Renewable Carbon Source Research Articles

Biosynthesis of C4-C8 3-Hydroxycarboxylic Acids from Glucose through the Inverted Fatty Acid β-Oxidation by Metabolically Engineered Escherichia coli.

Inverted fatty acid β-oxidation represents a versatile biochemical platform for biosynthesis by the engineered microbial strains of numerous value-added chemicals from convenient and abundant renewable carbon sources, including biomass-derived sugars. Although, in recent years, significant progress has been made in the production through this pathway of n-alcohols, 1,3-diols, and carboxylic acids and its 2,3-unsaturated derivatives, the potential of the pathway for the biosynthesis of 3-hydroxycarboxylic acids remained almost undisclosed. In this study, we demonstrate the microaerobic production of even-chain-length C4-C8 3-hydroxycarboxylic acids from glucose through the inverted fatty acid β-oxidation by engineered E. coli strains. The notable accumulation of target compounds was achieved upon the strong constitutive expression of the genes atoB, fadA, fadB, fadE/fabI, and tesB, which code for the key enzymes catalysing reactions of aerobic fatty acid β-oxidation and thioesterase II, in strains devoid of mixed-acid fermentation pathways and lacking nonspecific thioesterase YciA. The best performing recombinants were able to synthesise up to 14.5 mM of 3-hydroxycarboxylic acids from glucose with a total yield of 0.34 mol/mol and a C4/C6/C8 ratio averaging approximately 63/28/9. The results provide a framework for the development of highly efficient strains and processes for the bio-based production of valuable 3-hydroxycarboxylates from renewable raw materials.

From waste to raw chemicals: Catalytic transformation of fusel oil by mixed metal oxides

In this study, we detail a catalytic process for the conversion of fusel oil (FO), a by-product of ethanol production considered as waste, into valuable organic compounds, including aldehydes, alkenes, ketones, and alkylbenzenes spanning a carbon chain length from C6 to C12. The reaction was carried out in a fixed bed flow reactor at atmospheric pressure, employing mixed metal oxides (MMO) derived from hydrotalcites (HTC) as catalysts. The HTC were readily synthesized by co-precipitation method involving Mg2+ and Al3+, wherein 20 mol% Mg2+ was substituted by M2+ = Mn, Fe, Co, Ni, Cu, and Zn, and MMO obtained by HTC calcination. These catalysts offer cost-effectiveness compared to noble metal-containing ones while exhibiting remarkable activity in dehydrogenation, hydrogenation, and dehydration reactions pivotal for alcohol reactivity. The proposed methodology holds promise for large-scale industrial use and pioneers a fresh avenue for deriving value-added raw chemicals from a renewable carbon source.

Biobased Immiscible Polylactic Acid (PLA): Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) Blends: Impact of Rheological and Non-isothermal Crystallization on the Bead Foaming Behavior

Nowadays, bead foams are of great interest due to their high lightweight potential. The processing of such foams strongly depends on the crystallization and rheological behavior of the polymers used. By blending polymers, these properties can be tailored to obtain beaded foams with low density, small cell size and high cell density. As a bio-based polymer, PLA is of great interest due to its renewable carbon source. PLA suffers from its low thermal and rheological properties, which can be compensated by using blends. The correlation between the PLA/PHBV ratio and the rheological as well as the crystallization behavior was investigated. The use of PHBV as a minor phase significantly changes the rheological properties and increases the crystallization behavior of PLA. These findings were applied to the foam extrusion process to obtain low density bead foams. Bead foams with densities below 100 kg/m3, mean cell sizes below 50 µm and cell densities of 1 × 107 cells/cm3 were obtained.

