Corynespora cassiicola phaeohyphomycosis and found that he carried biallelic deleterious mutations in CLEC7A encoding the CARD9-coupled, β-glucan–binding receptor, Dectin-1. The patient’s PBMCs failed to produce TNF-α and IL-1β in response to β-glucan and/or C. cassiicola. To confirm the cellular and molecular requirements for immunity against C. cassiicola, we developed a mouse model of this infection. Mouse macrophages required Dectin-1 and CARD9 for IL-1β and TNF-α production, which enhanced fungal killing in an interdependent manner. Deficiency of either Dectin-1 or CARD9 was associated with more severe fungal disease, recapitulating the human observation. Because these data implicated impaired Dectin-1 responses in susceptibility to phaeohyphomycosis, we evaluated 17 additional unrelated patients with severe forms of the infection. We found that 12 out of 17 carried deleterious CLEC7A mutations associated with an altered Dectin-1 extracellular C-terminal domain and impaired Dectin-1–dependent cytokine production. Thus, we show that Dectin-1 and CARD9 promote protective TNF-α– and IL-1β–mediated macrophage defense against C. cassiicola. More broadly, we demonstrate that human Dectin-1 deficiency may contribute to susceptibility to severe phaeohyphomycosis by certain dematiaceous fungi.Subcutaneous phaeohyphomycosis typically affects immunocompetent individuals following traumatic inoculation. Severe or disseminated infection can occur in CARD9 deficiency or after transplantation, but the mechanisms protecting against phaeohyphomycosis remain unclear. We evaluated a patient with progressive, refractory   Phaeohyphomycosis is an invasive fungal infection caused by dematiaceous fungi, which are characterized by melanin production and filamentous growth. Phaeohyphomycosis typically affects the subcutaneous tissues following traumatic inoculation and is treatable with antifungal therapy and/or surgical resection. Phaeohyphomycosis may also manifest as cerebral or disseminated disease in immunosuppressed patients or, infrequently, in putatively immunocompetent individuals, and its prognosis is usually poor , 2. BettCARD9, which encodes an adaptor protein that relays fungus-sensing signals by multiple C-type lectin receptors (CLRs), such as Dectin-1 . Fungi grew as sterile mycelia and were identified molecularly as C. cassiicola  with a nonhealing growth on his nose following an injury he sustained while working in construction . Biopsy ssiicola . The minssiicola .CLEC7A  . The p.Y238* and p.I223S mutations have population allelic frequencies of 6.0% and 0.33%, respectively .A dihydrorhodamine test revealed normal phagocyte respiratory burst. We performed whole-exome sequencing to search for genes underlying human fungal susceptibility . We idenCLEC7A , which e, 2021.) . When in, 2021.) , 25. TheCLEC7A mutations for Dectin-1 expression. The patient’s monocytes had significantly reduced Dectin-1 surface expression by flow cytometry with an antibody that recognizes the extracellular, β-glucan–binding, C-terminal domain where the Dectin-1 mutations reside  mice exhibited swollen footpads during the first week, which resolved over the next 2–3 weeks  by myeloid cells in the footpads of –/–Clec7a and –/–Card9 mice relative to WT mice , 32C. ca WT mice . Althoug WT mice , A and Bfootpads , collectapalumab , 33, 34.–/–Clec7a and –/–Card9 mice compared with WT mice , IL-1β–, and TNF-α–deficient mice and found them to exhibit increased footpad swelling after C. cassiicola infection  in 12 of them (70.9%)  patients with severe phaeohyphomycosis carried deleterious CLEC7A mutations: 5 with biallelic and 8 with heterozygous CLEC7A mutations. This represents a significantly greater frequency than that seen at the population level, evidenced by comparing to healthy individuals recorded in 1000 Genomes    . Combinect test) . Moreovepatients . Impairepatients , similar scores > in 12 ofpatients –38, suggC. cassiicola phaeohyphomycosis, an infection previously reported in inherited CARD9 deficiency. Therefore, Dectin-1 deficiency may phenocopy CARD9 deficiency with respect to susceptibility to this dematiaceous fungus in the setting of traumatic fungal inoculation. We show that Dectin-1 binds to C. cassiicola and promotes proinflammatory cytokine production by human PBMCs. In C. cassiicola–infected mice, Dectin-1 and CARD9 deficiencies impaired fungal control and decreased production of TNF-α and IL-1β, which contribute to protective immunity via macrophage fungal killing. Moreover, in an additional 12 out of 17 unrelated patients with severe forms of phaeohyphomycosis, we identified heterozygous or biallelic deleterious CLEC7A mutations associated with an altered Dectin-1 extracellular β-glucan–binding domain, impairing TNF-α production in response to fungal stimulation. Collectively, these data underscore the importance of Dectin-1 in antifungal immunity to phaeohyphomycetes.We report a patient with Dectin-1 deficiency with severe C. cassiicola causes multiple diseases in plants  mutations should be sought in affected patients.n plants . It alson plants , CARD9-dn plants , 21, 41,n plants –44. Human plants . This sund TNF-α . The p.Ypatients , 38. We C. cassiicola phaeohyphomycosis is notable relative to the frequency of human Dectin-1 deficiency, as approximately 5 and approximately 0.1 out of 1,000 individuals are homozygous for p.Y238* and p.I223S, respectively. Therefore, these deleterious CLEC7A variants in themselves do not represent a primary immunodeficiency per se. By contrast, these population CLEC7A variants are relatively benign unless the carrier is traumatically exposed to C. cassiicola or certain other phaeohyphomycetes to instigate disease, after which impaired Dectin-1–dependent immune responses appear to contribute to suboptimal infection control. The contribution of common population variants to infection susceptibility has been previously shown. For example, common heterozygous CFTR mutations are associated with increased risk of respiratory infections , or inhalational exposure to highly virulent fungi in certain geographic areas (Coccidioides).The rarity of fections , 46. Furfections –49. Morerculosis . Indeed, patient . In addihan 30°C  and its han 30°C  further e common , 52. Whee common , imply tC. cassiicola phaeohyphomycosis, we modeled it in mice. We used footpad swelling as a surrogate measure of infection control, which we found correlated to tissue fungal burden, although differential swelling in different gene-deficient mice may also be due to inappropriate inflammation and inability to form or resolve granulomas. Using this model, we found that Dectin-1 and CARD9 were each required for anti–C. cassiicola defense, which depended on macrophages, and TNF-α and IL-1β were critical for protection and macrophage fungal killing. The mechanism by which macrophages mediate C. cassiicola killing remains to be determined, although our data indicated that ROS was either not involved or could be compensated by additional nonoxidative mechanisms. These data are consistent with the normal oxidative burst of our index patient and the lack of reported phaeohyphomycosis in patients with chronic granulomatous disease who lack phagocyte oxidative burst , following the NimbleGen SeqCap EZ Library SR User’s Guide, v4.0 . Briefly, 1 μg of each gDNA was sheared using the Covaris instrument and Covaris microTUBEs . The following shearing conditions were used: duty cycle, 10%; intensity, 5.0; bursts per second, 200; duration, 