Thermodynamics of a hyperthermostable carboxylesterase from Anoxybacillus geothermalis D9.

Hyperthermostable enzymes are highly desirable biocatalysts due to their exceptional stability at extreme temperatures. Recently, a hyperthermostable carboxylesterase EstD9 from Anoxybacillus geothermalis D9 was biochemically characterized. The enzyme exhibited remarkable stability at high temperature. In this study, we attempted to probe the conformational adaptability of EstD9 under extreme conditions via in silico approaches. Circular dichroism revealed that EstD9 generated new ß-sheets at 80 °C, making the core of the hydrolase fold more stable. Interestingly, the profiles of molecular dynamics simulation showed the lowest scores of radius of gyration and solvent accessible surface area (SASA) at 80 °C. Three loops were responsible for protecting the catalytic site, which resided at the interface between the large and cap domains. To further investigate the structural adaptation in extreme conditions, the intramolecular interactions of the native structure were investigated. EstD9 revealed 18 hydrogen bond networks, 7 salt bridges, and 9 hydrophobic clusters, which is higher than the previously reported thermostable Est30. Collectively, the analysis indicates that intramolecular interactions and structural dynamics play distinct roles in preserving the overall EstD9 structure at elevated temperatures. This work is relevant to both fundamental and applied research involving protein engineering of industrial thermostable enzymes.

Characterization of a highly thermostable recombinant xylanase from Anoxybacillus ayderensis.

Xylanases are the main enzymes to hydrolyze xylan, the major hemicellulose found in lignocellulose. Xylanases also have a wide range of industrial applications. Therefore, the discovery of new xylanases has the potential to enhance efficiency and sustainability in many industries. Here, we report a xylanase with thermophilic character and superior biochemical properties for industrial use. The new xylanase is discovered in Anoxybacillus ayderensis as an intracellular xylanase (AAyXYN329) and recombinantly produced. While AAyXYN329 shows significant activity over a wide pH and temperature range, optimum activity conditions were determined as pH 6.5 and 65 °C. The half-life of the enzyme was calculated as 72 h at 65 °C. The enzyme did not lose activity between pH 6.0-9.0 at +4 °C for 75 days. Km, kcat and kcat/Km values of AAyXYN329 were calculated as 4.09824 ± 0.2245 µg/µL, 96.75 1/sec, and 23.61/L/g.s -1, respectively. In conclusion, the xylanase of A. ayderensis has an excellent potential to be utilized in many industrial processes.

Untangling the adaptive strategies of thermophilic bacterium Anoxybacillus rupiensis TPH1 under low temperature.

The present study investigates the low temperature tolerance strategies of thermophilic bacterium Anoxybacillus rupiensis TPH1, which grows optimally at 55 °C , by subjecting it to a temperature down-shift of 10 °C (45 °C) for 4 and 6 h followed by studying its growth, morphophysiological, molecular and proteomic responses. Results suggested that although TPH1 experienced increased growth inhibition, ROS production, protein oxidation and membrane disruption after 4 h of incubation at 45 °C yet maintained its DNA integrity and cellular structure through the increased expression of DNA damage repair and cell envelop synthesizing proteins and also progressively alleviated growth inhibition by 20% within two hours i.e., 6 h, by inducing the expression of antioxidative enzymes, production of unsaturated fatty acids, capsular and released exopolysaccharides and forming biofilm along with chemotaxis proteins. Conclusively, the adaptation of Anoxybacillus rupiensis TPH1 to lower temperature is mainly mediated by the synthesis of large numbers of defense proteins and exopolysaccharide rich biofilm formation.

Genome-Based Reclassification of Anoxybacillus geothermalis Filippidou et al. 2016 as a Later Heterotypic Synonym of Anoxybacillus rupiensis Derekova et al. 2007.

