that were sham-irradiated or irradiated with 8 Gy, one metabolite was related to behavioral and cognitive measures. In HU animals irradiated with 3 Gy, six metabolites were related to behavioral and cognitive measures. These data suggest that it will be possible to develop stable plasma biomarkers of behavioral and cognitive performance, following environmental challenges like HU and radiation exposure.

59 BASIC BIOLOGICAL SCIENCES↗

Oxygen and the evolution of metabolic pathways

While a considerable amount of evidence has been accumulated about the history of oxygen on this planet, little is known about the relative amounts to which primitive cells might have been exposed. One clue may be found in the metabolic pathways of extant microorganisms. While eucaryotes are principally aerobic organisms, a number are capable of anaerobic growth by fermentation. One such eucaryotic microorganism, Saccharomyces cerevisiae, will grow in the complete absence of oxygen when supplemented with unsaturated fatty acid and sterol. Oxygen-requiring enzymes are involved in the synthesis of both of these compounds. Studies have demonstrated that the oxidative desaturation of palmitic acid and the conversion of squalene to sterols occur in the range of 10-(3) to 10(-2) PAL. Thus, if the oxygen requirements of these enzymatic processes are an indication, eucaryotes might be more primitive than anticipated from the microfossil record. Results of studies on the oxygen requirements for sterol and unsaturated fatty acid synthesis in a more primitive procaryotic system are also discussed.

Jahnke, L. L.↗

Development of high throughput light-sheet fluorescence lifetime imaging microscopy for 3D functional imaging of metabolic pathways in plant and microorganisms (Final Technical Report)

This research program will enable new biochemical contrast in the nanosecond lifetime domain through use of the recently demonstrated electro-optic fluorescence lifetime imaging technique (EO-FLIM) for wide-field lifetime imaging. The Stanford/Stanford Linear Accelerator Center multidisciplinary collaboration -- physics, applied physics, and structural biology -- will develop a light-sheet fluorescence lifetime imaging microscope for functional studies of microbial and plant metabolic pathways and dynamic interactions between plants and microorganisms in the rhizosphere. The proposed approach overcomes the imaging time bottleneck associated with existing fluorescence lifetime imaging methods. Initial demonstrations have shown a factor of 100,000 improvement in photon throughput compared to existing methods. High photon efficiency allowed the first wide-field fluorescence lifetime imaging of single molecules. Recent work has improved the technique’s repetition rate to enable compatibility with mode-locked lasers and demonstrated the combination of wide-field fluorescence lifetime imaging with super-resolution localization microscopy, observations of single molecule dynamics, and observation of donor lifetime quenching in single-molecule imaging. These results were achieved on standard camera sensors and would not have been possible with other wide-field approaches. The throughput and photon economy of the EO-FLIM method enables new BER-relevant imaging opportunities. In particular, scanned single- and two-photon light-sheet excitation will be used to achieve volumetric imaging with time-domain contrast.

47 OTHER INSTRUMENTATION↗

Enhancing isoprenol production by systematically tuning metabolic pathways using CRISPR interference in E. coli

Regulation of metabolic gene expression is crucial for maximizing bioproduction titers. Recent engineering tools including CRISPR/Cas9, CRISPR interference (CRISPRi), and CRISPR activation (CRISPRa) have enabled effective knock-out, knock-down, and overexpression of endogenous pathway genes, respectively, for advanced strain engineering. CRISPRi in particular has emerged as a powerful tool for gene repression through the use of a deactivated Cas9 (dCas9) protein and target guide RNA (gRNA). By constructing gRNA arrays, CRISPRi has the capacity for multiplexed gene downregulation across multiple orthogonal pathways for enhanced bioproduction titers. In this study, we harnessed CRISPRi to downregulate 32 essential and non-essential genes in E. coli strains heterologously expressing either the original mevalonate pathway or isopentenyl diphosphate (IPP) bypass pathway for isoprenol biosynthesis. Isoprenol remains a candidate bioproduct both as a drop-in blend additive and as a precursor for the high-performance sustainable aviation fuel, 1,4-dimethylcyclooctane (DMCO). Of the 32 gRNAs targeting genes associated with isoprenol biosynthesis, a subset was found to vastly improve product titers. Construction of a multiplexed gRNA library based on single guide RNA (sgRNA) performance enabled simultaneous gene repression, yielding a 3 to 4.5-fold increase in isoprenol titer (1.82 ± 0.19 g/L) on M9-MOPS minimal medium. We then scaled the best performing CRISPRi strain to 2-L fed-batch cultivation and demonstrated translatable titer improvements, ultimately obtaining 12.4 ± 1.3 g/L isoprenol. Our strategy further establishes CRISPRi as a powerful tool for tuning metabolic flux in production hosts and that titer improvements are readily scalable with potential for applications in industrial bioproduction.