Designing biomass policy: The political economy of renewable energy for net zero

AbstractThe climate, ecological, and energy crises require change in our political, economic, and societal systems to ensure we decouple humanity from a reliance on fossil fuels, prevent rising carbon dioxide emissions, and develop sustainable solutions for people and the planet. As well as technical processes, renewable energy transitions are processes of social, environmental, and economic change which have the potential to challenge the status quo. This status quo determines who benefits from energy, where wealth is created, and the level of inequality between stakeholders within our energy systems. The politicization of energy transitions motivates stakeholders to engage in the policymaking process to ensure any trade‐offs associated with policy changes benefit them. Bioenergy is unique amongst renewable energy sources as it is inherently linked to biomass extraction from our natural environment and because biomass is the only source of renewable carbon. However, this further politicizes its use and is a source of controversy in public debate. Polarized perspectives in the public debate on biomass policy allow stakeholders to assert themselves as experts on the topic and to make authoritative claims that further their interests to influence policy development. Therefore, political and economic drivers shape and influence the sustainability and success of proposed policies. Despite this, there is little research into the nontechnical factors influencing the design of sustainable biomass policy for net zero. This research highlights how political economy impacts the success of renewable energy technologies in replacing fossil fuels and the implications for using bioenergy.This article is categorized under: Sustainable Energy > Bioenergy Policy and Economics > Energy Transitions Climate and Environment > Net Zero Planning and Decarbonization

Engineering Terpene Production Pathways in Methylobacterium extorquens AM1.

Terpenes are diverse specialized metabolites naturally found within plants and have important roles in inter-species communication, adaptation and interaction with the environment. Their industrial applications span a broad range, including fragrances, flavors, cosmetics, natural colorants to agrochemicals and therapeutics, yet formal chemical synthesis is economically challenging due to structural complexities. Engineering terpene biosynthesis could represent an alternative in microbial biotechnological workhorses, such as Saccharomyces cerevisiae or Escherichi coli, utilizing sugars or complex media as feedstocks. Host species that metabolize renewable and affordable carbon sources may offer unique sustainable biotechnological alternatives. Methylotrophs are bacteria with the capacity to utilize one-carbon feedstocks, such as methanol or formate. They colonize the phyllosphere (above-ground area) of plants, and many accumulate abundant carotenoid pigments. Methylotrophs have the capacity to take up and use a subset of the rare earth elements known as lanthanides. These metals can enhance one-carbon (methylotrophic) metabolism. Here, we investigated whether manipulating the metabolism enables and enhances terpene production. A carotenoid-deficient mutant potentially liberates carbon, which may contribute to bioproduct accumulation. To test this hypothesis, terpene-producing bacterial strains regulated by two distinct promoters were generated. Wildtype Methylobacterium extorquens, ∆Meta1_3665, a methylotrophic mutant lacking the carotenoid pathway, and an E. coli strain were transformed with an exogenous terpene pathway and grown both in the presence and absence of lanthanides. The extraction, and the comparison of analytical profiles, provided evidence that engineered cultured M. extorquens under control of a native, inducible methylotrophic promoter can yield the sesquiterpene patchoulol when supplemented with lanthanide. In contrast, using a moderate-strength constitutive promoter failed to give production. We demonstrated colonization of the phyllosphere with the engineered strains, supporting the future engineering of selected species of the plant microbiome and with promising implications for the synthetic biology of small molecules.

Tailored polyhydroxyalkanoate production from renewable non-fatty acid carbon sources using engineered Cupriavidus necator H16

As thermoplastic, nontoxic, and biocompatible polyesters, polyhydroxyalkanoates (PHAs) are considered promising biodegradable plastic candidates for diverse applications. Short-chain-length/medium-chain-length (SCL/MCL) PHA copolymers are flexible and versatile PHAs that are typically produced from fatty acids, which are expensive and toxic. Therefore, to achieve the sustainable biosynthesis of SCL/MCL-PHAs from renewable non-fatty acid carbon sources (e.g., sugar or CO2), we used the lithoautotrophic bacterium Cupriavidus necator H16 as a microbial platform. Specifically, we synthesized tailored PHA copolymers with varying MCL-3-hydroxyalkanoate (3HA) compositions (10–70 mol%) from fructose by rewiring the MCL-3HA biosynthetic pathways, including (i) the thioesterase-mediated free fatty acid biosynthetic pathway coupled with the beta-oxidation cycle and (ii) the hydroxyacyl transferase-mediated fatty acid de novo biosynthetic pathway. In addition to sugar-based feedstocks, engineered strains are also promising platforms for the lithoautotrophic production of SCL/MCL-PHAs from CO2. The set of engineered C. necator strains developed in this study provides greater opportunities to produce customized polymers with controllable monomer compositions from renewable resources.

Recent Advances in Microbial Metabolic Engineering for Production of Natural Phenolic Acids.