In this study, our aim was to elucidate the relationship between Anoxybacillus rupiensis DSM 17127T and Anoxybacillus geothermalis GSsed3T through whole-genome phylogenetic analysis. The obtained 16S rRNA gene sequence from the genome of A. rupiensis DSM 17127T exhibited a 99.8% similarity with A. geothermalis GSsed3T. In the phylogenetic trees constructed using whole-genome sequences and 16S rRNA gene sequences, A. rupiensis DSM 17127T and A. geothermalis GSsed3T were observed to form a clade, indicating a close relationship between them. Moreover, the average amino acid identity, average nucleotide identity, and digital DNA-DNA hybridization values calculated between A. rupiensis DSM 17127T and A. geothermalis GSsed3T exceeded the threshold values typically used for species demarcation. Furthermore, the phylogenomic analysis based on the core genome of the strains in question provided additional support for the formation of a monophyletic clade by A. rupiensis DSM 17127T and A. geothermalis GSsed3T. Most phenotypic and chemotaxonomic features between both strains were almost identical except for a few exceptions. These findings suggest that both strains should be classified as belonging to the same species, and we propose that A. geothermalis GSsed3T is a later heterotypic synonym of A. rupiensis DSM 17127T.

Novel extracellular synthesized silver nanoparticles using thermophilic Anoxybacillus flavithermus and Geobacillus stearothermophilus and their evaluation as nanodrugs.

In this investigation, two new thermophilic bacteria were isolated. The new isolates were characterized by 16S rRNA, biochemical, morphological, and physiological analyzes and the isolates were identified as Geobacillus stearothermophilus strain Gecek20 and thermophilic Anoxybacillus flavithermus strain Gecek19. Various biological activities of extracellular Ag-NPs synthesized from thermophilic G. stearothermophilus strain Gecek20 and thermophilic A. flavithermus strain Gecek19 were evaluated. The produced NPs were analyzed by SEM, SEM-EDX, and XRD analyses. The antioxidant abilities of new synthesized Ag-NPs from thermophilic G. stearothermophilus strain Gecek20 (T1-Ag-NPs) and new synthesized Ag-NPs from thermophilic A. flavithermus strain Gecek19 (T2-Ag-NPs) were studied by DPPH inhibition and metal chelating ability. The highest DPPH and metal chelating abilities of T1-Ag-NPs and T2-Ag-NPs at 200 mg/L concentration were 93.17 and 90.85%, and 75.80 and 83.64%, respectively. The extracellular green synthesized T1-Ag-NPs and T2-AgN-Ps showed DNA nuclease activity at all tested concentrations. Moreover, both new synthesized Ag-NPs had antimicrobial activity against the strains studied, especially on Gram positive bacteria. T1-Ag-NPs and T2-AgNPs also showed powerful Escherichia coli growth inhibition. The highest biofilm inhibition percentages of T1-Ag-NPs and T2-Ag-NPs against Pseudomonas aeruginosa and Staphylococcus aureus were 100.0%, respectively, at 500 mg/L.

The genus Anoxybacillus: an emerging and versatile source of valuable biotechnological products.

Thermophilic and alkaliphilic microorganisms are unique organisms that possess remarkable survival strategies, enabling them to thrive on a diverse range of substrates. Anoxybacillus, a genus of thermophilic and alkaliphilic bacteria, encompasses 24 species and 2 subspecies. In recent years, extensive research has unveiled the diverse array of thermostable enzymes within this relatively new genus, holding significant potential for industrial and environmental applications. The biomass of Anoxybacillus has demonstrated promising results in bioremediation techniques, while the recently discovered metabolites have exhibited potential in medicinal experiments. This review aims to provide an overview of the key experimental findings related to the biotechnological applications utilizing bacteria from the Anoxybacillus genus.

Cardinal models to describe the effect of temperature and pH on the growth of Anoxybacillus flavithermus & Bacillus licheniformis.

Anoxybacillus flavithermus and Bacillus licheniformis are among the predominant spore-formers of heat-processed foods. To our knowledge, no systematic analysis of growth kinetic data of A. flavithermus or B. licheniformis is currently available. In the present study, the growth kinetics of A. flavithermus and B. licheniformis in broth at various temperature and pH conditions were studied. Cardinal models were used to model the effect of the above-mentioned factors on the growth rates. The estimated values for the cardinal parameters Tmin,Topt,Tmax,pHmin and pH1/2 for A. flavithermus were 28.70 ± 0.26, 61.23 ± 0.16 and 71.52 ± 0.32 °C, 5.52 ± 0.01 and 5.73 ± 0.01, respectively, while for B. licheniformis they were 11.68 ± 0.03, 48.05 ± 0.15, 57.14 ± 0.01 °C, 4.71 ± 0.01 and 5.670 ± 0.08, respectively. The growth behaviour of these spoilers was also investigated in a pea beverage at 62 and 49 °C, respectively, to adjust the models to this product. The adjusted models were further validated at static and dynamic conditions and demonstrated good performance with 85.7 and 97.4% of predicted populations for A. flavithermus and B. licheniformis, respectively, being within the -10%-10% relative error (RE) zone. The developed models can be useful tools in assessing the potential of spoilage of heat-processed foods including plant-based milk alternatives.