120 seconds; mode, frequency sweeping; temperature, 5.5°C to 6.0°C. Each sample was prepared with a specific indexing adapter to facilitate multiplex pooling in the exon enrichment procedure. Exome enrichment was performed using the SeqCap EZ Human v3.0 Exome Enrichment kit on 200 ng of each TruSeq library. Libraries were combined to create a 6-plex reaction for the enrichment, yielding a total DNA library mass of 1,200 ng. One microgram of the 6-plex pooled libraries was hybridized to capture target oligonucleotides for 48 hours, as specified in the manufacturer’s protocol.Exome-enriched libraries were quantified using the KAPA Library Quantification Kit (KAPA Biosystems), clustered on the cBot Cluster Station, and sequenced as 2 × 100 bp reads on the HiSeq 2500 instrument, according to the manufacturer’s protocol (Illumina).sequence alignment/map)  using Bowtie 2 and the default parameters with mixed mode disabled . Multiplent/map) . AdditioGenomic DNA was isolated from peripheral blood leukocytes using a Gentra Puregene DNA isolation kit (Qiagen) and amplified using Platinum Taq PCR SuperMix High Fidelity (Thermo Fisher Scientific) following the manufacturers’ protocols with primers and cycling conditions listed in C. cassiicola strain used in all experiments was isolated from our Dectin-1–deficient patient. C. cassiicola was stored in 50% glycerol at –80°C, and routinely grown on potato dextrose agar  plates at 20°C for 7 days, harvested, and replated onto fresh PDA plates and grown for an additional 7 days before use. To harvest C. cassiicola, 10 mL sterile tissue-culture grade water (Gibco) was added to each plate, and a soft paint brush (sterilized in 70% ethanol and rinsed in sterile water) used to brush the colonies and lift fungal cells. The fungi and water were pipetted off the plate, and a further wash/brush repeated with 5 mL of water. The resulting suspension was centrifuged  to pellet C. cassiicola, which was resuspended in sterile PBS. Each suspension was adjusted to an OD600 of 2.0 for use in experiments. Microscopy analysis and analysis of Dectin-1 ligand exposure was performed as outlined below.The C. cassiicola was prepared as above and 50 μL used for staining. Pelleted fungal cells were resuspended in 1 mL 2% paraformaldehyde and incubated at room temperature for 10 minutes. Fungi were centrifuged again and supernatants discarded, before washing in 1 mL PBS. Cell pellets were resuspended in 400 μL 10% mouse serum/PBS and incubated for 15 minutes at room temperature to block, and then washed in 1 mL PBS. Cells were resuspended in 100 μL PBS and 1 μL Fc-Dectin-1  were used as a positive control, and secondary-only–stained samples used as a negative control.Dectin-1  added toPBMCs from the Dectin-1–deficient patients, CARD9-deficient patients, or healthy donors, were harvested from whole blood by gradient centrifugation using Lymphocyte Separation Media (Lonza), according to the manufacturer’s instructions. PBMCs were washed in PBS and resuspended in 10% DMSO/FBS, and slowly frozen before storing in liquid nitrogen. Frozen PBMCs were defrosted in a 37°C water bath and washed in RPMI 1640 supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin (hereafter referred to as “complete RPMI”) prior to counting with trypan blue exclusion. Washed PBMCs were used for stimulation assays or flow cytometry.5) were incubated in a round-bottom 96-well plate (Corning) at 37°C in a 5% CO2 incubator in complete RPMI, supplemented with either 200 ng/mL LPS (Sigma-Aldrich), 1 μg/mL Pam3CSK4 (Sigma-Aldrich), α-mannan (see below), β-glucan (see below), or heat-killed (incubation at 70°C for 1 hour) C. cassiicola, which was added at a 1:12 dilution to the cells. After 48 hours, PBMCs were pelleted and the supernatant was collected and stored at –80°C until analysis. Cytokine levels were analyzed via conventional ELISA , and then prepared for PBMC stimulation. In brief, 5 mg of particulate β-glucan was added to 1 mL water, sonicated for 30 seconds, pelleted by centrifugation at 10,000PBMCs were isolated as above, resuspended in PBS, and Fc receptors blocked with anti-CD16/32 (BD Biosciences) on ice for 10 minutes prior to staining with anti–human CD14–FITC  and anti–human Dectin-1–PE , or the appropriate isotype control , on ice for 30 minutes. Stained samples were washed in PBS and acquired using a BD LSR, equipped with BD FACSDiva software. FlowJo (BD) was used for the final analysis.g for 15 minutes at 4°C. Supernatants were collected and protein concentrations were determined using the Bradford Protein Assay (Bio-Rad), according to the manufacturer’s protocol. The proteins (20 μg) were resolved in 12% SDS-PAGE and electrotransferred onto 0.2 μm PVDF. Membranes were blocked in 5% BSA and incubated with primary antibody against Dectin-1  and β-actin , followed by secondary anti–rabbit IgG, HRP-linked antibodies . Chemiluminescence detection was performed with Clarity Western ECL Blotting Substrate (Bio-Rad), using the ChemiDoc MP Imaging System (Bio-Rad). Quantification was obtained by densitometry image analysis using Image Lab 5.2 software (Bio-Rad).Whole-cell lysates were suspended in 1× RIPA buffer containing protease and phosphatase inhibitors (Thermo Fisher Scientific). The lysates were centrifuged at approximately 14,0005 cells/well in a 6-well plate and allowed to rest overnight. The following day, cells were transfected using 2 μg WT or p.Y238* plasmid and Fugene HD reagent (Promega) at a 3:1 ratio. Twenty-four hours later, cells were harvested for immunoblot analysis.WT Dectin-1 expression plasmid  was used for site-directed mutagenesis to generate the patient variant, c.714T>G; p.Y238*. HEK293 cells (ATCC) were plated at 5 × 10–/–Rag1, –/–Il1r1, –/–Tnfa, and –/–Ifng. All other strains and their respective controls/littermates were bred in-house at the NIH . In experiments using Cre-expressing mouse lines, mice were pretreated with 3 mg tamoxifen  daily by intraperitoneal injection for 5 days prior to infection. Animals were infected and analyses performed as outlined below. Animals were euthanized by cervical dislocation following administration of ketamine/xylazine cocktail at defined time points after infection (see figure legends).Eight- to 12-week-old female mice were maintained in individually ventilated cages under specific pathogen–free conditions at the NIH. The following strains (and their respective WT controls/littermates) were obtained from the NIAID Taconic contract; C. cassiicola was prepared as above and 50 μL injected into the left hind footpad of each mouse using a 21-G needle. Footpad swelling was measured once per week using calipers, measuring the height of the foot just below the toes at the widest part of the foot. The right uninfected footpad was measured and swelling expressed as a percentage relative to the uninfected footpad for each individual mouse. For C. albicans footpad infections, C. albicans SC5314 was grown in YPD broth overnight at 30°C with shaking at 200 rpm, washed in PBS, and counted with a hemocytometer. Yeast cells (5 × 106) were injected into the left hind footpad in a 50 μL volume and footpad swelling measured every 2 days as described above. C. albicans footpad burdens were measured by removing the infected footpad with a scalpel, homogenizing in 0.5 mL sterile PBS, and plating on YPD agar plates. Viable colonies were counted after incubation at 37°C for 24 hours and expressed as CFU per gram of footpad tissue.