Phenolic acids are important natural bioactive compounds with varied physiological functions. They are extensively used in food, pharmaceutical, cosmetic, and other chemical industries and have attractive market prospects. Compared to plant extraction and chemical synthesis, microbial fermentation for phenolic acid production from renewable carbon sources has significant advantages. This review focuses on the structural information, physiological functions, current applications, and biosynthesis pathways of phenolic acids, especially advances in the development of metabolically engineered microbes for the production of phenolic acids. This review provides useful insights concerning phenolic acid production through metabolic engineering of microbial cell factories.

Identifying and charactering a 4-aminobutyryl-CoA ligase for the production of butyrolactam

Butyrolactam, a crucial four-carbon molecule, serves as building block in synthesis of polyamides. While biosynthesis of butyrolactam from renewable carbon sources offers a more sustainable approach, it has faced challenges in achieving high product titer and yield. Here, an efficient microbial platform for butyrolactam production was constructed by elimination of rate-limiting step and systematic pathway optimization. Initially, a superior 4-aminobutyryl-CoA ligase was discovered and characterized among six acyl-CoA ligases from different sources, which greatly improved the pathway efficiency. Subsequent optimizations were implemented to further enhance butyrolactam production, including promoter engineering, the elimination of competing pathways, transporter engineering and improving the availability of precursors. There efforts resulted in achieving approximately 2 g/L butyrolactam in shake flask experiments. Finally, the biosynthesis of butyrolactam was scaled up in a 3-L bioreactor in 84 hours, resulting in a significantly increased production of 45.2 g/L, with a carbon yield of 0.34 g/g glucose. This study highlights the construction of a microbial platform with the capability to achieve elevated levels of butyrolactam production and unlocks its potential in sustainable manufacturing processes.

The chemical logic of enzymatic lignin degradation.

Lignin is an aromatic heteropolymer, found in plant cell walls as 20-30% of lignocellulose. It represents the most abundant source of renewable aromatic carbon in the biosphere, hence, if it could be depolymerised efficiently, then it would be a highly valuable source of renewable aromatic chemicals. However, lignin presents a number of difficulties for biocatalytic or chemocatalytic breakdown. Research over the last 10 years has led to the identification of new bacterial enzymes for lignin degradation, and the use of metabolic engineering to generate useful bioproducts from microbial lignin degradation. The aim of this article is to discuss the chemical mechanisms used by lignin-degrading enzymes and microbes to break down lignin, and to describe current methods for generating aromatic bioproducts from lignin using enzymes and engineered microbes.

Identification and characterization of a potential strain for the production of polyhydroxyalkanoate from glycerol.

While poly (3-hydroxybutyrate) (PHB) holds promise as a bioplastic, its commercial utilization has been hampered by the high cost of raw materials. However, glycerol emerges as a viable feedstock for PHB production, offering a sustainable production approach and substantial cost reduction potential. Glycerol stands out as a promising feedstock for PHB production, offering a pathway toward sustainable manufacturing and considerable cost savings. The identification and characterization of strains capable of converting glycerol into PHB represent a pivotal strategy in advancing PHB production research. In this study, we isolated a strain, Ralstonia sp. RRA (RRA). The strain exhibits remarkable proficiency in synthesizing PHB from glycerol. With glycerol as the carbon source, RRA achieved a specific growth rate of 0.19 h-1, attaining a PHB content of approximately 50% within 30 h. Through third-generation genome and transcriptome sequencing, we elucidated the genome composition and identified a total of eight genes (glpR, glpD, glpS, glpT, glpP, glpQ, glpV, and glpK) involved in the glycerol metabolism pathway. Leveraging these findings, the strain RRA demonstrates significant promise in producing PHB from low-cost renewable carbon sources.

Lignin Extraction by Using Two-Step Fractionation: A Review.

Lignocellulosic biomass represents the most abundant renewable carbon source on earth and is already used for energy and biofuel production. The pivotal step in the conversion process involving lignocellulosic biomass is pretreatment, which aims to disrupt the lignocellulose matrix. For effective pretreatment, a comprehensive understanding of the intricate structure of lignocellulose and its compositional properties during component disintegration and subsequent conversion is essential. The presence of lignin-carbohydrate complexes and covalent interactions between them within the lignocellulosic matrix confers a distinctively labile nature to hemicellulose. Meanwhile, the recalcitrant characteristics of lignin pose challenges in the fractionation process, particularly during delignification. Delignification is a critical step that directly impacts the purity of lignin and facilitates the breakdown of bonds involving lignin and lignin-carbohydrate complexes surrounding cellulose. This article discusses a two-step fractionation approach for efficient lignin extraction, providing viable paths for lignin-based valorization described in the literature. This approach allows for the creation of individual process streams for each component, tailored to extract their corresponding compounds.