Anoxybacillus: an overview of a versatile genus with recent biotechnological applications.

The Bacillaceae family members are considered to be a good source of microbial factories for biotechnological processes. In contrast to Bacillus and Geobacillus, Anoxybacillus, which would be thermophilic and spore-forming group of bacteria, is a relatively new genus firstly proposed in the year of 2000. The development of thermostable microbial enzymes, waste management and bioremediation processes would be a crucial parameter in the industrial sectors. There has been increasing interest in Anoxybacillus strains for biotechnological applications. Therefore, various Anoxybacillus strains isolated from different habitats have been explored and identified for biotechnological and industrial purposes such as enzyme production, bioremediation and biodegradation of toxic compounds. Certain strains have ability to produce exopolysaccharides possessing biological activities including antimicrobial, antioxidant and anticancer. This current review provides past and recent discoveries regarding Anoxybacillus strains and their potential biotechnological applications in enzyme industry, environmental processes and medicine.

Genome-based reclassification of Anoxybacillus karvacharensis Panosyan et al. as a later heterotypic synonym of Anoxybacillus kestanbolensis Dulger et al. 2004; A. flavithermus subsp. yunnanensis CCTCC AB2010187T Dai et al. 2011 as a later heterotypic synonym of A. tengchongensis DSM 23211T Zhang et al. 2011.

In the present study, we attempted to clarify the taxonomic positions of Anoxybacillus karvacharensis K1T, Anoxybacillus kestanbolensis NCIMB 13971T, Anoxybacillus flavithermus subsp. yunnanensis CCTCC AB2010187T, and Anoxybacillus tengchongensis DSM 23211T using whole-genome phylogenetic analysis. The genome sequence of A. kestanbolensis NCIMB13971T was not available in any database, so it was sequenced in this study. The 16S rRNA gene sequence obtained from the genome of A. kestanbolensis NCIMB13971T had 99.93% similarity with A. karvacharensis K1T. The average nucleotide identity (ANI), average amino acid identity (AAI), and digital DNA-DNA hybridization (DDH) values between A. karvacharensis K1T and A. kestanbolensis NCIMB13971T and between A. flavithermus subsp. yunnanensis CCTCCAB 2010187T and A. tengchongensis DSM 23211T were greater than the threshold values for species demarcation. The present results indicate that A. karvacharensis K1T is a later heterotypic synonym of A. kestanbolensis NCIMB13971T; A. flavithermus subsp. yunnanensis CCTCCAB 2010187T is a later heterotypic synonym of A. tengchongensis DSM 23211T.

Genome-based reclassification of Anoxybacillus kamchatkensis Kevbrin et al. 2005 as a later heterotypic synonym of Anoxybacillus ayderensis Dulger et al. 2004.

In this study, we aimed to clarify the taxonomic positions of Anoxybacillus kamchatkensis DSM 14988T and Anoxybacillus ayderensis AB04T using whole-genome phylogenetic analysis, biochemical and chemotaxonomic characteristics. In phylogenetic trees drawn using whole-genome sequences and 16S rRNA gene sequences, A. kamchatkensis DSM 14988T and A. ayderensis AB04T clade together and showed high sequence similarity (99.6%) based on 16S rRNA gene. The average amino acid identity, average nucleotide identity and digital DNA-DNA hybridization values between A. kamchatkensis DSM 14988T and A. ayderensis AB04T were found to be greater than the threshold values for species demarcation. Most phenotypic and chemotaxonomic features between both species were almost identical except for a few exceptions. The present results show that A. kamchatkensis DSM 14988T is a later heterotypic synonym of A. ayderensis AB04T.

tRNA modification profiles in obligate and moderate thermophilic bacilli.