+ cells, the cells were stimulated with PMA (50 ng/mL) and ionomycin (2.5 μg/mL) for 3.5 hours at 37°C and 5% CO2 in a humidified chamber, in the presence of 1 mg/mL brefeldin A. Cells were then fixed/permeabilized using the Foxp3 Staining Buffer Set (eBioscience) prior to labeling with anti–IL-1β , anti–TNF-α , or anti–IFN-γ .Footpads were removed with a scalpel, finely minced, and digested for 30 minutes at 37°C in digestion medium (RPMI supplemented with 250 μg/mL Liberase TL and 50 μg/mL DNase [both from Roche]), which was supplemented with 10 μg/mL brefeldin A (Sigma-Aldrich) for intracellular cytokine staining experiments involving detection of IL-1β and TNF-α. The resulting cellular suspension was filtered and washed in PBS. Cells were stained with fluorophore-labeled antibodies against the following proteins: CD45 , Ly6G , CD11b , CD11c , Ly6C , MHCII , F4/80 , CD3 , TCRβ , CD90.2 , CD4 , CD8 , CD19 , NKp46 , and IFN-γR1 , and data were acquired on a BD LSR Fortessa equipped with BD FACSDiva software. FlowJo (Tree Star) was used for the final analysis. For intracellular cytokine staining of IL-1β and TNF-α, mice were first injected with 250 μg brefeldin A 6 hours prior to sacrifice. To stain for IFN-γ2 tissue culture flasks (Corning) for 5 days, replacing the media on day 3. On day 5, the media were replaced with ice-cold 2 mM EDTA/PBS and flasks incubated on ice for 10 minutes. Adherent cells (macrophages) were gently lifted using a cell scraper (Gibco) and counted using trypan blue exclusion. Macrophages were then seeded into flat-bottom 96-well plates (4 × 105 per well) in RPMI supplemented with 10% FBS and 1% penicillin/streptomycin. Some wells were additionally supplemented with 10 ng/mL recombinant murine TNF-α (Biolegend) and/or 10 ng/mL recombinant murine IL-1β (Biolegend), or 5 mM N-acetyl cysteine (Sigma-Aldrich). Macrophages were incubated overnight at 37°C and 5% CO2. The following day, C. cassiicola (prepared as above to OD600 = 2.0) was added to the macrophages  and the incubation at 37°C continued for a further 3 hours. Culture supernatants were then collected and frozen at –20°C. Concentrations of β-D-glucan in the supernatants were measured using the Glucatell (1→3)-β-D-glucan detection reagent kit (Cape Cod Associates), as per the manufacturer’s instructions.Bone marrow cells from the femurs/tibias of C57BL/6 females were flushed out with sterile 2 mM EDTA/PBS, washed, and resuspended in RPMI  with 20% FBS (Sigma-Aldrich), 1% penicillin/streptomycin (Invitrogen), and 40 ng/mL M-CSF (Biolegend). Bone marrow cells were incubated in 75 cm–/–Card9, and –/–Clec7a mice was removed and fixed in 10% formalin for 24 hours before embedding in paraffin wax. Tissue sections were stained with periodic acid–Schiff (PAS). To estimate C. cassiicola burden in the footpad, pictures were taken of the entire footpad section (at least 2 per mouse) and the total area covered by fungal cells measured using ImageJ software (NIH). For cytokine analysis, infected footpads were removed with a scalpel and added to 0.5 mL PBS supplemented with 0.05% Tween 20 and a protease inhibitor cocktail (Roche). Footpads were homogenized and cell debris removed by centrifugation at 1,100g for 5 minutes at 4°C. The resulting supernatant was snap-frozen on dry ice and stored at –80°C until analysis. Samples were analyzed by Luminex array as described above, and cytokine concentrations were determined per milligram of footpad tissue.The infected left hind foot of WT, –/–Card9, –/–Clec7a, and –/–Ifng mice were harvested and homogenized in 1 mL PBS using Omni tip homogenizers . To isolate and quantify DNA from pure C. cassiicola, the fungus was grown as described above and the fungal suspension was adjusted to an OD600 of 2.0 in 1 mL PBS. The footpad homogenates and pure fungal suspensions were centrifuged at 5,000g, supernatants were discarded, and the pellets were utilized to isolate DNA as described previously  or 1-way ANOVA with Dunnett’s multiple corrections . See figure legends for details of tests used for each figure panel. To compare the frequency of deleterious CLEC7A mutations with high CADD scores (>20) in patients with severe phaeohyphomycosis relative to healthy individuals recorded in 1000 Genomes, Fisher’s exact was used. In all cases, P values of less than 0.05 were considered significant.Statistical analyses were performed using GraphPad Prism 9.0 software. Details of individual tests are included in the figure legends. In general, data were tested for normal distribution by the Kolmogorov-Smirnov normality test and analyzed accordingly by unpaired Guide for the Care and Use of Laboratory Animals , under the auspices of protocol LCIM14E approved by the Animal Care and Use Committee of the NIAID. The patients and healthy donors were enrolled in protocols approved by the NIH Institutional Board Review and provided written informed consent for participation in the study. Patients or their parents provided written consent for their photographs to be published as part of this study and the record for the consent has been retained. This study was conducted in accordance with the Declaration of Helsinki. The Dectin-1–deficient index patient was enrolled in ClinicalTrials.gov study number NCT00001355. The CARD9-deficient patients and the additional putatively immunocompetent patients with severe forms of phaeohyphomycosis (Animal studies were performed in accordance with the recommendations in the NIH omycosis  were enrRAD, JVD, APH, VO, JAA, SLA, MS, SV, DY, AK, NSPV, SP, AMZ, DBK, and MSL performed experiments and analyzed the data. RAD, JVD, APH, DCV, KDMB, AMZ, CMM, KJKC, JKL, DBK, SMH, and MSL designed experiments. MSA, MAW, DB, BD, KH, CSZ, EMNF, MMS, JAB, YRS, CAA, CA, MM, ZT, AGT, M Natarajan, M Nigo, VM, KCB, SGK, MSW, GR, CJW, NM, JGB, LCK, WRM, AG, AGJ, MS, AFF, JEB, SMH, KB, RH, and MSL provided clinical care and referred patients. AJO analyzed the genomic data. TD provided regulatory support to patient enrollment and consenting. KDMB, YI, KJKC, YB, and GDB provided key reagents and expertise. MSL conceived and supervised the project. RAD and MSL wrote the final manuscript.


Salvia miltiorrhiza root/Danshen) of DanHong and many other Danshen-containing injections. Our earlier pharmacokinetic investigation of DanHong suggested that hepatic and/or renal uptake of the Danshen compounds was the crucial steps in their systemic elimination. This investigation was designed to survey the molecular basis underlying hepatobiliary and renal excretion of the Danshen compounds, i.e., protocatechuic acid, tanshinol, rosmarinic acid, salvianolic acid D, salvianolic acid A, lithospermic acid, and salvianolic acid B. A large battery of human hepatic and renal transporters were screened for transporting the Danshen compounds and then characterized for the uptake kinetics and also compared with associated rat transporters. The samples were analyzed by liquid chromatography/mass spectrometry. Because the Danshen phenolic acids are of poor or fairly good membrane permeability, their elimination via the liver or kidneys necessitates transporter-mediated hepatic or renal uptake from blood. Several human transporters were found to mediate hepatic and/or renal uptake of the Danshen compounds in a compound-molecular-mass-related manner. Lithospermic acid and salvianolic acid B (both >500 Da) underwent systemic elimination, initiated by organic anion-transporting polypeptide (OATP)1B1/OATP1B3-mediated hepatic uptake. Rosmarinic acid and salvianolic acids D (350–450 