Flame resistant cotton lines generated by synergistic epistasis in a MAGIC population.

Textiles made from cotton fibers are flammable and thus often include flame retardant additives for consumer safety. Transgressive segregation in multi-parent populations facilitates new combinations of alleles of genes and can result in traits that are superior to those of any of the parents. A screen of 257 recombinant inbred lines from a multi-parent advanced generation intercross (MAGIC) population for naturally enhance flame retardance (FR) was conducted. All eleven parents, like all conventional white fiber cotton cultivars produce flammable fabric. MAGIC recombinant inbred lines (RILs) that produced fibers with significantly lower heat release capacities (HRC) as measured by microscale combustion calorimetry (MCC) were identified and the stability of the phenotypes of the outliers were confirmed when the RILs were grown at an additional location. Of the textiles fabricated from the five superior RILs, four exhibited the novel characteristic of inherent flame resistance. When exposed to open flame by standard 45° incline flammability testing, these four fabrics self-extinguished. To determine the genetic architecture of this novel trait, linkage, epistatic and multi-locus genome wide association studies (GWAS) were conducted with 473k SNPs identified by whole genome sequencing (WGS). Transcriptomes of developing fiber cells from select RILs were sequenced (RNAseq). Together, these data provide insight into the genetic mechanism of the unexpected emergence of flame-resistant cotton by transgressive segregation in a breeding program. The incorporation of this trait into global cotton germplasm by breeding has the potential to greatly reduce the costs and impacts of flame-retardant chemicals.

Intrafibrillar Dispersion of Cuprous Oxide (Cu2O) Nanoflowers within Cotton Cellulose Fabrics for Permanent Antibacterial, Antifungal and Antiviral Activity.

With increasingly frequent highly infectious global pandemics, the textile industry has responded by developing commercial fabric products by incorporating antibacterial metal oxide nanoparticles, particularly copper oxide in cleaning products and personal care items including antimicrobial wipes, hospital gowns and masks. Current methods use a surface adsorption method to functionalize nanomaterials to fibers. However, this results in poor durability and decreased antimicrobial activity after consecutive launderings. In this study, cuprous oxide nanoparticles with nanoflower morphology (Cu2O nanoflowers) are synthesized in situ within the cotton fiber under mild conditions and without added chemical reducing agents from a copper (II) precursor with an average maximal Feret diameter of 72.0 ± 51.8 nm and concentration of 17,489 ± 15 mg/kg. Analysis of the Cu2O NF-infused cotton fiber cross-section by transmission electron microscopy (TEM) confirmed the internal formation, and X-ray photoelectron spectroscopy (XPS) confirmed the copper (I) reduced oxidation state. An exponential correlation (R2 = 0.9979) between the UV-vis surface plasmon resonance (SPR) intensity at 320 nm of the Cu2O NFs and the concentration of copper in cotton was determined. The laundering durability of the Cu2O NF-cotton fabric was investigated, and the superior nanoparticle-leach resistance was observed, with the fabrics releasing only 19% of copper after 50 home laundering cycles. The internally immobilized Cu2O NFs within the cotton fiber exhibited continuing antibacterial activity (≥99.995%) against K. pneumoniae, E. coli and S. aureus), complete antifungal activity (100%) against A. niger and antiviral activity (≥90%) against Human coronavirus, strain 229E, even after 50 laundering cycles.

Thermosensitive textiles made from silver nanoparticle-filled brown cotton fibers.

Filling fibers with nanomaterials can create new functions or modify the existing properties. However, as nanocomposite formation for natural cellulosic fibers has been challenging, little information is available on how the embedded nanomaterials alter the properties of cellulosic fibers. Here we filled brown cotton fibers with silver nanoparticles (Ag NPs) to examine their thermosensitive properties. Using naturally present tannins in brown cotton fibers as a reducing agent, Ag NP-filled brown cotton fibers (nanoparticle diameter of about 28 nm, weight fraction of 12 500 mg kg-1) were produced through a one-step process without using any external agents. The in situ formation of Ag NPs was uniform across the nonwoven cotton fabric and was concentrated in the lumen of the fibers. The insertion of Ag NPs into the fibers shifted the thermal decomposition of cellulose to lower temperatures with increased activation energy and promoted heat release during combustion. Ag NPs lowered the thermal effusivity of the fabric, causing the fabric to feel warmer than the control brown cotton. Ag NP-filled brown cotton was more effectively heated to higher temperatures than control brown cotton under the same heating treatments.

