Role of Soil Microbiota Enzymes in Soil Health and Activity Changes Depending on Climate Change and the Type of Soil Ecosystem.

The extracellular enzymes secreted by soil microorganisms play a pivotal role in the decomposition of organic matter and the global cycles of carbon (C), phosphorus (P), and nitrogen (N), also serving as indicators of soil health and fertility. Current research is extensively analyzing these microbial populations and enzyme activities in diverse soil ecosystems and climatic regions, such as forests, grasslands, tropics, arctic regions and deserts. Climate change, global warming, and intensive agriculture are altering soil enzyme activities. Yet, few reviews have thoroughly explored the key enzymes required for soil fertility and the effects of abiotic factors on their functionality. A comprehensive review is thus essential to better understand the role of soil microbial enzymes in C, P, and N cycles, and their response to climate changes, soil ecosystems, organic farming, and fertilization. Studies indicate that the soil temperature, moisture, water content, pH, substrate availability, and average annual temperature and precipitation significantly impact enzyme activities. Additionally, climate change has shown ambiguous effects on these activities, causing both reductions and enhancements in enzyme catalytic functions.

Rational and random mutagenesis of GDEst-95 carboxylesterase: New functionality insights.

Lipolytic enzymes are important contributors in industrial processes from lipid hydrolysis to biofuel production or even polyester biodegradation. While these enzymes can be used in numerous applications, the genotype-phenotype space of certain promising enzymes is still poorly explored. This limits the effective application of such biocatalysts. In this work the genotype space of a 55 kDa carboxylesterase GDEst-95 from Geobacillus sp. 95 was explored using site-directed mutagenesis and directed evolution methods. In this study four site-directed mutants (Gly108Arg, Ala410Arg, Leu226Arg, Leu411Ala) were created based on previous analysis of GDEst-95 carboxylesterase. Error-prone PCR resulted three mutants: two of them with distal mutations: GDEst-RM1 (Arg75Gln), GDEst-RM2 (Gly20Ser Arg75Gln) and the third, GDEst-RM3, with a distal (Ser210Gly) and Tyr317Ala (amino acid position near to the active site) mutation. Mutants with Ala substitution displayed approximately twofold higher specific activity. Arg mutations lead a reduced specific activity, retaining 2.86 % (Gly108Arg), 10.95 % (Ala410Arg), and 44.23 % (Leu226Arg) of lipolytic activity. All three random mutants displayed increased specific activity as well as improved catalytic properties. This research provides the first deeper insights into the functionality of understudied Geobacillus spp. carboxylesterases with 55 kDa in size.

Engineered Geobacillus lipolytic enzymes - Attractive polyesterases that degrade polycaprolactones and simultaneously produce esters.

Plastic pollution is one of the biggest environmental problems plaguing the modern world. Polyester-based plastics contribute significantly to this ecological safety concern. In this study, lipolytic biocatalysts GD-95RM and GDEst-lip developed based on lipase/esterase produced by Geobacillus sp. 95 strain were applied for the degradation of polycaprolactone films (Mn 45.000 (PCL45000) and Mn 80.000 (PCL80000)). The degradation efficiency was significantly enhanced by the addition of short chain alcohols. Lipase GD-95RM (1 mg) can depolymerize 264.0 mg and 280.7 mg of PCL45000 and PCL80000, films respectively, in a 24 h period at 30 °C, while the fused enzyme GDEst-lip (1 mg) is capable of degrading 145.5 mg PCL45000 and 134.0 mg of PCL80000 films in 24 h. The addition of ethanol (25 %) improves the degradation efficiency ~2.5 fold in the case of GD-95RM. In the case of GDEst-lip, 50 % methanol was found to be the optimal alcohol solution and the degradation efficiency was increased by ~3.25 times. The addition of alcohols not only increased degradation speeds but also allowed for simultaneous synthesis of industrially valuable 6-hydroxyhexonic acid esters. The suggested system is an attractive approach for removing of plastic waste and supports the principles of bioeconomics.

Insights in MICP dynamics in urease-positive Staphylococcus sp. H6 and Sporosarcina pasteurii bacterium.