Da) underwent systemic elimination, initiated by OATP1B1/OATP1B3/organic anion transporter (OAT)2-mediated hepatic uptake and by OAT1/OAT2-mediated renal uptake. Protocatechuic acid and tanshinol (both <200 Da) underwent systemic elimination, initiated by OAT1/OAT2-mediated renal uptake and OAT2-mediated hepatic uptake. A similar scenario was observed with the rat orthologs. The investigation findings advance our understanding of the disposition of the Danshen phenolic acids and could facilitate pharmacokinetic research on other Danshen-containing injections.Phenolic acids are cardiovascular constituents (originating from the Chinese medicinal herb  Salvia miltiorrhiza roots (Danshen in Chinese) are a commonly used Chinese cardiovascular herb which, alone or in combination with other medicinal herbs, is formulated for oral or intravenous administration. Hydrophilic phenolic acids and lipophilic diterpene quinones are believed to be responsible for most of the cardiovascular effects of Danshen (Carthamus tinctorius flowers (Honghua) ( Danshen . DanHongHonghua) . It is aHonghua) . It is rDue to the aqueous extraction for preparing DanHong, the constituents originating from Danshen were the hydrophilic acids, rather than the lipophilic diterpene quinones. The major Danshen hydrophilic phenolic acids present in DanHong are caffeic acid derivatives, occurring as monomers (tanshinol), dimers , trimers , and tetramers . In addition, these compounds contain one or more catechol moieties and have one or more carboxylic acid groups . Protoca217βG), estrone-3-sulfate (E1S), prostaglandin F2α (PGF2α), tetraethylammonium (TEA), taurocholic acid (TCA), para-aminohippuric acid (PAH), glycylsarcosine (GS), methotrexate (MTX), and glycyrrhizin (GL) were obtained from Sigma-Aldrich . Adenosine 5′-triphosphate  and other reagents were obtained from Sinopharm Chemical Reagent Co., Ltd. .The test Danshen compounds protocatechuic acid, tanshinol, rosmarinic acid, salvianolic acid D, salvianolic acid A, lithospermic acid, and salvianolic acid B were obtained from the Tongtian Biotechnology  and their purity exceeded 98%. The positive substrates for testing validity of transporter systems, i.e., estradiol-17β-D-glucuronide (E+/taurocholate cotransporting polypeptide (NTCP), peptide transporter (PEPT) 1, and PEPT2 and rat Oatp1a1, Oatp1b2, Oat1, Oat2, Oat3, Oct1, Oct2, Oct3, carnitine/organic cation transporter (Octn) 2, Ntcp, Pept1, and Pept2 were synthesized and subcloned into pcDNA 3.1 (+) expression vectors by Invitrogen Life Technologies . Prior to the study, all expression plasmids were sequence-verified according to their GeneBank accession numbers, i.e., NM_006446 (human OATP1B1), NM_019844 (human OATP1B3), NM_007256 (OATP2B1), NM_004790 (OAT1), NM_006672 (OAT2), NM_004254 (OAT3), NM_018484 (OAT4), NM_003057 (OCT1), NM_003058 (OCT2), NM_021977 (OCT3), and NM_003049 (NTCP) and NM_017111 (Oatp1a1), NM_031650 (Oatp1b2), NM_017224 (Oat1), NM_053537 (Oat2), NM_031332 (Oat3), NM_012697 (Oct1), NM_031584 (Oct2), NM_019230 (Oct3), NM_019269 (Octn2), and NM_017047 (Ntcp).Human embryonic kidney 293 (HEK-293) cells were obtained from the American Type Culture Collection . Full open reading frames of cDNA for human organic anion-transporting polypeptide (OATP) 1B1, OATP1B3, OATP2B1, organic anion transporter (OAT) 1, OAT2, OAT3, OAT4, organic cation transporter (OCT) 1, OCT2, OCT3, NaInside-out membrane vesicles (5 mg protein/mL), prepared from insect cells expressing human multidrug resistance-associated protein (MRP) 2, MRP3, MRP4, breast cancer resistance protein (BCRP), bile salt export pump (BSEP), and multidrug resistance protein (MDR) one and rat Mrp2, Mrp4, Bcrp, and Bsep, and vesicles  were obtained from Genomembrane .2, in Dulbecco’s modified Eagle’s medium, which was fortified with 10% fetal bovine serum, 1% minimal essential medium nonessential amino acids, and 1% antibiotic-antimycotic solution. A day before transfection, cells were seeded onto 24-well poly-d-lysine-coated plates at a density of 2 × 105 cells per well. After incubation at 37°C and 5% CO2 for 24 h, the expression plasmid and the empty vector were introduced separately into the HEK293 cells with Lipofectamine 2000 transfection reagent , according to the manufacturer’s protocol to yield transporter-expressing cells and mock cells, respectively. Before use, the transfected cells were validated functionally using the positive substrate GL , E217βG (for Oatp1a1), PAH (OAT1 and Oat1), PGF2α (OAT2 and Oat2), E1S , TEA , TCA (NTCP and Ntcp), and GS  (Cell cultures and transfection were performed as described previously . Brieflyd Pept2) .2, 0.5 mM MgCl2, 5 mM glucose, and 5 mM HEPES, pH 7.4) for preincubation and cell washing and buffer B  for incubation. The transport rate (pmol/mg protein/min) of the test Danshen phenolic acids was calculated using CL, VL, T, and WL are the concentration of the test Danshen compound in the cellular lysate (μM), the volume of the lysate (μL), the incubation time (10 min), and the protein amounts measured in the lysate (mg), respectively. Differential uptake between the transfected cells and mock cells was defined as net transport ratio (TransportTC/TransportMC ratio), where TransportTC and TransportMC are the transport rates of a test Danshen compound into transfected and mock cells, respectively. A net transport ratio >3, with a statistically significant difference between TransportTC and TransportMC, demonstrated enough substrate activity in the screen to warrant determination of the compound for the Michaelis constant (Km), maximum velocity (Vmax), and intrinsic clearance (CLint). The determination was performed under linear uptake conditions by incubation for 5 min. Transport studies were performed 48 h after the transfection. The test Danshen compounds, i.e., protocatechuic acid, tanshinol, rosmarinic acid, salvianolic acid D, salvianolic acid A, lithospermic acid, and salvianolic acid B at 100 µM final concentration each, were separately incubated with the transfected cells for 10 min. Krebs-Henseleit buffer was used for preincubation, incubation, and cell washing, except for the transporters PEPT1, PEPT2, Pept1, and Pept2 using buffer A  was used for preincubation and incubation, while buffer D  was used for vesicle washing. The transport rate (pmol/mg protein/min) of the test Danshen compound was calculated using CV, VV, T, and WV represent concentration of the compound in vesicular lysates supernatant (μM), volume of the lysates (μL), incubation time (10 min), and amount of vesicle protein amount per well (0.05 mg), respectively. Differential transport of the test Danshen compound between ATP-containing vesicles and AMP-containing vesicles was defined as net transport ratio (TransportATP/TransportAMP ratio). Based on the experience in our laboratory, a net transport ratio >3, with a statistically significant difference between TransportATP and TransportAMP, often suggests a positive result. All the incubation samples were analyzed by liquid chromatography/mass spectrometry.Inside-out membrane vesicles expressing one of the ABC transporters MRP2, MRP3, MRP4, BCRP, BSEP, MDR1, Mrp2, Mrp4, Bcrp, and Bsep were used to assess the transport of the test Danshen compound, i.e., protocatechuic acid, tanshinol, rosmarinic acid, salvianolic acid D, salvianolic acid A, lithospermic acid, and salvianolic acid B, using a rapid filtration method . Before via a HESI source with an Agilent 1290 infinity LC system . The chromatographic separation