Application of Lignin-Containing Cellulose Nanofibers and Cottonseed Protein Isolate for Improved Performance of Paper.

There is current interest in replacing petroleum-based additives in consumer paper products with abundantly available, renewable and sustainable biopolymers such as lignin-containing cellulose nanofibers (LCNFs) and cottonseed protein. This research characterized the performance of cottonseed protein isolate with/without LCNFs to increase the dry strength of filter paper. The application of 10% protein solution with 2% LCNFs as an additive improved the elongation at break, tensile strength and modulus of treated paper products compared to the improved performance of cottonseed protein alone. Improvements in tensile modulus and tensile strength were greatest for samples containing larger amounts of lignin and a greater degree of polymerization than for those with less lignin from the same biomass sources.

Silver Nanoparticle-Intercalated Cotton Fiber for Catalytic Degradation of Aqueous Organic Dyes for Water Pollution Mitigation.

Azo dyes are commonly used in textile color processing for their wide array of vibrant colors. However, in recent years these dyes have become of concern in wastewater management given their toxicity to humans and the environment. In the present work, researchers remediated water contaminated with azo dyes using silver nanoparticles (Ag NPs) intercalated within cotton fabric as a catalyst, for their enhanced durability and reusability, in a reductive degradation method. Three azo dyes­methyl orange (MO), Congo red (CR), and Chicago Sky Blue 6B (CSBB)­were investigated. The azo degradation was monitored by UV/vis spectroscopy, degradation capacity, and turnover frequency (TOF). The Ag NP−cotton catalyst exhibited excellent degradation capacity for the dyes, i.e., MO (96.4% in 30 min), CR (96.5% in 18.5 min), and CSBB (99.8% in 21 min), with TOFs of 0.046 min−1, 0.082 min−1, and 0.056 min−1, respectively, using a 400 mg loading of catalyst for 100 mL of 25 mg L−1 dye. To keep their high reusability while maintaining high catalytic efficiency of >95% degradation after 10 cycles, Ag NPs immobilized within cotton fabric have promising potential as eco-friendly bio-embedded catalysts.

Structure/Function Analysis of Truncated Amino-Terminal ACE2 Peptide Analogs That Bind to SARS-CoV-2 Spike Glycoprotein.

The global burden of the SARS-CoV-2 pandemic is thought to result from a high viral transmission rate. Here, we consider mechanisms that influence host cell-virus binding between the SARS-CoV-2 spike glycoprotein (SPG) and the human angiotensin-converting enzyme 2 (ACE2) with a series of peptides designed to mimic key ACE2 hot spots through adopting a helical conformation analogous to the N-terminal α1 helix of ACE2, the region experimentally shown to bind to the SARS-CoV-2 receptor-binding domain (RBD). The approach examines putative structure/function relations by assessing SPG binding affinity with surface plasmon resonance (SPR). A cyclic peptide (c[KFNHEAEDLFEKLM]) was characterized in an α-helical conformation with micromolar affinity (KD = 500 µM) to the SPG. Thus, stabilizing the helical structure of the 14-mer through cyclization improves binding to SPG by an order of magnitude. In addition, end-group peptide analog modifications and residue substitutions mediate SPG binding, with net charge playing an apparent role. Therefore, we surveyed reported viral variants, and a correlation of increased positive charge with increased virulence lends support to the hypothesis that charge is relevant to enhanced viral fusion. Overall, the structure/function relationship informs the importance of conformation and charge for virus-binding analog design.

Detection of Human Neutrophil Elastase by Fluorescent Peptide Sensors Conjugated to TEMPO-Oxidized Nanofibrillated Cellulose.