Transfer RNAs (tRNAs) are the most ancient RNA molecules in the cell, modification pattern of which is linked to phylogeny. The aim of this study was to determine the tRNA modification profiles of obligate (Anoxybacillus, Geobacillus, Paragebacillus) and moderate (Bacillus, Brevibacillus, Ureibacillus, Paenibacillus) thermophilic aerobic bacilli strains to find out its linkage to phylogenetic variations between species. LC-MS was applied for the quantification of modified nucleosides using both natural and isotopically labeled standards. The presence of m2A and m7G modifications at high levels was determined in all species. Relatively high level of i6A and m5C modification was observed for Paenibacillus and Ureibacillus, respectively. The lowest level of Cm modification was found in Bacillus. The modification ms2i6A and m1G were absent in Brevibacillus and Ureibacillus, respectively, while modifications Am and m22G were observed only for Ureibacillus. While both obligate and moderate thermophilic species contain Gm, m1G and ms2i6A modifications, large quantities of them (especially Gm and ms2i6A modification) were detected in obligate thermophilic ones (Geobacillus, Paragebacillus and Anoxybacillus). The collective set of modified tRNA bases is genus-specific and linked to the phylogeny of bacilli. In addition, the dataset could be applied to distinguish obligate thermophilic bacilli from moderate ones.

Exopolysaccharide of Anoxybacillus pushchinoensis G11 has antitumor and antibiofilm activities.

Exopolysaccharides (EPS/EPSs) possess several various applications in the food and pharmaceutical industries. This study was performed to investigate the biological (antibiofilm and antitumor), rheological (temperature, shear rate, and density) and chemical (solubility, carbohydrate and protein content, composition, molecular weight, functional group analysis, thermal analysis, X-ray diffraction pattern and scanning electron microscopy) properties of the EPS, which was purified from the locally isolated thermophilic bacterium Anoxybacillus pushchinoensis G11 (MN720646). EPS was found to have antibiofilm and antitumor [lung (A-549) and colon (Caco-2 and HT-29) cancer] activities. The viscosity of EPS showing Newtonian flow was temperature dependent. As chemical properties, the EPS was found to be a heteropolysaccharide containing arabinose (57%), fructose (26%), glucose (12%), and galactose (5%). EPS contained 93% carbohydrates and 1.08% protein. The molecular weight of EPS was determined as 75.5 kDa. The FTIR analysis confirmed the presence of sulfate ester (band at 1217 cm-1), an indication of the antitumor effect. The EPS was semi-crystalline. It could maintain 36% of its weight at 800 °C and crystallization and melting temperatures were 221 and 255.6 °C. This is the first report on the EPS production potential and the biological activity of A. pushchinoensis.

Anoxybacillus karvacharensis sp. nov., a novel thermophilic bacterium isolated from the Karvachar geothermal spring in Nagorno-Karabakh.

Twelve thermophilic Anoxybacillus strains were isolated from sediment and water samples from a Karvachar hot spring located in the northern part of Nagorno-Karabakh. Based on phenotypic, chemotaxonomic and phylogenetic characteristics, one of the isolates, designated strain K1T, was studied in detail. The cells are straight, motile rods that are 0.2-0.4×2.3-7.2 µm in size. The strain is a Gram-stain-positive, moderately thermophilic facultative anaerobe with an optimum growth temperature of 60-65 °C and a growth temperature range of 45-70 °C. Growth of strain K1T was observed at pH 6-11 (optimum, pH 8-9) and was inhibited in the presence of NaCl concentrations above 2.5 % (optimum, 1-1.5 %). The isolate could utilize a wide variety of carbon sources, including d-arabinose, d-ribose, d-galactose, d-fructose, d-mannitol, maltose, aesculin, melibiose, sucrose, trehalose, raffinose, amidone, glycogen, turanose, d-lyxose, d-tagatose, potassium gluconate and 2-keto-gluconate. The strain was able to hydrolyse starch, casein and gelatin, was positive for oxidase and catalase, and reduced nitrate to nitrite, but was negative for H2S production. Production of urease and indole was not observed. The major cellular fatty acids were C15 : 0 iso, C16 : 0 and C17 : 0 iso (52.5, 13.6 and 19.6 % of total fatty acids, respectively). Strain K1T shares >99 % 16S rRNA sequence similarity and a genomic average nucleotide identity value of 94.5 % with its closest relative, Anoxybacillus flavithermus DSM 2641T, suggesting that it represents a separate and novel species, for which the name Anoxybacillus karvacharensis sp. nov. is proposed. The type strain of Anoxybacillus karvacharensis is K1T (=DSM 106524T=KCTC 15807T).