was achieved on Phenomenex Gemini 5-μm C18 column . The mobile phases, which consisted of solvent A  and solvent B , was delivered at 0.3 ml/min. A 6-min gradient elution method was used as follows: 0–3.5 min, from 1% solvent B to 40% solvent B; 3.5–4.5 min, at 98% solvent B; and 4.6–6 min, at 1% solvent B. The mass spectrometry measurement was performed in the negative ion mode with precursor-product ion pairs for selected-reaction-monitoring of protocatechuic acid, tanshinol, rosmarinic acid, salvianolic acid D, salvianolic acid A, lithospermic acid, and salvianolic acid B at m/z 153→108, 197→123, 359→133, 417→197, 493→185, 537→185, and 717→321, respectively. Matrix-matched calibration curves for quantification of these test Danshen compounds  were constructed using weighted (1/X) linear regression of the peak area (Y) against the corresponding nominal analyte concentration . The sample preparation was performed using methanol-based protein precipitation and centrifugation; the supernatant was analyzed by liquid chromatography/mass spectrometry. Although no internal standard was used, assay validation, implemented according to the European Medicines Agency Guideline on bioanalytical method validation , demonstrated that the developed assays were reliable and reproducible for the intended use. The assays’ lower limit of quantification was 4.1 nM for all the test Danshen compounds. The intra-batch accuracy and precision were within 94.1–105% and 2.41–10.1%, respectively, while associated inter-batch values were within 86.6–104% and 2.91–11.3%, respectively. The coefficients of variation reflecting matrix effects on assays were 2.23–10.7%. The analyte stability under conditions mimicking the analytical process was also evaluated, including storage at 24°C for 3 h, storage at 8°C for 24 h, and three freeze-and-thaw cycles. The analytes were stable under the test conditions, as indicated by the measured mean concentrations fluctuating within 86.9–113% of the nominal concentrations. All the preceding validation results were within the acceptable ranges.Validated bioanalytical assays were used to measure the test Danshen compounds in cell- and vesicle-based biomatrices. Analyses were performed on a TSQ Vantage mass spectrometer  interfaced 217βG, E1S, PGF2α, TEA, TCA, PAH, GS, MTX, and GL was achieved using liquid chromatography/mass spectrometry-based methods described previously  was used to determine the Km and Vmax values by nonlinear regression analysis of initial transport rates as a function of substrate concentration. Km and Vmax values of the test Danshen compound were calculated using the following V is the initial transport of the test Danshen compound in transfected cells (pmol/min/mg protein), Vmax is the maximal transport rate (pmol/min/mg protein), S is the concentration of the test Danshen compound (μM) and Pdif is the clearance of the test Danshen compound via passive diffusion in mock cells.GraFit software . A value of Km, Vmax, and CLint values summarized in Km, Vmax, and CLint values in Rosmarinic acid (molecular mass: 360 Da), salvianolic acid D (418 Da), lithospermic acid (538 Da), and salvianolic acid B (718 Da) were found to be substrates of the human hepatic sinusoidal uptake solute carrier (SLC) transporters OATP1B1 and OATP1B3; their net transport ratios are summarized in Km, Vmax, and CLint values summarized in Km, Vmax, and CLint values in Similar to the situation for human hepatic transporters, rosmarinic acid, salvianolic acid D, lithospermic acid, and salvianolic acid B were substrates of the rat hepatic sinusoidal uptake SLC transporter Oatp1b2 (the closest ortholog of both human hepatic OATP1B1 and OATP1B3) ; Figure Km, Vmax, and CLint values in Protocatechuic acid , tanshinol (198 Da), rosmarinic acid (360 Da), and salvianolic acid D (418 Da) were found to be substrates of the human OAT1 and OAT2, two renal basolateral uptake SLC transporters. The compounds’ net transport ratios for OAT1 are shown in Km, Vmax, and CLint values in Similar to the situation for human renal transporters, protocatechuic acid, tanshinol, and rosmarinic acid were substrates of the rat renal basolateral uptake SLC transporters Oat1 and Oat3  ; Figure via the liver or kidneys necessitates transporter-mediated hepatic or renal uptake from blood, and this serves as the crucial elimination step. In this investigation, we found both hepatic and renal SLC transporters that could mediate uptake of the Danshen phenolic acids into the hepatocytes and/or renal proximal tubular epithelia, respectively, in a molecular-mass-related manner , tanshinol (198 Da), rosmarinic acid (360 Da), salvianolic acid D (418 Da), salvianolic acid A (494 Da), lithospermic acid (538 Da), and salvianolic acid B (718 Da) are believed to be pharmacologically important for the injection due to their cardiovascular properties . Becaused manner . The reld manner .via bile and/or urine. Understanding drug compounds’ tendency for excretion into the bile and/or urine is important, because the excretion can be a major factor influencing drugs’ therapeutic and toxicological properties. The belief that molecular mass influences the excretion tendency of a drug compound is implied in several early publications  studies.For pharmacokinetic research on Chinese herbal medicines , rat is in vitro transporter kinetic data . Here, the DDI indices were estimated based on their (Cmax × fu-plasma)/Km ratios, where the Cmax and fu-plasma data were obtained from our earlier human pharmacokinetic study of DanHong (50) of 38 µM for OATP1B1  have been found to mediate potential pharmacokinetic DDIs with herbal medicines, comprising the herb-drug interaction  and the tic data  and 5 in DanHong . Another OATP1B1 ; the ass OATP1B1 , pharmacIn this investigation, we identified human transporters responsible for hepatobiliary and renal excretion of Danshen phenolic acids. Lithospermic acid and salvianolic acid B (both >500 Da) underwent systemic elimination, initiated by OATP1B1/OATP1B3-mediated hepatic uptake. Rosmarinic acid and salvianolic acid D (350–450 Da) underwent systemic elimination, initiated by OATP1B1/OATP1B3/OAT2-mediated hepatic uptake and by OAT1/2-mediated renal uptake. Protocatechuic acid and tanshinol (both <200 Da) underwent systemic elimination, initiated by OAT1/OAT2-mediated renal uptake and OAT2-mediated hepatic uptake. A similar scenario was observed with the rat orthologs, including the rat SLC transporters acting on the Danshen compounds, also in a compound-molecular-mass-related manner. The findings of this investigation improve our understanding of disposition of the Danshen phenolic acids and could facilitate pharmacokinetic research on other Danshen-containing injections. Future investigations are planned to elucidate whether any new transporter(s) could mediate hepatic uptake of salvianolic acid A and how efflux of many Danshen phenolic acids from hepatocytes and renal proximal tubular epithelia is achieved after the SLC transporter-mediated hepatic and renal uptake, respectively.