Microbially induced calcite precipitation (MICP) is an efficient and eco-friendly technique that has attracted significant interest for resolving various problems in the soil (erosion, improving structural integrity and water retention, etc.), remediation of heavy metals, production of self-healing concrete or restoration of different concrete structures. The success of most common MICP methods depends on microorganisms degrading urea which leads to the formation of CaCO3 crystals. While Sporosarcina pasteurii is a well-known microorganism for MICP, other soil abundant microorganisms, such as Staphylococcus bacteria have not been thoroughly studied for its efficiency in bioconsolidation though MICP is a very important proccess which can ensure soil quality and health. This study aimed to analyze MICP process at the surface level in Sporosarcina pasteurii and a newly screened Staphylococcus sp. H6 bacterium as well as show the possibility of this new microorganism to perform MICP. It was observed that Staphylococcus sp. H6 culture precipitated 157.35 ± 3.3 mM of Ca2+ ions from 200 mM, compared to 176 ± 4.8 mM precipitated by S. pasteurii. The bioconsolidation of sand particles was confirmed by Raman spectroscopy and XRD analysis, which indicated the formation of CaCO3 crystals for both Staphylococcus sp. H6 and S. pasteurii cells. The water-flow test suggested a significant reduction in water permeability in bioconsolidated sand samples for both Staphylococcus sp. H6 and S. pasteurii. Notably, this study provides the first evidence that CaCO3 precipitation occurs on the surface of Staphylococcus and S. pasteurii cells within the initial 15-30 min after exposure to the biocementation solution. Furthermore, Atomic force microscopy (AFM) indicated rapid changes in cell roughness, with bacterial cells becoming completely coated with CaCO3 crystals after 90 min incubation with a biocementation solution. To our knowledge, this is the first time where atomic force microscopy was used to visualize the dynamic of MICP on cell surface.

Effect of Biosynthesized Silver Nanoparticles on the Growth of the Green Microalga Haematococcus pluvialis and Astaxanthin Synthesis.

Silver nanoparticles (AgNPs) are widely known for their antimicrobial activity in various systems from microorganisms to cell cultures. However, the data on their effects on microalgae are very limited. Unicellular green algae Haematococcus pluvialis is known for its ability to accumulate large amounts of astaxanthin under stress conditions. Therefore, it can be used as a suitable model system to test the influence of AgNPs on stress induction in unicellular algae, with visible phenotypic effects, such as astaxanthin synthesis and cell morphology. This study tested different AgNP concentrations (0-8 mg/L) effects on different growth stages (red and green) of H. pluvialis culture. Effects on cell morphology, culture productivity, and astaxanthin synthesis were evaluated. Data showed that the addition of high concentrations of AgNPs to the growing culture had a significant negative impact on culture productivity. Green-stage (HpG) cultures productivity was reduced by up to 85% by increasing AgNPs concentration to 8 mg/L while the impact on red-stage (HpR) culture was lower. Astaxanthin concentration measurements showed that AgNPs do not have any effect on astaxanthin concentration in HpG culture and caused decreased astaxanthin production rate in HpR culture. HpG culture astaxanthin concentration stayed constant at ~0.43% dry weight, while HpR culture astaxanthin concentration was significantly reduced from 1.89% to 0.60% dry weight by increasing AgNP concentration. AgNPs in the media lead to significant changes in cell morphology in both HpG and HpR cultures. Cell deformations and disrupted cytokinesis, as well as AgNPs and induced sexual reproduction, were observed.

Biosynthesis of Silver Nanoparticles Produced Using Geobacillus spp. Bacteria.

Silver nanoparticles (AgNPs) are well known for their unique physical and chemical properties, which can be incorporated into a wide range of applications. The growing resistance of microorganisms to antimicrobial compounds promoted the use of AgNPs in antimicrobial therapy. AgNPs can be obtained using physical and chemical methods, but these technologies are highly unfriendly to nature and produce large amounts of side compounds (for example, sodium borohydride and N,N-dimethylformamide). Therefore, alternative technologies are required for obtaining AgNPs. This report focuses on the biosynthesis of silver nanoparticles through the reduction of Ag+ with the cell-free secretomes of four Geobacillus bacterial strains, namely, 18, 25, 95, and 612. Only a few studies that involved Geobacillus bacteria in the synthesis of metal nanoparticles, including AgNPs, have been reported to date. The silver nanoparticles synthesized through bio-based methods were characterized using UV-Vis spectroscopy, scanning electron microscopy (SEM), dynamic light scattering (DLS), and zeta potential measurements. UV-Vis spectroscopy showed a characteristic absorbance peak at 410-425 nm, indicative of AgNPs. SEM analysis confirmed that most nanoparticles were spherical. DLS analysis showed that the sizes of the obtained AgNPs were widely distributed, with the majority less than 100 nm in diameter, while the zeta potential values ranged from -25.7 to -31.3 mV and depended on the Geobacillus spp. strain.