59 BASIC BIOLOGICAL SCIENCES↗

Phylogenetic sequence of metabolic pathways in Precambrian cellular life

A sequence of major metabolic events is presented as they may have appeared during prokaryote evolution. This is based on (1) the phylogenetic schema derived from sequences of bacterial ferredoxin, 2Fe-2S ferredoxin, 5S ribosomal RNA, and c-type cytochromes; (2) metabolic settings in which these macromolecules are found; and (3) metabolic capabilities of the prokaryotes that carry these molecules.

Barnabas, J.↗

Evaluating the contribution of plant metabolic pathways in the light to the ATP:NADPH demand using a meta-analysis of isotopically non-stationary metabolic flux analyses

Abstract Balancing the ATP: NADPH demand from plant metabolism with supply from photosynthesis is essential for preventing photodamage and operating efficiently, so understanding its drivers is important for integrating metabolism with the light reactions of photosynthesis and for bioengineering efforts that may radically change this demand. It is often assumed that the C3 cycle and photorespiration consume the largest amount of ATP and reductant in illuminated leaves and as a result mostly determine the ATP: NADPH demand. However, the quantitative extent to which other energy consuming metabolic processes contribute in large ways to overall ATP: NADPH demand remains unknown. Here, we used the metabolic flux networks of numerous recently published isotopically non-stationary metabolic flux analyses (INST-MFA) to evaluate flux through the C3 cycle, photorespiration, the oxidative pentose phosphate pathway, the tricarboxylic acid cycle, and starch/sucrose synthesis and characterize broad trends in the demand of energy across different pathways and compartments as well as in the overall ATP:NADPH demand. These data sets include a variety of species including Arabidopsis thaliana , Nicotiana tabacum , and Camelina sativa as well as varying environmental factors including high/low light, day length, and photorespiratory levels. Examining these datasets in aggregate reveals that ultimately the bulk of the energy flux occurred in the C3 cycle and photorespiration, however, the energy demand from these pathways did not determine the ATP: NADPH demand alone. Instead, a notable contribution was revealed from starch and sucrose synthesis which might counterbalance photorespiratory demand and result in fewer adjustments in mechanisms which balance the ATP deficit.

59 BASIC BIOLOGICAL SCIENCES↗

Engineering of xylose metabolic pathways in Rhodotorula toruloides for sustainable biomanufacturing

Abstract The oleaginous yeast Rhodotorula toruloides is a promising microbial cell factory for the sustainable production of biofuels and value-added chemicals from renewable carbon sources. Unlike the conventional yeast Saccharomyces cerevisiae, R. toruloides can naturally metabolize xylose, the second most abundant sugar in lignocellulosic hydrolysates. However, its native xylose metabolism is inefficient, characterized by slow xylose uptake and accumulation of D-arabitol. Moreover, despite its phenotype, research on the enzymes involved in xylose metabolism has yet to reach a consensus. Therefore, this review provides a comprehensive analysis of the non-canonical xylose metabolism in R. toruloides, focusing on the properties of key enzymes involved in xylose metabolism. Native xylose reductase and xylitol dehydrogenase exhibit broad substrate promiscuity compared to their counterparts in the xylose-fermenting Scheffersomyces stipitis. Additionally, the absence of xylulokinase expression under xylose-utilizing conditions redirects metabolism toward D-arabitol accumulation. Consequently, D-arabitol dehydrogenases and ribulokinase play essential roles in the xylose metabolism of R. toruloides. These findings highlight the fundamental differences between R. toruloides xylose metabolism and the oxidoreductase pathways observed in other xylose-fermenting yeast, providing insights for metabolic engineering strategies to improve xylose utilization and enhance bioconversion of cellulosic hydrolysates to different bioproducts by R. toruloides.

Biotechnology & Applied Microbiology↗

Light‐Driven Metabolic Pathways in Non‐Photosynthetic Biohybrid Bacteria

Biomanufacturing via microorganisms relies on carbon substrates for molecular feedstocks and a source of energy to carry out enzymatic reactions. This creates metabolic bottlenecks and lowers the efficiency for substrate conversion. Nanoparticle biohybridization with proteins and whole cell surfaces can bypass the need for redox cofactor regeneration for improved secondary metabolite production in a non-specific manner. Here we propose using nanobiohybrid organisms (Nanorgs), intracellular protein-nanoparticle hybrids formed through the spontaneous coupling of core-shell quantum dots (QDs) with histidine-tagged enzymes in non-photosynthetic bacteria, for light-mediated control of bacterial metabolism. Furthermore, this proved to eliminate metabolic constrictions and replace glucose with light as the source of energy in Escherichia coli, with an increase in growth by 1.7-fold in 75 % reduced nutrient media. Metabolomic tracking through carbon isotope labeling confirmed flux shunting through targeted pathways, with accumulation of metabolites downstream of respective targets. Finally, application of Nanorgs with the Ehrlich pathway improved isobutanol titers/yield by 3.9-fold in 75 % less sugar from E. coli strains with no genetic alterations. These results demonstrate the promise of Nanorgs for metabolic engineering and low-cost biomanufacturing.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