Peptide-cellulose conjugates designed for use as optical protease sensors have gained interest for point-of-care (POC) detection. Elevated serine protease levels are often found in patients with chronic illnesses, necessitating optimal biosensor design for POC assessment. Nanocellulose provides a platform for protease sensors as a transducer surface, and the employment of nanocellulose in this capacity combines its biocompatibility and high specific surface area properties to confer sensitive detection of dilute biomarkers. However, a basic understanding of the spatiotemporal relationships of the transducer surface and sensor disposition is needed to improve protease sensor design and development. Here, we examine a tripeptide, fluorogenic elastase biosensor attached to TEMPO-oxidized nanofibrillated cellulose via a polyethylene glycol linker. The synthetic conjugate was found to be active in the presence of human neutrophil elastase at levels comparable to other cellulose-based biosensors. Computational models examined the relationship of the sensor molecule to the transducer surface. The results illustrate differences in two crystallite transducer surfaces ((110) vs. (1-10)) and reveal preferred orientations of the sensor. Finally, a determination of the relative (110) vs. (1-10) orientations of crystals extracted from cotton demonstrates a preference for the (1-10) conformer. This model study potentiates the HNE sensor results for enhanced sensor activity design.

Effect of Nanocellulose on the Properties of Cottonseed Protein Isolate as a Paper Strength Agent.

Currently, there is an increasing interest in the use of biopolymers in industrial applications to replace petroleum-based additives, since they are abundantly available, renewable and sustainable. Cottonseed protein is a biopolymer that, when used as a modifier, has shown improved performance for wood adhesives and paper products. Thus, it would be useful to explore the feasibility of using cellulose nanomaterials to further improve the performance of cottonseed protein as a paper strength agent. This research characterized the performance of cottonseed protein isolate with/without cellulose nanofibers (CNFs) and cellulose nanocrystals (CNCs) to increase the dry strength of filter paper. An application of 10% protein solution with CNCs (10:1) or CNFs (50:1) improved the elongation at break, tensile strength and modulus of treated paper products compared to the improved performance of cottonseed protein alone. Further analysis using scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) indicated that the cottonseed protein/nanocellulose composites interacted with the filter paper fibers, imparting an increased dry strength.

Silver Nanoparticle-Infused Cotton Fiber: Durability and Aqueous Release of Silver in Laundry Water.

Although the application of silver nanoparticles to commercial antibacterial items is well-established, there have been increasing concerns that such particles might leach out, particularly into laundry water from textile products. A recently developed process wherein silver nanoparticles are synthesized in situ within the cotton fiber itself promises, however, to achieve the desired washing durability. In this study, the silver release behavior of the silver nanoparticle-infused cotton fabric during consecutive launderings in water and a detergent solution was analyzed. Silver nanoparticles (12 ± 3 nm in diameter) were uniformly produced throughout the entire volume of cotton fiber with a concentration of 3017 ± 56 mg/kg. A combination of colorimetric, spectroscopic, and elemental analyses showed (1) nonlinear silver release behavior, with a rapid release from externally formed nanoparticles during the initial washing and a plateau-like release from internally formed nanoparticles during extended washing, and (2) superior nanoparticle-leach resistance compared to those in commercial and laboratory-prepared textiles analyzed in the literature. The internal nanoparticles immobilized within cotton fiber exhibited persistent antibacterial activity after 50 home laundering cycles.

Cellulose hydrolysis using ionic liquids and inorganic acids under dilute conditions: morphological comparison of nanocellulose.

The preparation of cellulose nanocrystals (CNCs) from cellulose extracted from cotton gin motes (CGM) using an ionic liquid (1-butyl-3-methylimidazolium chloride, [BMIm]Cl) under dilute conditions is reported. The concurrent process involves minimal swelling of cellulose with an ionic liquid and hydrolysis of the cellulose initiated by the addition of either phosphoric (H3PO4), hydrochloric (HCl), or sulfuric (H2SO4) acid. The obtained nanocrystals had similar physical properties (e.g. crystallinity) to the counterparts prepared under conventional conditions and exhibited superior thermal properties for sulfate CNCs. Additionally, the obtained CNCs had low surface functionalization, yet were colloidally stable for >90 days, which is a desirable trait for post-functionalization of CNCs. This process represents a general strategy utilizing dilute ionic liquids in the preparation of nanocellulose under mildly acidic conditions.

Thermal properties and surface chemistry of cotton varieties mineralized with calcium carbonate polymorphs by cyclic dipping.