One-Pot Reversible Complexation-Mediated Polymerization (RCMP) from Benzylic Alcohols for Facile Access to Polymer-Grafted Lignin.

One-pot synthesis of methacrylic and acrylic polymers from benzylic alcohols (R-OH) used as initiating moieties was developed. R-OH was converted to alkyl iodide (R-I), and the generated R-I was used as an initiator without purification or isolation in the subsequent reversible complexation mediated polymerization (RCMP), leading to one-pot RCMP from R-OH. As a useful application, this technique was exploited for one-pot polymer-grafting from lignin that is the second most abundant renewable carbon-source on earth and bears benzylic alcohols. The direct initiation from lignin eliminates tedious initiator attachment and purification, offering a facile access to polymer-grafted lignin. The obtained polymer-grafted lignin was utilized to form an efficient UV-absorbing film with high transparency in visible region. One-pot RCMP may serve as a practical method to obtain value-added functional lignin-polymer composites.

Sustainable bio-manufacturing of D-arabitol through combinatorial engineering of Zygosaccharomyces rouxii, bioprocess optimization and downstream separation

Biosynthesis of D-arabitol, a high value-added platform chemical, from renewable carbon sources provides a sustainable and eco-friendly alternative to the chemical industry. Here, a robust brewing yeast, Zygosaccharomyces rouxii, capable of naturally producing D-arabitol was rewired through genome sequencing-based metabolic engineering. The recombinant Z. rouxii obtained by reinforcing the native D-xylulose pathway, improving reductive power of the rate-limiting step, and inhibiting the shunt pathway, produced 73.61% higher D-arabitol than the parent strain. Subsequently, optimization of the fermentation medium composition for the engineered strain provided 137.36 g/L D-arabitol, with a productivity of 0.64 g/L/h in a fed-batch experiment. Finally, the downstream separation of D-arabitol from the complex fermentation broth using an ethanol precipitation method provided a purity of 96.53%. This study highlights the importance of D-xylulose pathway modification in D-arabitol biosynthesis, and pave a complete and efficient way for the sustainable manufacturing of this value-added compound from biosynthesis to preparation.

Facile synthesis of ultrathin carbon nanosheets from waste cellulose

Ultrathin carbon nanosheets were fabricated using renewable carbon sources. Cellulose, an important component in the food industry, was processed to form a food byproduct and used to synthesize carbon nanosheets. Both bacterial and nonbacterial cellulose from kombucha byproducts and apple pomace, respectively, were processed via purification and pyrolysis. An inert argon atmosphere and elevated temperatures of 600 °C–800 °C for 20 min were maintained during pyrolysis. Under these conditions, the apple pomace produced a higher yield of nanosheets than the kombucha byproduct. The nanosheets with the thickness of 4 nm were characterized using different spectroscopic techniques such as Fourier transform infrared spectroscopy and Raman spectroscopy as well as microscopic techniques such as scanning electron microscopy and atomic force microscopy. This sustainable, simple, and green method of carbon nanosheet production is a promising alternative to conventional methods of production.

Ni Nanoparticles Supported Over Triazine Based Porous Organic Polymer for Selective CO2 Photo‐Reduction to Methanol

AbstractPorous organic polymers (POPs) have attracted substantial attentions over the years due to their exceptionally high specific surface areas, high chemical stability of the organic network and ease of surface functionalization with the desired organic groups. In this work, a triazine based POP (TrzPOP) was synthesized through Schiff base polycondensation reaction between a tetramine bearing triazine rings and phenolic–OH group rich dialdehyde. Ni nanoparticles (NiNP) synthesized independently were immobilized over TrzPOP to obtain the NiNP@TrzPOP composite catalyst. This TrzPOP possesses a high BET surface area of 1494 m2 g−1 and low band gap, which facilitates its role as visible light absorbent. NiNP@TrzPOP with N‐rich surfaces and phenolic–OH moieties displayed excellent photocatalytic activity in the CO2 photoreduction under mild reaction conditions. NiNP@TrzPOP composite selectively reduces CO2 to methanol. The turn over number (TON) for this photoreduction of CO2 under optimized reaction conditions is 270, which is considerably high comparing to other reported photocatalytic systems. Moreover, NiNP@TrzPOP composite catalytic system showed high recycling efficiency without noticeable decrease in its performance over five consecutive reaction cycles, suggesting its huge potential for large‐scale methanol synthesis from the renewable carbon source.