An in silico assessment of gene function and organization of the phenylpropanoid pathway metabolic networks in Arabidopsis thaliana and limitations thereof

The Arabidopsis genome sequencing in 2000 gave to science the first blueprint of a vascular plant. Its successful completion also prompted the US National Science Foundation to launch the Arabidopsis 2010 initiative, the goal of which is to identify the function of each gene by 2010. In this study, an exhaustive analysis of The Institute for Genomic Research (TIGR) and The Arabidopsis Information Resource (TAIR) databases, together with all currently compiled EST sequence data, was carried out in order to determine to what extent the various metabolic networks from phenylalanine ammonia lyase (PAL) to the monolignols were organized and/or could be predicted. In these databases, there are some 65 genes which have been annotated as encoding putative enzymatic steps in monolignol biosynthesis, although many of them have only very low homology to monolignol pathway genes of known function in other plant systems. Our detailed analysis revealed that presently only 13 genes (two PALs, a cinnamate-4-hydroxylase, a p-coumarate-3-hydroxylase, a ferulate-5-hydroxylase, three 4-coumarate-CoA ligases, a cinnamic acid O-methyl transferase, two cinnamoyl-CoA reductases) and two cinnamyl alcohol dehydrogenases can be classified as having a bona fide (definitive) function; the remaining 52 genes currently have undetermined physiological roles. The EST database entries for this particular set of genes also provided little new insight into how the monolignol pathway was organized in the different tissues and organs, this being perhaps a consequence of both limitations in how tissue samples were collected and in the incomplete nature of the EST collections. This analysis thus underscores the fact that even with genomic sequencing, presumed to provide the entire suite of putative genes in the monolignol-forming pathway, a very large effort needs to be conducted to establish actual catalytic roles (including enzyme versatility), as well as the physiological function(s) for each member of the (multi)gene families present and the metabolic networks that are operative. Additionally, one key to identifying physiological functions for many of these (and other) unknown genes, and their corresponding metabolic networks, awaits the development of technologies to comprehensively study molecular processes at the single cell level in particular tissues and organs, in order to establish the actual metabolic context.

NASA Program Fundamental Space Biology↗

Continuous cropping of potato changed the metabolic pathway of root exudates to drive rhizosphere microflora

For potato production, continuous cropping (CC) could lead to autotoxicity buildup and microflora imbalance in the field soil, which may result in failure of crops and reduction in yield. In this study, non-targeted metabolomics (via liquid chromatography with tandem mass spectrometry (LC–MS/MS)) combined with metagenomic profiling (via high-throughput amplicon sequencing) were used to evaluate correlations between metabolomics of potato root exudates and communities of bacteria and fungi around potato plants to illustrate the impacts of CC. Potato plants were grown in soil collected from fields with various CC years (0, 1, 4, and 7 years). Metabolomic analysis showed that the contents and types of potential autotoxins in potato root exudates increased significantly in CC4 and CC7 plants (i.e., grown in soils with 4 and 7 years of CC). The differentially expressed metabolites were mainly produced via alpha-linolenic acid metabolism in plant groups CC0 and CC1 (i.e., no CC or 1 year CC). The metabolomics of the groups CC4 and CC7 became dominated by styrene degradation, biosynthesis of siderophore group non-ribosomal peptides, phenylpropanoid biosynthesis, and biosynthesis of various plant secondary metabolites. Continuous cropping beyond 4 years significantly changed the bacterial and fungal communities in the soil around the potato crops, with significant reduction of beneficial bacteria and accumulation of harmful fungi. Correlations between DEMs and microflora biomarkers were established with strong significances. These results suggested that continuous cropping of potato crops changed their metabolism as reflected in the plant root exudates and drove rhizosphere microflora to directions less favorable to plant growth, and it needs to be well managed to assure potato yield.