Bioprocesses for the recovery of bioenergy and value-added products from wastewater: A review.

Wastewater and activated sludge present a major challenge worldwide. Wastewater generated from large and small-scale industries, laundries, human residential areas and other sources is emerging as a main problem in sanitation and maintenance of smart/green cities. During the last decade, different technologies and processes have been developed to recycle and purify the wastewater. Currently, identification and fundamental consideration of development of more advanced microbial-based technologies that enable wastewater treatment and simultaneous resource recovery to produce bioenergy, biofuels and other value-added compounds (organic acids, fatty acids, bioplastics, bio-pesticides, bio-surfactants and bio-flocculants etc.) became an emerging topic. In the last several decades, significant development of bioprocesses and techniques for the extraction and recovery of mentioned valuable molecules and compounds from wastewater, waste biomass or sludge has been made. This review presents different microbial-based process routes related to resource recovery and wastewater application for the production of value-added products and bioenergy. Current process limitations and insights for future research to promote more efficient and sustainable routes for this under-utilized and continually growing waste stream are also discussed.

Immobilized GDEst-95, GDEst-lip and GD-95RM lipolytic enzymes for continuous flow hydrolysis and transesterification reactions.

In this study lipolytic biocatalysts GD-95RM, GDEst-95 and GDEst-lip were immobilized by encapsulation in calcium alginate beads. All three immobilized biocatalysts demonstrated significantly increased thermal stability at 60-70 °C temperatures and the activity of GD-95RM lipase increased by 50% at 70-80 °C following the immobilization. Moreover, encapsulated GDEst-95 esterase retained higher than 50% lipolytic activity after 3 months of incubation with butanol (25%) and ethanol (50%); GDEst-lip enzyme possessed 50% activity after 2 months of treatment with ethanol (25%) and methanol (25%); and GD-95RM lipase displayed higher that 50% activity after two-week incubation with methanol (50%). All three immobilized enzymes displayed long-term storage capability (>50% activity) at least until 3 months at 4 °C. It was also detected that immobilized GD-95RM and GDEst-lip can perform flow hydrolysis of both avocado oil and p-NP dodecanoate in prototype packed-bed column reactor. The analysis of continuous transesterification of avocado or sunflower oil with ethanol or methanol as substrates confirmed that encapsulated GD-95RM and GDEst-lip enzymes is a useful approach to produce fatty acid alkyl esters.

Study of individual domains' functionality in fused lipolytic biocatalysts based on Geobacillus lipases and esterases.

The prospects of industrial uses of microbial enzymes have increased greatly during the 21st century. Fused lipolytic enzymes (where one or both fused domains possess lipolytic activity) is a rapidly growing group of industrial biocatalysts. However, the most effective fusion strategy, catalytic behavior of each domain and influence of added linkers on physicochemical and kinetic characteristics of such biocatalysts has not been yet explored. In this study the functionality of individual domains in fused lipolytic enzymes, while using GDEst-lip, GDLip-lip and GDEst-est enzymes as a model system, is analyzed for the first time. Analysis of mutant GDEst-lip, GDLip-lip and GDEst-est variants, where one domain is inactive, showed that both domains retained their activity, although the reduction in specific activity of individual domains has been detected. Moreover, experimental data proposed that the N-terminal domain mostly influenced the thermostability, while the C-terminal domain was responsible for thermal activity. GDEst-lip variants fused by using rigid (EAAELAAE) and flexible (GGSELSGG) linkers indicated that a unique restriction site or a rigid linker is the most preferable fusion strategy to develop new chimeric biocatalysts with domains of Geobacillus lipolytic enzymes.

New engineered Geobacillus lipase GD-95RM for industry focusing on the cleaner production of fatty esters and household washing product formulations.