Preparation of high-performance nitrogen doped porous carbon from cork biomass by K2CO3 activation for adsorption of rhodamine B

Biomass waste, an attractive renewable carbon source, has great potential for carbon reduction. In recent years, high-performance porous carbon materials prepared from agricultural and forestry waste have demonstrated significant promise for applications in the field of dye wastewater purification. In this study, cork waste from Quercus variabilis was utilized as a raw material, melamine as a nitrogen source, and K2CO3 as a green activator to prepare nitrogen-doped porous cork activated carbon (NCAC) with a honeycomb structure. The results showed that NCAC-900, activated at 900 °C, exhibited a highly porous structure with an optimal specific surface area of 2051.35 m2/g and a pore volume of 1.18 cm3/g. To evaluate its adsorption performance towards rhodamine B (RhB), collaborative activation and post-treatment were employed to enhance the adsorption capacity of the dyes. NCAC-900 demonstrated excellent removal efficiency, with a theoretical maximum adsorption capacity of 2552.52 mg/g (at an adsorbent dosage of 0.2 g/L) in a 300 mg/L solution. Nitrogen doping effectively improved the adsorption capacity for RhB, fitting well to the quasi-secondary kinetic model (R2 = 0.9973–0.9999) and Langmuir isotherm (R2 = 0.7697–0.9523) for the adsorption kinetics and equilibrium data. A comparison with the existing literature also revealed that NCAC-900 possessed a remarkably fast adsorption rate of 12.82 mg/g/min. Moreover, the resorption efficiency of NCAC-900 for RhB reached approximately 66.78% after five consecutive adsorption-desorption cycles. Additionally, the obtained nitrogen-doped cork activated carbon exhibited impressive adsorption capabilities of anionic dye Congo red (CR, qm=1499.14 mg/g) and cationic dye methylene blue (MB, qm=571.54 mg/g). In conclusion, our work presented a promising method for preparing excellent NCAC as an efficient adsorbent for wastewater dyes, offering new ideas and theoretical foundations for the high-value utilization of cork materials and the development of cork activated carbon.

Engineering the synthetic β-alanine pathway in Komagataella phaffii for conversion of methanol into 3-hydroxypropionic acid

BackgroundMethanol is increasingly gaining attraction as renewable carbon source to produce specialty and commodity chemicals, as it can be generated from renewable sources such as carbon dioxide (CO2). In this context, native methylotrophs such as the yeast Komagataella phaffii (syn Pichia pastoris) are potentially attractive cell factories to produce a wide range of products from this highly reduced substrate. However, studies addressing the potential of this yeast to produce bulk chemicals from methanol are still scarce. 3-Hydroxypropionic acid (3-HP) is a platform chemical which can be converted into acrylic acid and other commodity chemicals and biopolymers. 3-HP can be naturally produced by several bacteria through different metabolic pathways.ResultsIn this study, production of 3-HP via the synthetic β-alanine pathway has been established in K. phaffii for the first time by expressing three heterologous genes, namely panD from Tribolium castaneum, yhxA from Bacillus cereus, and ydfG from Escherichia coli K-12. The expression of these key enzymes allowed a production of 1.0 g l−1 of 3-HP in small-scale cultivations using methanol as substrate. The addition of a second copy of the panD gene and selection of a weak promoter to drive expression of the ydfG gene in the PpCβ21 strain resulted in an additional increase in the final 3-HP titer (1.2 g l−1). The 3-HP-producing strains were further tested in fed-batch cultures. The best strain (PpCβ21) achieved a final 3-HP concentration of 21.4 g l−1 after 39 h of methanol feeding, a product yield of 0.15 g g−1, and a volumetric productivity of 0.48 g l−1 h−1. Further engineering of this strain aiming at increasing NADPH availability led to a 16% increase in the methanol consumption rate and 10% higher specific productivity compared to the reference strain PpCβ21.ConclusionsOur results show the potential of K. phaffii as platform cell factory to produce organic acids such as 3-HP from renewable one-carbon feedstocks, achieving the highest volumetric productivities reported so far for a 3-HP production process through the β-alanine pathway.