Xing, Yanhong↗

Fundamental Research Aimed at Diverting Excess Reducing Power in Photosynthesis to Orthogonal Metabolic Pathways

Photosystems are incredible biological machines that use sunlight to drive the conversion of carbon dioxide to sugar. The amount of sunlight available for photosynthesis sometimes exceeds the amount of energy plants can use. This excess energy has to be safely dissipated through non-productive biological processes. The ultimate goal of this project is to understand whether we can utilize that otherwise unused excess energy. In our previous work, we showed that, in principle, it is possible to attach a catalyst to photosystem I and generate H2 using light. Our current strategy is to genetically fuse parts of the photosystem I complex with a recently discovered oxygen-tolerant [FeFe] hydrogenase. Our rationale is that such chimeric proteins may potentially result in the natural incorporation of the photosystem I-hydrogenase link using the inherent genetic machinery of the cell. In this project, we aim to verify that light-driven hydrogen production in this construction is possible. Throughout the project, we designed nanoconstructs that showcase the plausibility of this technology, at least in vitro. We take advantage of these constructs to investigate details of the coupling between photosystem I and a H2-producing enzyme called [FeFe] hydrogenase. This part of the project reveals details of the electron transfer between photosystem I and the attached hydrogenase, providing information that can lead to new strategies for improved biological photocatalysis. We also researched efficient and robust tethering of the [FeFe] hydrogenase to photosystem I in cyanobacteria. This work will highlight successful design strategies to guide the future development of photosynthetic biohybrids. Uncovering the principles governing the utilization of otherwise unusable energy significantly further our understanding of cyanobacterial photosynthesis. The work proposed establishes the feasibility of diverting excess energy under high light conditions to orthogonal enzymatic pathways and set design rules for efficient utilization of such a strategy for scientific and industrial applications in biosensing, renewable energy, and high-value chemicals production. The work addresses the DOE-BES Photosynthetic Systems program goal to develop a multidimensional understanding of photosystems that would provide specific metrics that instruct strategies for improving biological photosynthesis and for guiding the future development of bioreactors and biomimetic energy systems.

Photosynthetic systems, hydrogenase, cyanobacteria↗

Anaerobic benzene oxidation in Geotalea daltonii involves activation by methylation and is regulated by the transition state regulator AbrB

ABSTRACT Benzene is a widespread groundwater contaminant that persists under anoxic conditions. The aim of this study was to more accurately investigate anaerobic microbial degradation pathways to predict benzene fate and transport. Preliminary genomic analysis of Geotalea daltonii strain FRC-32, isolated from contaminated groundwater, revealed the presence of putative aromatic-degrading genes. G. daltonii was subsequently shown to conserve energy for growth on benzene as the sole electron donor and fumarate or nitrate as the electron acceptor. The hbs gene, encoding for 3-hydroxybenzylsuccinate synthase (Hbs), a homolog of the radical-forming, toluene-activating benzylsuccinate synthase (Bss), was upregulated during benzene oxidation in G. daltonii , while the bss gene was upregulated during toluene oxidation. Addition of benzene to the G. daltonii whole-cell lysate resulted in toluene formation, indicating that methylation of benzene was occurring. Complementation of σ 54 - (deficient) E. coli transformed with the bss operon restored its ability to grow in the presence of toluene, revealing bss to be regulated by σ 54 . Binding sites for σ 70 and the transition state regulator AbrB were identified in the promoter region of the σ 54 -encoding gene rpoN, and binding was confirmed. Induced expression of abrB during benzene and toluene degradation caused G. daltonii cultures to transition to the death phase. Our results suggested that G. daltonii can anaerobically oxidize benzene by methylation, which is regulated by σ 54 and AbrB. Our findings further indicated that the benzene, toluene, and benzoate degradation pathways converge into a single metabolic pathway, representing a uniquely efficient approach to anaerobic aromatic degradation in G. daltonii . IMPORTANCE The contamination of anaerobic subsurface environments including groundwater with toxic aromatic hydrocarbons, specifically benzene, toluene, ethylbenzene, and xylene, has become a global issue. Subsurface groundwater is largely anoxic, and further study is needed to understand the natural attenuation of these compounds. This study elucidated a metabolic pathway utilized by the bacterium Geotalea daltonii capable of anaerobically degrading the recalcitrant molecule benzene using a unique activation mechanism involving methylation. The identification of aromatic-degrading genes and AbrB as a regulator of the anaerobic benzene and toluene degradation pathways provides insights into the mechanisms employed by G. daltonii to modulate metabolic pathways as necessary to thrive in anoxic contaminated groundwater. Our findings contribute to the understanding of novel anaerobic benzene degradation pathways that could potentially be harnessed to develop improved strategies for bioremediation of groundwater contaminants.

Bullows, James E.↗