This study presents a new microbial lipolytic enzyme GD-95RM designed via random mutagenesis using previously characterized GD-95 lipase as a template. The improvement in activity of GD-95 lipase was caused by E100K, F154V and V174I mutations. Compared with GD-95 lipase, the GD-95RM lipase had 1.3-fold increased specific activity (2000 U/mg), demonstrated resistance to higher temperatures (75-85 °C), had fourfold increased Vmax towards p-NP dodecanoate and showed 2.5-fold lower KM for p-NP butyrate. It retained > 50% of its lipolytic activity when hydrolyzing short, medium and long acyl chain substrates at 30 °C and 55 °C reaction temperatures after 20 days' incubation with 25% of ethanol. GD-95RM also displayed long-term tolerance (40 d) to 5% NaCl, trisodium citrate, sodium perborate, urea, 0.1% boric acid, citric acid and Triton X-100. Moreover, oil hydrolysis and transesterification results revealed the capability of GD-95RM lipase to produce fatty acids or fatty acid esters through eco-friendly hydrolysis and transesterification reactions using a broad range of vegetable and fish oils, animal fat and different alcohols as substrates. GD-95RM lipase was successfully applied in synthesis reactions for ethyl oleate, octyl oleate and isoamyl oleate without giving to use additional reaction compounds or special reaction conditions.

Development of a new Geobacillus lipase variant GDlip43 via directed evolution leading to identification of new activity-regulating amino acids.

In this study three lipases GD-28, GD-95 and GD-66 (all 43 kDa in size), isolated from Geobacillus spp. were subjected to directed evolution experiments to yield a new synthetic lipolytic enzyme. This new lipase, obtained by DNA shuffling and epPCR, was named GDlip43 (also 43 kDa in size). It demonstrated increased thermoactivity, thermostability, an ability to hydrolyze short and long acyl chain p-NP esters and was activated by different organic solvents. Different activity of GDlip43 raised the hypothesis of new candidate amino acids which could be important for the activity of Geobacillus lipases. Based on the sequence alignment of parental and GDlip43 lipase, three candidate amino acids were selected. The importance of these amino acids, localized at positions 153, 154 and 247 (all of which are distant from the catalytic center of Geobacillus lipases) was investigated using site-directed mutagenesis. Directed evolution experiments also yielded another new lipase - GDlip30 (30 kDa in size). This low molecular mass derivative of GDlip43 had clearly detectable lipolytic activity (40 U/mg) and is the smallest currently known active Geobacillus lipase variant.

Nanosecond duration pulsed electric field together with formic acid triggers caspase-dependent apoptosis in pathogenic yeasts.

Antifungal substances that are used for the treatment of candidiasis have considerable side effects and Candida yeasts are known to obtain drug resistance. The multidrug resistance cases are promoting the search for the new alternative methods and pulsed electric field (PEF) treatment could be the alternative or could be used in combination with conventional therapy for the enhancement of the effect. We have shown that nanosecond range PEF is capable to induce apoptosis in the S. cerevisiae as well as in the drug resistant C. lusitaniae and C. guilliermondii. Supplementing the PEF procedure with formic acid (final concentration 0.05%) resulted in improvement of the inactivation efficacy and the induction of apoptosis in the majority of the yeast population. After the treatment yeast were displaying the DNA strand brakes, activation of yeast metacaspase and externalization of phosphatidylserine. Apoptotic phenotypes were registered already after 30 kV/cm × 250 ns × 50 pulses treatment. The highest number of apoptotic yeast cells (>60%) was obtained during the 30 kV/cm × 750 ns × 50 pulses protocol when combined with 0.05% formic acid. The results of our study are useful for development of new non-toxic and effective protocols to induce programed cell death in different yeast species and thus minimize inflammation of the tissue.

Usage of GD-95 and GD-66 lipases as fusion partners leading to improved chimeric enzyme LipGD95-GD66.