The surfactant marketrepresents a key sector of the chemical industryand encompasses many diverse applications. Their sustainability interms of feedstock used, synthetic procedure, biodegradability, andformulation are crucial parameters to assessing the environmentalimpact of the surfactant. The anionic surfactant linear alkyl benzenesulfonates have proven successful to date because of their high performance,low cost, and extensive studies within formulations to optimize performance,allowing usage in a large variety of applications, especially in cleaning.Due to their advantageous properties and extensive research and development,their substitution with a biobased surfactant such as sodium dodecylsulfate has struggled to succeed. Furan surfactants have been reportedas valuable candidates for the implementation of green alternativesto traditional anionic sulfonated surfactants with a perfect trade-offbetween performances and green credentials. However, their implementationsuffers of scalability and high cost in producing the final productdue to feedstock availability and low yields of the final product.Herein, we report a new class of furan surfactants, sulfonated alkylfuroates, which are derived from the esterification of furoic acidand fatty alcohols, followed by a sulfonation step. Compared to traditionalsurfactants, they showed more favorable behavior in basic proprieties, which gives a good prospective for theintroduction of a new biobased chemical with superior performances. SAF is a new biobased surfactant thatcan replace oil-basedanalogues providing superior performances by exploiting fatty alcoholand furoic acid. The market size ofsurfactants was 15 million tons/years in 2014 and is expected to continueexpanding by 4.3% per year through 2022.2Surfactantsare one of the most used commodity chemicals, commercializedin high volumes and finding applications in many different fields.An optimum surfactant design for a specific application isbasedon the careful selection of the chemical structure of the hydrophilicand hydrophobic parts as these structures strongly affect the surfactant’sproperties. However, desirable surfactant properties differ accordingto the application. For example, it is desired that surfactants usedfor home care products have a low critical micelle concentration (CMC),a low Krafft temperature (KP), high solubility in water, high resistanceto hard water, low toxicity, and high biodegradability.5Emulsifyingproperties are also important parameters for detergentsand applications in enhanced oil recovery, while foaming and mildnessare important factors for personal care. The increasing demand formore sustainable and greener products has added an extra requirementto the surfactant industry since product formulators are demandingbiobased surfactants with equal performance to the current traditionalpetrochemical derivatives in the final application and remove allthe petrochemical additives currently used to improve surfactant performance,which cause harm to the environment.6 particularly linear alkyl benzenesulfonates (LASs) and sodium dodecyl sulfate (SDS). LAS is a petroleum-basedsurfactant, while SDS is a biobased surfactant with several limitationssuch as low water solubility, low resistance to hard water, and lowperformances due to high CMC.7 A solutionto improve detergent formulations in terms of sustainability and competitivenessis needed. This solution should be focused on synthesizing surfactantsderived from low cost, biorenewable raw materials. These surfactantsshould also ideally exhibit high performance with a minimum use ofadditives. Consequently, researchers have sought biobased and biodegradableanionic surfactants that have performance and costs comparable tothose of LAS.Currently, themost used surfactants in industrial applicationsare anionic surfactants, and they account about 20% of a typical householdproduct,9 Different paths have also been reported, which use furans to makesurfactants.10 Lee and co-workers reporteda 5-hydroxymethyl furfural (HMF)-derived sulfonated surfactant,11 but the product suffers from a complex and expensivesynthetic route associated with HMF12 andlow stability of the final product. Hoffman and co-workers13 have proposed a new class of biodegradable surfactantsderived from photo-oxygenation of furfural with air, leading to afuranone head group. The author demonstrated the high biodegradabilityof these surfactants, but scalability of the process is limited bythe photo-oxygenation step.Sugar derivatives have attracted much attention,and many catalyticroutes have been developed to valorize sugars into furanic intermediates.7 reported a new class of sulfonated furan alkylsurfactants basedon the Friedel–Crafts acylation of furan. In their work, theauthors evaluated the effect of substituting the benzene ring of LASwith a furan ring and analyzed different structural variants basedon the hydrophobic chains. With the same alkyl chain, the new surfactantproved to have superior properties compared with the conventionalLAS, including higher resistance to hard water. The authors demonstrateda strong correlation between the final proprieties of the surfactantwith the furan linker and alkyl chain linker. A ketone linker provedto be detrimental for the final surfactant properties in terms ofCMC, wettability, and resistance to hard water, requiring a reductionstep of the ketone group to have good final surfactant properties.The performance of the furan alkyl surfactants after reduction provedto be higher than that of LAS, but major improvements are still requiredto decrease the Krafft point. Further studies for the efficient productionof these surfactants were reported by implementing a high-temperatureprocess by Vlachos and co-workers which used an iron-based catalystto produce the ketone-based surfactant starting from furoic acid (FA)and fatty alcohol. However, the overall yield could not reach morethan 50%.14 The group of Palkovits andco-workers15 has further reported anotherclass of surfactants derived from condensation of furfuryl alcoholand fatty alcohol forming an ether linker, but this has proved tobe unstable upon sulfonation, leading to complete degradation of thefinal product; therefore, the authors were not able to characterizethe surfactant. Corma and co-workers proposed a highly efficient methodto produce the carboxylate ether surfactant from HMF in a two-stepprocess where the alcohol group is condensed with a fatty alcoholand the aldehyde group is oxidized into a carboxylic acid.16 However, the authors did not characterize itsproperties. Another group proposed an ester surfactant with the carboxylatehead group derived from 2,5-furandicarboxylic acid (FDCA), provinggood behavior of the surfactant toward biodegradation and aggregationproperties.17Recently, Dauenhauer and co-workers18These efforts havedemonstrated that the nature of the linker andthe position of the sulfonate group strongly impact the propertiesof furan-based surfactants. Moreover, the choice of the furan sourceplays an important role on the feasibility of the process due to challengesin obtaining HMF and (biobased) furan at reasonable cost, which underminesthe feasibility of the process at large scale.15 While different linkers havebeen reported in the literature , the esterlinkage in combination with the sulfonated group has not been previouslyreported.In this work, we introduce a new highly scalableand biobased familyof anionic surfactants called sulfonated alkyl furoates SAFs, 1 based oA property packagewas created in Aspen Properties v9.0., where the compounds used weredefined as pseudo-components. The normal boiling point, density, andmolecular weight were imported from COSMOtherm to create the pseudo-component.The COSMOSAC model was selected as the thermodynamic model, and agamma method was modified to use COSMOSAC-Mathias modification. Theσ-profile was specified as the pure component properties SGPRF1-5,and the COSMO volume was specified