In this study, hydroentangled cotton nonwovens were identified as effective hosts for mineralization of calcium carbonate (CaCO3) polymorphs to modify and improve their properties. All cotton varieties studied, including raw white cotton, scoured white cotton, and raw brown cotton, readily crystallized CaCO3 via a simple cyclic dipping process. A combination of analyses agreed that the surface chemistry of cotton fibers influenced the formation of different CaCO3 polymorphs. Scoured white cotton that consisted of almost pure cellulose predominantly produced the most stable calcite, whereas raw white and raw brown cottons that contain proteins facilitated the production of partial metastable vaterite. The morphology of calcite was better defined on the scoured cotton. The mineralization altered the hydrophobic surface of raw cottons to be hydrophilic, i.e., two-fold increase in moisture regain and decrease in water contact angle from 130 to 0 degrees. The mineralized cottons also exhibited improved thermal resistance, i.e., slower thermal decomposition with decreased activation energies and reduction in heat release capacity by up to 40%.

Method for identifying the triple transition (glass transition-dehydration-crystallization) of amorphous cellulose in cotton.

Although having a full picture of the heat-induced alterations in the fine structure of cellulosic materials is essential for designing their thermal processing, there have been no reliable methods to identify thermal transition temperatures. This study shows that colorimetric, thermogravimetric, and thermal kinetic parameters were sensitive to the thermochemical and thermo-structural changes in cotton fiber at low temperatures. Among these parameters, the activation energy for the thermal decomposition, evolving two local maxima against the preheating temperature, identified sequential thermal transformations in amorphous cellulose: 1) glass transition at 160-180 °C (Tg), 2) cellulose dehydration at 200-220 °C, and 3) crystallization at 240-260 °C. These results indicated that results of the mechanical tests or other methods discussed in the literature, which have produced inconsistent and higher Tg values (200-240 °C) for cellulosic materials, might have been misled by the dehydration of amorphous cellulose.

Alkali Hydrolysis of Sulfated Cellulose Nanocrystals: Optimization of Reaction Conditions and Tailored Surface Charge.

Cellulose nanocrystals (CNCs) are a biorenewable resource, which may be chemically modified to impart specific properties. Modified CNCs have found use in imaging applications, as rheology modifiers, polymer reinforcements, barrier and/or optical films, and nanocomposites. Nanoparticle dimensions of CNCs are typically 5-10 nm in width, with lengths of <100-300 nm. However, the physical properties are dependent upon the number and nature of the surface charge groups imparted during preparation. In the case of CNCs produced from sulfuric acid hydrolysis, the sulfated surface groups may be partially removed prior to further functionalization. This gives more available hydroxyls yet renders the CNCs less colloidally stable. Furthermore, conditions vary significantly and there is no consensus about the optimal conditions for partial removal of sulfate functionality or conditions developed to give specific surface charge. In the following, alkali hydrolysis of sulfate half-esters was quantified by conductometric titration of the strong acid groups, and using a design of experiments (DOE), optimal conditions were determined to produce CNCs with tailored surface charge.

Nanocellulose as a colorimetric biosensor for effective and facile detection of human neutrophil elastase.

Nanocellulose has functionalities suitable for efficient sensor transducer surface design including crystallinity, biocompatible and high specific surface area. Here we explore two forms of nanocellulose as transducer surfaces to enable colorimetric detection of human neutrophil elastase (HNE), and a wide range of inflammatory diseases. A deep eutectic solvent (DES) was utilized to mediate formation of cotton cellulose nanocrystals (DCNCs) employed to prepare a peptide-cellulose conjugate as a protease sensor of HNE. The tetrapeptide-cellulose analog on DCNC is contrasted with an analogous derivative of TEMPO-oxidized wood cellulose nanofibrils (WCNFs). DCNCs showed greater degree of substitution of HNE tetrapeptide and sensitivity to the elastase than WCNFs, despite the smaller surface area and pore sizes. XRD models revealed the higher crystallinity and larger crystallite sizes of DCNCs, indicating the well-arranged cellulose chains for immobilization of the tetrapeptide on (110) lattice reflections of cellulose crystals. The sensitivity of DCNCs-based colorimetric sensor was less than 0.005 U/mL, which would provide a convenient, sensitive sensor applicable for improved colorimetric point of care protease biomarker detection.

A reinforced thermal barrier coat of a Na-tannic acid complex from the view of thermal kinetics.