A new method for the preconcentrations of U(VI) and Th(IV) by magnetized thermophilic bacteria as a novel biosorbent.

This paper proposes the use of Anoxybacillus flavithermus SO-15 immobilized on iron oxide nanoparticles (NPs) as a novel magnetized biosorbent for the preconcentrations of uranium (U) and thorium (Th). The SPE procedure was based on biosorption of U(VI) and Th(IV) on a column of iron oxide NPs loaded with dead and dried thermophilic bacterial biomass prior to U(VI) and Th(IV) measurements by ICP-OES. The biosorbent characteristicswere explored using FT-IR, SEM, and EDX. Significant operational factors such as solution pH, volume and flow rate of the sample solution, amounts of dead bacteria and iron oxide nanoparticles, matrix interference effect, eluent type, and repeating use of the biosorbent on process yield were studied. The biosorption capacities were found as 62.7 and 56.4 mg g-1 for U(VI) and Th(IV), respectively. The novel extraction process has been successfullyapplied to the tap, river, and lake water samples for preconcentrations of U(VI) and Th(IV).

Potential of Thermolysin-like Protease A69 in Preparation of Bovine Collagen Peptides with Moisture-Retention Ability and Antioxidative Activity.

Bovine bone is rich in collagen and is a good material for collagen peptide preparation. Although thermolysin-like proteases (TLPs) have been applied in different fields, the potential of TLPs in preparing bioactive collagen peptides has rarely been evaluated. Here, we characterized a thermophilic TLP, A69, from a hydrothermal bacterium Anoxybacillus caldiproteolyticus 1A02591, and evaluated its potential in preparing bioactive collagen peptides. A69 showed the highest activity at 60 °C and pH 7.0. We optimized the conditions for bovine bone collagen hydrolysis and set up a process with high hydrolysis efficiency (99.4%) to prepare bovine bone collagen peptides, in which bovine bone collagen was hydrolyzed at 60 °C for 2 h with an enzyme-substrate ratio of 25 U/g. The hydrolysate contained 96.5% peptides that have a broad molecular weight distribution below 10000 Da. The hydrolysate showed good moisture-retention ability and a high hydroxyl radical (•OH) scavenging ratio of 73.2%, suggesting that the prepared collagen peptides have good antioxidative activity. Altogether, these results indicate that the thermophilic TLP A69 has promising potential in the preparation of bioactive collagen peptides, which may have potentials in cosmetics, food and pharmaceutical industries. This study lays a foundation for the high-valued utilization of bovine bone collagen.

Detoxification of azo dye Direct Black G by thermophilic Anoxybacillus sp. PDR2 and its application potential in bioremediation.

Direct Black G (DBG) is a highly toxic synthetic azo dye which is difficult to degrade. Biological treatment seems to be a promising option for the treatment of azo dye containing effluent. A thermophilic bacterial strain (Anoxybacillus sp. PDR2) previously isolated from the soil can effectively remove DBG. However, the molecular underpinnings of DBG degradation and the microbial detoxification ability remains unknown. In the present study, the genetic background of PDR2 for the efficient degradation of DBG and its adaptation to azo dye-contaminated environments was revealed by bioinformatics. Moreover, the possible biodegradation pathways were speculated based on the UV-vis spectral analysis, FTIR, and intermediates identified by LC-MS. Additionally, phytotoxicity and the comet experiment studies clearly indicated that PDR2 converts toxic azo dye (DBG) into low toxicity metabolites. The combination of biodegradation pathways and detoxification analysis were utilized to explore the molecular degradation mechanism and bioremediation of azo dye for future applications. These findings will provide a valuable theoretical basis for the practical treatment of azo dye wastewater.

Identification and Characterization of a Thermostable GH36 α-Galactosidase from Anoxybacillusvitaminiphilus WMF1 and Its Application in Synthesizing Isofloridoside by Reverse Hydrolysis.