as the CSACVL component parameter.18 Estimationof the capital costs was done using the integrated Aspen Economicpackage and the purchase and installation costs annualized over 10years. Process simulations were done for a plant capacity for 2232tons/year for alkyl furoate (AF) from furoic acid, 1948 tons/yearfor LAB evaluation, and 1948 tons/year for AF starting from furfural.The minimum selling price (MSP) and CO2 emissions werenormalized per Kg of product.Prices for technoeconomic assessment are reported in 2 emissions were estimated as the methanerequired to generate steam at medium pressure (12 bar) with a thermalefficiency of 0.8. Heat integration was done respecting a pinch pointof 5 °C. The energy required to reach the reaction temperatureis considered supplied by external heating.MSP was estimated as the sum ofthe feedstock, utility, and annualizedcapital costs. CODodecyl furoateis produced according to the following process:two streams of dodecanol (DOD) were split to favor heat integrationwith the downstreams. After heat integration, FA is added at roomtemperature (B1) at a stoichiometric amount. The stream is preheatedwith the product stream and sent to the stoichiometric reactor (B3)at 150 °C. The water vapor generated is separated (B5) with agas–liquid separator, condensed with heat recovery (B9), andsent to the wastewater treatment (WWT) system. The process is reportedin 2). The heat of reactionis removed with cooling water, and the cost is taken into accountin the economics. The vapor is separated in B6, and the product iscooled at B8 with heat recovery. The process is reported in Furfural(FUR) and dodecanol are mixed at room temperature. The stream is preheatedwith the product stream (S3), then heated at 130 °C (B3) andreacted in B2 with pure oxygen (ODodecene (DODEC) and benzene aremixed at room temperature and preheated with heat recovery (B2). Thereaction is carried out in reactor B3. The heat of reaction is removedwith cooling water, and the cost is integrated in the economics. Theprocess is reported in Catalyst screeningwas conducted with the following procedure: a stoichiometric amountof furoic acid (FA) and dodecanol (DOD) was placed in a 3 mL vial at room temperature. The catalyst was thenadded. In case sulfuric acid was used, 1 mol % was added, while incase Nafion, Amberlyst, and Purolite were used, 300 mg was added.The vial was placed in a preheated heating block at 150 °C for1.5 h. During the reaction, the vials were vented through a needleto allow the removal of the water generated during the reaction.For the kinetic experiments, 500 mg of FA was mixed with a stoichiometricamount of DOD (830 mg) and different amounts of sulfuric acid . The reaction mixture was placed in a preheated heatingblock at 150 °C for the desired time. This was repeated for differenttimes. The mixture was analyzed by gas chromatography–massspectrometry–flame ionization detection (GC–MS–FID)using naphthalene as the internal standard.2SO4 was added. The water was collectedwith a Dean Stark apparatus connected to a vacuum line with a pressurecontroller at 800 mbar. AF was obtained as a yellowish liquid. Theproduct was confirmed by NMR and GC–MS. The residual FA contentwas assessed to be below 2% by HPLC.The reaction was performedwith a Dean Stark apparatus to collect the water generated. FA (1equiv) was mixed with a slight excess of DOD (1.05 equiv) and heateduntil the reaction mixture reaches a temperature of 150 °C. Then1 mol % H1H NMR (DMSO-d6): δ8 ,7.3, 6.7 , 4.23 , 1.66, 1.4–1.8 , 0.86 . 13C{1H} NMR (DMSO-d6): δ159.9 (C=O), 147.8 (CH–C–C=O),144.6 (O–CH–CH), 119, 113 (−C–CH–CH–C), 65 (O–CH2–CH2), 31.7, 31.1, 29.1,28.7, 25.8, 22.7 , 14.4 (−CH2–CH3) ppm. MS : m/z = 225.1486. FT-IR: 1705 cm–1 (O–C=O stretch).1H NMR (DMSO-d6): δ7.9 ,7.2, 6.7 , 4.23 ,1.66, 1.4–1.8 , 0.86 . 13C{1H} NMR (DMSO-d6): δ 159.9 (C=O), 148.2 (CH–C–C=O), 144.5 (O–CH–CH), 119, 112.5 (−C–CH–CH–C), 65.7 (O–CH2–CH2), 31.8, 29.5, 29.2, 29.7, 28.8, 25.9,22.6 , 14.1(−CH2–CH3) ppm. MS : m/z = 281.2109. FT-IR: 1705 cm–1 (O–C=Ostretch).1HNMR (benzene-d6): δ 7.0, 6.9 , 5.8 , 4.1 , 1.44, 1.37–1.05 , 0.85 . 13C{1H} NMR (benzene-d6): δ 158.5(C=O), 145.7 (CH–C–C=O),145.5 (O–CH–CH), 117, 111 (−C–CH–CH–C), 64.4 (O–CH2–CH2), 32, 29.9–29.3, 28.8, 26, 23 , 14.1 (−CH2–CH3) ppm. MS : m/z = 337.2735.FT-IR: 1705 cm–1 (O–C=O stretch).1H NMR spectroscopy to confirm that the reaction was completewith full conversion of the ester and consumption of most of the CSA(CSA shift: 10.9 ppm). Chloroform was removed under vacuum to afforda dark-green solution, which was diluted with water and neutralizedto pH 7. The mixture was then placed in a fridge, where most of thesurfactant precipitated as a white solid. The product was collectedby precipitation and washed with cold water. Analysis by ion chromatographyconfirmed that residual sulfate and chloride salts are below 0.2%.The furoate ester (10 g) was addedto dry chloroform (5 mL) and mixed at room temperature until fulldissolution was achieved. Chloroform was needed to avoid foaming,which for large batches represented an operative problem. The reactionflask was then connected to a water-filled bubbler through which theproduced HCl was vented. Chlorosulfonic acid (CSA) was then added(1.05 equiv). The water was first saturated by HCl and then bubblingbegan, indicating consumption of CSA. The reaction was left untilno further bubbling was observed, and the mixture analyzed by 1H NMR (D2O): 6.9, 6.7, 4.2 , 1.6, 1.34–1.0 , 0.8 . 13C{1H} NMR (D2O): δ 158.5(C=O), 155.8(O–CH–CH)144.5 (CH–C–C=O), 118.2–112.2(−C–CH–CH–C), 66.1 (O–CH2–CH2), 31.7, 29.2, 29.1, 28.3, 25.7, 22.6 , 13.8 (−CH2–CH3) ppm. MS : m/z (abundance) = 303.087(C13H19O6S– 100%).FT-IR: 1250–1300 cm–1, (S=O stretching),1705 cm–1 (O–C=O stretching).1H NMR (DMSO-d6): δ 7.9 , 7.2, 6.7 , 4.23 , 1.66, 1.4–1.8, 0.86 . 13C{1H} NMR (DMSO-d6): δ159.9 (C=O), 148.2 (CH–C–C=O),144.5 (O–CH–CH), 119, 112.5 (−C–CH–CH–C), 65.7 (O–CH2–CH2), 31.8, 29.5, 29.2,29.7, 28.8, 25.9, 22.6 , 14.1 (−CH2–CH3) ppm. MS : m/z (abundance) = 359.0865 (C17H27O6S– 100%). FT-IR: 1100–1300cm–1, (S=O stretching), 1705 cm–1 (O–C=O stretching).1H NMR (D2O): δ 6.9, 6.7 ,4.1 , 1.54, 1.26–0.95 , 0.8 . 13C{1H} NMR (D2O):δ 159.5 (C=O), 156.5(O–CH–CH) 144.6 (CH–C–C=O),118.9–112.9 (−C–CH–CH–C), 66.1 (O–CH2–CH2), 32.2, 31–29.3, 28.5, 26, 22.8,13.8 (−CH2–CH3) ppm. MS : m/z (abundance) = 415.1465 (C21H35O6S– 100%). FT-IR: 1100–1300 cm–1, (S=O stretching), 1705 cm–1 (O–C=O stretching).13 however, a technoeconomic analysis which estimatesthe environmental impact and price viability was never reported forthese compounds, even though purification techniques and feedstockprice are crucial parameters to define the success that these surfactantscan have in the market.The usage of furanbuilding blocks has attracted much interest in the last 20 years forthe production of different biobased chemicals which can substitutebulk petrochemicals. For example, different catalytic pathways usingHMF, furfural, or furan have been used;2 emissions, energy requirements,and waste generation.18 The developmentof an efficient catalytic pathway should be focused on high yieldsat short reaction times, minimizing the requirements of purificationsteps (such as distillation or solvent washing) to avoid high operatingcosts which would compromise the feasibility of the process.In order to establish a sustainableprocess, the synthesis methodology is extremely important since itdirectly impacts the COIn our first analysis in performing the first step