Lipases are used as biocatalysts in industrial processes mainly because of their stability at broad temperature and pH range, resistance to organic solvents and wide spectrum of substrates. The usage of several lipolytic domains, each with different activity and resistance profiles, enables both the flexibility and efficiency of industrial processes. In this study, GD-95 and GD-66 lipases produced by Geobacillus sp. 95 and Geobacillus sp. 66, respectively, were used as fusion partners to create a new fused lipolytic enzyme LipGD95-GD66. Chimeric LipGD95-GD66 lipase displayed tenfold increase in activity (200 U/mg) compared to parental GD-66 lipase, improved Vmax (10 µmol/min mg-1) and catalytic efficiency (2 ∗ 105 min-1 mM-1) for p-NP palmitate as a substrate and increased activity at 70-75 °C compared to both parental lipases. All three lipases also retained >50% of their lipolytic activity after incubation with methanol, n-hexane, ethanol and DMF for longer than three weeks, highlighting a great prospect for application in industrial processes. Moreover, transesterification results revealed the capability of parental GD-95 lipase to be the most promising biocatalyst for production of methyl and ethyl esters through eco-friendly transesterification using argan oil and ethanol/methanol as acceptors of acyl group.

Characterization and application of keratinolytic paptidases from Bacillus spp.

Solid keratin-rich waste management is one of essential research area in nowadays. Conventional chemical and high thermal keratin waste decomposition methods are fully explored and not enough effective for future biotechnology perspectives. However, traditional keratin-rich waste decomposition methods could be replaced by environmentally-friendly and economical microbial keratin waste biodegradation methods without energy wastage and essential amino acids and nutrition elements loss. In this study BPKer and BAKer keratinolytic peptidases from Bacillus sp. AD-W and Bacillus sp. AD-AA3 strains, respectively, were successfully produced, purified and biochemically characterized. Physical and chemical characterization of native BPKer and BAKer suggested that new keratinolytic peptidases are powerful biocatalysts for efficient keratin waste biodegradation and can replace conventional insufficient non-biological hydrolysis processes without energy, important amino acids and nutritional elements loss. High value bio-active hydrolysis products - peptides obtained from keratin waste biodegradation by BPKer and BAKer are suitable for industrial applications in white and green biotechnology.

Microbial lipolytic fusion enzymes: current state and future perspectives.

Genetic fusion of coding ORFs or connection of proteins in a post translational process are rather novel techniques to build products called fusion proteins that possess combined characteristics of their parental biomolecules. This attractive strategy used to create new enzymes not only diversifies their functionality by improving thermostability, thermo- and catalytic activity, substrate specificity, regio- or enantio-selectivity but also facilitates their purification and increases their yield. Many examples of microbial synthetic fusion biocatalysts are associated with fused enzymes that are involved in biomass degradation. However, one of the leading production segments is occupied by microbial lipolytic enzymes (lipases and esterases). As powerful biocatalysts these enzymes found their application in detergent, food, oil and fat, pulp and paper, leather, textile, cosmetics, biodiesel production industries. Moreover, lipolytic enzymes market is predicted to maintain leadership up to the year of 2024 and exceed millions of dollars. Recently, creation of lipolytic fusion biocatalysts for industrial applications gained more attention since it is not only a way of achievement of enzymes with improved properties but also a way to reduce industrial energy costs and ensure other economic benefits. This paper provides a comprehensive review on current state of microbial lipolytic fusion enzymes and their future potential.

Construction of a novel lipolytic fusion biocatalyst GDEst-lip for industrial application.

The gene encoding esterase (GDEst-95) from Geobacillus sp. 95 was cloned and sequenced. The resulting open reading frame of 1497 nucleotides encoded a protein with calculated molecular weight of 54.7 kDa, which was classified as a carboxylesterase with an identity of 93-97% to carboxylesterases from Geobacillus bacteria. This esterase can be grouped into family VII of bacterial lipolytic enzymes, was active at broad pH (7-12) and temperature (5-85 °C) range and displayed maximum activity toward short acyl chain p-nitrophenyl (p-NP) esters. Together with GD-95 lipase from Geobacillus sp. strain 95, GDEst-95 esterase was used for construction of fused chimeric biocatalyst GDEst-lip. GDEst-lip esterase/lipase possessed high lipolytic activity (600 U/mg), a broad pH range of 6-12, thermoactivity (5-85 °C), thermostability and resistance to various organic solvents or detergents. For these features GDEst-lip biocatalyst has high potential for applications in various industrial areas. In this work the effect of additional homodomains on monomeric GDEst-95 esterase and GD-95 lipase activity, thermostability, substrate specificity and catalytic properties was also investigated. Altogether, this article shows that domain fusing strategies can modulate the activity and physicochemical characteristics of target enzymes for industrial applications.