A facile route to the synthesis of carbon replicas cast from narrow-mesoporous matrices

Carbon replicas find a variety of applications in chemical sciences and even in medicine. However, tedious synthesis hinders their application to the laboratory scale hitherto. Here, we develop a facile procedure for the synthesis of CMK-1 and CMK-2 materials cast from narrow-mesoporous hard templates, i.e. MCM-48 and SBA-1, respectively. The proposed recipe is reproducible, operates under mild conditions using basic laboratory equipment and a renewable carbon source (furfuryl alcohol). It relies on the solvent-polarity-controlled deposition of a polar carbon precursor on unmodified silica in a non-polar medium. Such a self-regulating process provides even coverage of the matrix with polymer leading to the formation of carbon mesostructures of exquisite replication fidelity and structural homogeneity while maintaining the primary morphology. The in-depth study of physisorption data employing the dual gas model (simultaneous N2 and CO2 fitting) disclosed the mixed, micro-mesoporous nature of the materials. This tool was found to provide a fair, comprehensive textural characterization, especially useful for materials with pore sizes on the boundary between micro- and mesopores. Therefore, this research paves the way for the manufacturing and application of CMK-1 and CMK-2 carbon replicas on a larger scale. Additionally, it provides certain hints for the robust textural characterization of such materials.

Designing a periplasmic photosynthetic biohybrid system for succinate and electric energy production

The emergence of photosynthetic biohybrid systems provides an environmentally friendly and sustainable approach to produce fuels and chemicals in microbial cell factories. However, the low efficiency of electron transfer between semiconductors and microorganisms hinders production efficiency. In this study, a periplasmic photosynthetic biohybrid system (PPB) was developed in E. coli through biological self-synthesis and optimized through engineering the periplasmic cytochrome network and cell membrane fluidity. The PPB significantly increased succinate production to a titer of 121.8 g/L in a 5 L fermenter by improving the reducing equivalents utilization. Additionally, the PPB was employed to establish a glucose-utilizing microbial consortium that exhibited robust performance, producing consistent electric energy output for a minimum of 17 days and achieving a maximum power density of 225.3 mW/m2. These findings demonstrate the capability of the PPB to enhance chemical production and generate electric energy from renewable carbon sources, thereby driving advances in green chemistry and promoting environmentally friendly industrial processes.

Biomass waste-derived carbon materials for sustainable remediation of polluted environment: A comprehensive review

In response to the growing global concern over environmental pollution, the exploration of sustainable and eco-friendly materials derived from biomass waste has gained significant traction. This comprehensive review seeks to provide a holistic perspective on the utilization of biomass waste as a renewable carbon source, offering insights into the production of environmentally benign and cost-effective carbon-based materials. These materials, including biochar, carbon nanotubes, and graphene, have shown immense promise in the remediation of polluted soils, industrial wastewater, and contaminated groundwater. The review commences by elucidating the intricate processes involved in the synthesis and functionalization of biomass-derived carbon materials, emphasizing their scalability and economic viability. With their distinctive structural attributes, such as high surface areas, porous architectures, and tunable surface functionalities, these materials emerge as versatile tools in addressing environmental challenges. One of the central themes explored in this review is the pivotal role that carbon materials play in adsorption processes, which represent a green and sustainable technology for the removal of a diverse array of pollutants. These encompass noxious organic compounds, heavy metals, and organic matter, encompassing pollutants found in soils, groundwater, and industrial wastewater. The discussion extends to the underlying mechanisms governing adsorption, shedding light on the efficacy and selectivity of carbon-based materials in different environmental contexts. Furthermore, this review delves into multifaceted considerations, spanning the spectrum from biomass and biowaste resources to the properties and applications of carbon materials. This holistic approach aims to equip researchers and practitioners with a comprehensive understanding of the synergistic utilization of these materials, ultimately facilitating effective and affordable strategies for combatting industrial wastewater pollution, soil contamination, and groundwater impurities.