The poor burning resistance of cotton necessitates the control of its pyrolytic reactions, but many approaches have relied on the use of synthetically engineered chemicals. Herein, we show how a natural polyphenol from plants-tannic acid-acts with sodium ions to create a robust thermal barrier coat on cotton, with a focus on thermal kinetics. The kinetic information, combined with thermal and spectral analyses, revealed that the outer layer of galloyl units in tannic acid decomposes via a two-step reaction, producing a multicellular char of crosslinked aromatic rings, followed by the blowing of carbonaceous cells into a further expanded structure. This intumescent function of tannic acid was found to be enhanced upon its complexation with sodium ions, which greatly increased the activation energy for the first step of the reaction of tannic acid, to promote the formation of a stable char. The resulting blown char coated the cotton fiber below the thermal decomposition temperature of cellulose and was sustained throughout the decomposition. The enhanced thermal barrier performance of the Na-tannic acid complex was demonstrated by the reduced heat release capacity of cotton, the value of which was only about one-third that of tannic acid itself, and the inhibition of flame generation on cotton.

Whole genome sequencing of a MAGIC population identified genomic loci and candidate genes for major fiber quality traits in upland cotton (Gossypium hirsutum L.).

KEY MESSAGE: Significant associations between candidate genes and six major cotton fiber quality traits were identified in a MAGIC population using GWAS and whole genome sequencing. Upland cotton (Gossypium hirsutum L.) is the world's major renewable source of fibers for textiles. To identify causative genetic variants that influence the major agronomic measures of cotton fiber quality, which are used to set discount or premium prices on each bale of cotton in the USA, we measured six fiber phenotypes from twelve environments, across three locations and 7 years. Our 550 recombinant inbred lines were derived from a multi-parent advanced generation intercross population and were whole-genome-sequenced at 3× coverage, along with the eleven parental cultivars at 20× coverage. The segregation of 473,517 single nucleotide polymorphisms (SNPs) in this population, including 7506 non-synonymous mutations, was combined with phenotypic data to identify seven highly significant fiber quality loci. At these loci, we found fourteen genes with non-synonymous SNPs. Among these loci, some had simple additive effects, while others were only important in a subset of the population. We observed additive effects for elongation and micronaire, when the three most significant loci for each trait were examined. In an informative subset where the major multi-trait locus on chromosome A07:72-Mb was fixed, we unmasked the identity of another significant fiber strength locus in gene Gh_D13G1792 on chromosome D13. The micronaire phenotype only revealed one highly significant genetic locus at one environmental location, demonstrating a significant genetic by environment component. These loci and candidate causative variant alleles will be useful to cotton breeders for marker-assisted selection with minimal linkage drag and potential biotechnological applications.

Hydrogen Peroxide Generation of Copper/Ascorbate Formulations on Cotton: Effect on Antibacterial and Fibroblast Activity for Wound Healing Application.

Greige cotton (unbleached cotton) is an intact plant fiber that retains much of the outer cotton fiber layers. These layers contain pectin, peroxidases, and trace metals that are associated with hydrogen peroxide (H2O2) generation during cotton fiber development. When greige cotton is subjected to a nonwoven hydroentanglement process, components of the outer cotton fiber layers are retained. When hydrated, this fabric can generate H2O2 (5⁻50 micromolar). This range has been characterized as inducing accelerated wound healing associated with enhanced cell signaling and the proliferation of cells vital to wound restoration. On the other hand, H2O2 levels above 50 micromolar have been associated with bacteriostatic activity. Here, we report the preparation and hydrogen peroxide activity of copper/ascorbate formulations, both as adsorbed and in situ synthesized analogs on cotton. The cooper/ascorbate-cotton formulations were designed with the goal of modulating hydrogen peroxide levels within functional ranges beneficial to wound healing. The cotton/copper formulation analogs were prepared on nonwoven unbleached cotton and characterized with cotton impregnation titers of 3⁻14 mg copper per gram of cotton. The copper/ascorbate cotton analog formulations were characterized spectroscopically, and the copper titer was quantified with ICP analysis and probed for peroxide production through assessment with Amplex Red. All analogs demonstrated antibacterial activity. Notably, the treatment of unbleached cotton with low levels of ascorbate (~2 mg/g cotton) resulted in a 99 percent reduction in Klebsiella pneumoniae and Staphylococcus aureus. In situ synthesized copper/ascorbate nanoparticles retained activity and did not leach out upon prolonged suspension in an aqueous environment. An assessment of H2O2 effects on fibroblast proliferation are discussed in light of the copper/cotton analogs and wound healing.