Detection of Asp371, Phe375, and Tyr376 Influence on GD-95-10 Lipase Using Alanine Scanning Mutagenesis.

GD-95-10 and GD-95-20 lipases are modified GD-95 lipase variants, which lack 10 and 20 C-terminal amino acids, respectively. Previous analysis showed that GD-95-10 lipase has higher activity than GD-95 lipase, while GD-95-20 lipase almost completely loses its activity. Analysis in silico suggested three conservative amino acids at region between 369 and 378 amino acids which can be relevant to the activity of GD-95-10 lipase. These amino acids have direct contacts with residues involved in substrate binding, stabilization of the serine loop or form oxyanion hole. In this work, the role of Asp371, Phe375, and Tyr376 on activity, functionality, and structure of GD-95-10 lipase was analyzed by Ala scanning mutagenesis. We showed that even a single mutation can impact the main structure and activity of Geobacillus lipases. Our experiments provide new knowledge about lipases from thermophilic Geobacillus bacteria and are important for protein engineering and synthetic biology. These enzymes and their engineering can be basis for future biocatalysts applied in production of biofuel or other industrial esters.

Keratinous waste decomposition and peptide production by keratinase from Geobacillus stearothermophilus AD-11.

A keratinolytic proteinase was cloned from thermophilic bacterium Geobacillus stearothermophilus AD-11 and was expressed in Escherichia coli BL21(DE3). Recombinant keratinolytic proteinase (RecGEOker) with an estimated molecular weight of 57 kDa was purified and keratinase activity was measured. RecGEOker showed optimal activity at pH 9 and 60 °C. Recombinant keratinolytic proteinase showed the highest substrate specificity toward keratin from wool > collagen > sodium caseinate > gelatin > and BSA in descending order. RecGEOker is applicable for efficient keratin waste biodegradation and can replace conventional non-biological hydrolysis processes. High-value small peptides obtained from enzymatic biodegradation by RecGEOker are suitable for industrial application in white and/or green biotechnology for use as major additives in various products.

Keratinolytic proteinase from Bacillus thuringiensis AD-12.

A new isolated strain noted to produce a novel detergent-stable serine keratinolytic proteinase and identified as Bacillus thuringiensis AD-12. Native keratinolytic proteinase from B. thuringiensis (BtKER) was purified and characterized. The purified BtKER enzyme is a monomer with a molecular mass of 39kDa. Biochemical characterization assays revealed that the BtKER attained optimal activity at pH 7 and 30°C. Residual activity after 1h incubation at 50°C was higher than 80%. The enzyme was activated and stabilized by Mn(2+) and Li(+) metal ions but inactivated by organic solvents. Purified BtKER showed the highest substrate specificity toward keratin from wool>sodium caseinate>collagen>BSA>gelatin in descending order. BtKER is the first reported keratinolytic proteinase from B. thuringiensis and obtained results suggested that new characterized enzyme can be a powerful biocatalyst in peptide production associated to hydrolysis of keratinous and/or keratin-like waste.

Influence of N- and/or C-terminal regions on activity, expression, characteristics and structure of lipase from Geobacillus sp. 95.

GD-95 lipase from Geobacillus sp. strain 95 and its modified variants lacking N-terminal signal peptide and/or 10 or 20 C-terminal amino acids were successfully cloned, expressed and purified. To our knowledge, GD-95 lipase precursor (Pre-GD-95) is the first Geobacillus lipase possessing more than 80% lipolytic activity at 5 °C. It has maximum activity at 55 °C and displays a broad pH activity range. GD-95 lipase was shown to hydrolyze p-NP dodecanoate, tricaprylin and canola oil better than other analyzed substrates. Structural and sequence alignments of bacterial lipases and GD-95 lipase revealed that the C-terminus forms an α helix, which is a conserved structure in lipases from Pseudomonas, Clostridium or Staphylococcus bacteria. This work demonstrates that 10 and 20 C-terminal amino acids of GD-95 lipase significantly affect stability and other physicochemical properties of this enzyme, which has never been reported before and can help create lipases with more specific properties for industrial application. GD-95 lipase and its modified variants GD-95-10 can be successfully applied to biofuel production, in leather and pulp industries, for the production of cosmetics or perfumes. These lipases are potential biocatalysts in processes, which require extreme conditions: low or high temperature, strongly acidic or alkaline environment and various organic solvents.