An α-galactosidase-producing strain named Anoxybacillus vitaminiphilus WMF1, which catalyzed the reverse hydrolysis of d-galactose and glycerol to produce isofloridoside, was isolated from soil. The α-galactosidase (galV) gene was cloned and expressed in Escherichia coli. The galV was classified into the GH36 family with a molecular mass of 80 kDa. The optimum pH and temperature of galV was pH 7.5 and 60 °C, respectively, and it was highly stable at alkaline pH (6.0-9.0) and temperature below 65 °C. The specificity for p-nitrophenyl α-d-galactopyranoside was 70 U/mg, much higher than that for raffinose and stachyose. Among the metals and reagents tested, galV showed tolerance in the presence of various organic solvents. The kinetic parameters of the enzyme towards p-nitrophenyl α-d-galactopyranoside were obtained as Km (0.12 mM), Vmax (1.10 × 10-3 mM s-1), and Kcat/Km (763.92 mM-1 s-1). During the reaction of reverse hydrolysis, the enzyme exhibited high specificity towards the glycosyl donor galactose and acceptors glycerol, ethanol and ethylene glycol. Finally, the isofloridoside was synthesized using galactose as the donor and glycerol as the acceptor with a 26.6% conversion rate of galactose. This study indicated that galV might provide a potential enzyme source in producing isofloridoside because of its high thermal stability and activity.

Genome and transcriptome analysis of a newly isolated azo dye degrading thermophilic strain Anoxybacillus sp.

Understanding azo dye degrading enzymes and the encoding of their functional genes is crucial for the elucidation of their molecular mechanisms. In this study, a thermophilic strain capable of degrading azo dye was isolated from the soil near a textile dye manufacturing factory. Based on its morphological, physiological and biochemical properties, as well as 16S rRNA gene sequence analysis, the strain was identified as Anoxybacillus sp. PDR2. The decolorization ratios of 100-600 mg/L Direct Black G (DBG) by strain PDR2 reached 82.12-98.39% within 48 h of dyes. Genome analysis revealed that strain PDR2 contains a circular chromosome of 3791144 bp with a G + C content of 42.48%. The genetic basis of azo dye degradation by strain PDR2 and its capacity to adapt to harsh environments, were further elucidated through bioinformatics analysis. RNA-Seq and qRT-PCR technology confirmed that NAD(P)H-flavin reductase, 2Fe-2S ferredoxin and NAD(P)-dependent ethanol dehydrogenase genes expressed by strain PDR2, were the key genes involved in DBG degradation. The combination of genome and transcriptome analysis was utilized to explore the key genes of strain PDR2 involved in azo dye biodegradation, with these findings providing a valuable theoretical basis for the practical treatment of azo dye wastewater.

The extremophile Anoxybacillus sp. PC2 isolated from Brazilian semiarid region (Caatinga) produces a thermostable keratinase.

The aim of this study was to select and identify thermophilic bacteria from Caatinga biome (Brazil) able to produce thermoactive keratinases and characterize the keratinase produced by the selected isolate. After enrichment in keratin culture media, an Anoxybacillus caldiproteolyticus PC2 was isolated. This thermotolerant isolate presents a remarkable feature producing a thermostable keratinase at 60°C. The partially purified keratinase, identified as a thermolysin-like peptidase, was active at a pH range of 5.0-10.0 with maximal activity at a temperature range of 50-80°C. The optimal activity was observed at pH 7.0 and 50-60°C. These characteristics are potentially useful for biotechnological purposes such as processing and bioconversion of keratin.

Parameter optimization for thermostable lipase production and performance evaluation as prospective detergent additive.

Lipase based formulations has been a rising interest to laundry detergent industry for their eco-friendly property over phosphate-based counterparts and compatibility with chemical detergents ingredients. A thermo-stable Anoxybacillus sp. ARS-1 isolated from Taptapani Hotspring, India was characterized for optimum lipase production employing statistical model central composite design (CCD) under four independent variables (temperature, pH, % moisture and bio-surfactant) by solid substrate fermentation (SSF) using mustard cake. The output was utilized to find the effect of parameters and their interaction employing response surface methodology (RSM). A quadratic regression with R2 = 0.955 established the model to be statically best fitting and a predicted highest lipase production of 29.4 IU/g at an optimum temperature of 57.5 °C, pH 8.31, moisture 50% and 1.2 mg of bio-surfactant. Experimental production of 30.3 IU/g lipase at above conditions validated the fitness of model. Anoxybacillus sp. ARS-1 produced lipase was found to resist almost all chemical detergents as well as common laundry detergent, proving it to be a prospective additive for incorporation.