Peptide-Cellulose Conjugates on Cotton-Based Materials Have Protease Sensor/Sequestrant Activity.

The growing incidence of chronic wounds in the world population has prompted increased interest in chronic wound dressings with protease-modulating activity and protease point of care sensors to treat and enable monitoring of elevated protease-based wound pathology. However, the overall design features needed for the combination of a chronic wound dressing that lowers protease activity along with protease detection capability as a single platform for semi-occlusive dressings has scarcely been addressed. The interface of dressing and sensor specific properties (porosity, permeability, moisture uptake properties, specific surface area, surface charge, and detection) relative to sensor bioactivity and protease sequestrant performance is explored here. Measurement of the material's zeta potential demonstrated a correlation between negative charge and the ability of materials to bind positively charged Human Neutrophil Elastase. Peptide-cellulose conjugates as protease substrates prepared on a nanocellulosic aerogel were assessed for their compatibility with chronic wound dressing design. The porosity, wettability and absorption capacity of the nanocellulosic aerogel were consistent with values observed for semi-occlusive chronic wound dressing designs. The relationship of properties that effect dressing functionality and performance as well as impact sensor sensitivity are discussed in the context of the enzyme kinetics. The sensor sensitivity of the aerogel-based sensor is contrasted with current clinical studies on elastase. Taken together, comparative analysis of the influence of molecular features on the physical properties of three forms of cellulosic transducer surfaces provides a meaningful assessment of the interface compatibility of cellulose-based sensors and corresponding protease sequestrant materials for potential use in chronic wound sensor/dressing design platforms.

Structure/Function Analysis of Cotton-Based Peptide-Cellulose Conjugates: Spatiotemporal/Kinetic Assessment of Protease Aerogels Compared to Nanocrystalline and Paper Cellulose.

Nanocellulose has high specific surface area, hydration properties, and ease of derivatization to prepare protease sensors. A Human Neutrophil Elastase sensor designed with a nanocellulose aerogel transducer surface derived from cotton is compared with cotton filter paper, and nanocrystalline cellulose versions of the sensor. X-ray crystallography was employed along with Michaelis-Menten enzyme kinetics, and circular dichroism to contrast the structure/function relations of the peptide-cellulose conjugate conformation to enzyme/substrate binding and turnover rates. The nanocellulosic aerogel was found to have a cellulose II structure. The spatiotemporal relation of crystallite surface to peptide-cellulose conformation is discussed in light of observed enzyme kinetics. A higher substrate binding affinity (Km) of elastase was observed with the nanocellulose aerogel and nanocrystalline peptide-cellulose conjugates than with the solution-based elastase substrate. An increased Km observed for the nanocellulosic aerogel sensor yields a higher enzyme efficiency (kcat/Km), attributable to binding of the serine protease to the negatively charged cellulose surface. The effect of crystallite size and ß-turn peptide conformation are related to the peptide-cellulose kinetics. Models demonstrating the orientation of cellulose to peptide O6-hydroxymethyl rotamers of the conjugates at the surface of the cellulose crystal suggest the relative accessibility of the peptide-cellulose conjugates for enzyme active site binding.

Water-based binary polyol process for the controllable synthesis of silver nanoparticles inhibiting human and foodborne pathogenic bacteria.

The polyol process is a widely used strategy for producing nanoparticles from various reducible metallic precursors; however, it requires a bulk polyol liquid reaction with additional protective agents at high temperatures. Here, we report a water-based binary polyol process using low concentrations of high-molecular-weight polyethylene glycol (100 000 g mol-1, 2 wt%) and ethylene glycol (5 wt%). The entangled conformation of the polyethylene glycol in water and the increased number of reducing sites by the ethylene glycol cooperatively contributed to the stability and effectiveness of reduction reaction and particle growth, producing uniformly sized silver nanoparticles (15.8 ± 2.2 nm) with no additional protective agents at a mild temperature of 80 °C. The measurement of particle size throughout the reaction and the dependence of the optical density of a silver colloidal solution on the concentration of ethylene glycol revealed three stages of particle growth. The minimum inhibitory concentrations of the purified silver nanoparticles against four representative human and foodborne pathogenic bacteria-S. aureus, P. aeruginosa, S. enterica, and E. coli-were 4.7, 2.3, 2.3, and 1.2 µg mL-1, respectively.