of the reactionscheme reported in 3 as a milder sulfonating agent in a falling film reactor whichtoday is used at large scale to minimize the cost and improve thequality of the final product, in addition to a better atom economywhich is equivalent to 100% for the SO3 process and 91%for the CSA option due to the formation of HCl as a side product.The main advantage of the falling film reactor is that SO3 can be stripped out the reaction mixture and neutralization canbe performed quicker, reducing the side products that can be formedunder acidic conditions.192 emissionsin producing AF (Supporting Information (Table S3).We alsoconducted an evaluation of the MSP and COucing AF 5d and co2 emissionsand a higher MSP since energy is required to remove the water throughevaporation with the further disadvantage of low heat of integrationdue to low heat of reaction. Remarkable advantages can be obtainedusing furfural as feedstock due to its lower price and the exothermicnature of the oxidation which provides heat to satisfy the energydemand of the plant, making the process more economically competitive.Moreover, the process starting from furfural has a similar atom economy(93.2%) compared to the process using FA (94%). The process modeledfor producing LAB yields a more competitive price, mainly due to thelow price of the feedstock which has a well-established supply chainin the commodity chemical market. However, further considerationsexist concerning the sustainability of the usage of petroleum-derivedfeedstocks since they need to undergo multiple transformations startingfrom paraffins and BTX  which are associatedwith high greenhouse gas emissions. Moreover, end of life emissionof LAS is the main contributor to the total emissions and estimatedto be 2.3 kgCO2/kgLAS.20 While the CO2 emissions estimated for the productionof SAF are low, the life cycle of furfural and FA are crucial to definethe overall environmental impact of SAF. However, currently, the supplychain for these feedstocks is not as fully developed as for petroleum-basedchemicals, and an exemplary, optimized process at large scale is stillnot constructed.The process which uses FA resulted in higher COTables S4 and S6), which suggests the potential for superiordetergency as low CMC is desirable in cleaning formulations. SAF-8exhibits a lower CMC than commercial SDS, while SAF-12 and SAF-16have a lower CMC than LAS, indicating that SAF could function as areplacement for traditional surfactants in a variety of home and personalcare products. While the CMC is a good estimation for micelle formation,it remains a rough estimation for the performance that the surfactantcan have in specific applications such as in oil recovery or detergency.A more rigorous estimation comes from measuring the decrease in surfacetension between water and hydrophobic solvents. A lower surface tensioncorresponds to a more favorable cleaning ability since it favors theformation of stable emulsions. The droplet formation of a water/surfactantsolution in octanol has been reported for SAF-8, SAF-12, and LAS (Table S7) at a surfactant concentration of twicethe CMC. SAF-8 shows the lowest surface tension (2.65 ± 0.32mN/cm) due mostly to the high concentration of the surfactant used(4298 ppm). SAF-12 shows a surface tension (3.42 ± 0.58 mN/cm)lower compared to that of LAS (4.48 ± 0.75 mN/cm), further indicatingthe potential for a high cleaning capability and emulsifying propertiesof this new category of sulfonated surfactants.The propertiesof the surfactant were evaluated under salt-free conditions (<100ppm) according to the procedure reported in the methodology. The resultsreported in T20 is observedwith longer alkyl chains. The solubility showed a strong dependencewith temperature, with the saturation of SAF-12 increasing from 2to 28% on increasing the temperature from 25 to 30 °C. The samebehavior was observed for SAF-16 at a higher temperature (47 °C).Attempts to stabilize SAF-12 at lower temperatures with the extensivelyused hydrotrope p-xylene sulfonic acid failed. However,we observed that the solubility increases remarkably by using SDSas a cosurfactant at a mass ratio of 6:1 (SAF-12/SDS). This allowedus to stabilize SAF at a lower temperature, reaching a solubilityof total surfactant concentration over 30% without the addition ofany hydrotrope. It is worth mentioning that this mixture exhibitsa lower CMC than SDS or SAF-12 alone, which is an indication of synergybetween the two surfactants.SAF-8 proved to be remarkably soluble in water, reachingsaturationat a temperature below 0 °C 6. We obs7 who showed that the linker between the aromatic head groupand the aliphatic chain has a strong influence on the surfactant precipitationin hard water. In that study, a functionalized linker such as a ketonesignificantly decreased the solubility of the surfactant in hard water,compared with C–C linkers. The fact that the ester linker utilizedhere proved beneficial for solubility in hard water eliminates theneed to perform a hydrogenation step to defunctionalize the linkage,representing a further advantage of SAF in terms of scalability. Themixture of SAF-12/SDS also proved to be highly stable in hard water,further confirming a synergetic effect of the mixture of these twocompounds in optimizing the CMC, solubility, and hard water resistance.SAF 8 and SAF-12 exhibit high resistanceto hard water, which isa noteworthy weakness for both LAS and SDS. This aspect was analyzedin a previous study by Dauenhauer and co-workers,Table S8), which is an important parameter for personal care formulations.LAS shows the highest foamability, but its use in the personal caresector is strongly limited because of its very low mildness and petrochemicalorigin. This is the reason why many current personal care productsuse SDS instead of LAS. SDS shows very good foamability in distilledwater, but this decreases significantly in hard water due to highinstability, which leads to precipitation. The stability of the foamis improved using SAF surfactants; specifically, SAF-12 shows higherfoamability compared with SDS, which is further improved in hard water,making this compound very suitable as a new biobased surfactant whichdoes not require any chelants or a foam boaster, which SDS does require.Further studies were performed to determine the foamability ofthe surfactant in distilled and hard water (Table S10). This testassesses skin irritation through the solubilization of the Zein proteinthrough denaturation when in contact with the surfactant. We useda methodology similar to that reported by Cohen and co-workers.22 Surprisingly, SAF-8 and SAF-12 showed better mildness values comparedwith the commercial LAS and SDS, suggesting a less irritating effectfor those applications which involve skin contact.The applicability of SAF in personal care applications was furtherstudied by analyzing the mildness of the surfactant through the Zeinsolubilization test . Our results indicate that the alkyl chain has a much higher influencecompared with the head group on the toxicity. SAF-8 proved to havea much lower toxicity compared with the 12-carbon chain surfactants.SAF-12 proved to have a higher toxicity than SDS, possibly due tothe presence of the ester group. However, SAF-12 exhibited lower toxicitycompared to LAS with both methodologies, indicating a more beneficialeffect of the furan ring compared with the aryl ring, further establishingSAF-12 as an eco-friendly alternative to LAS.The superiorproperties of SAF can greatly simplify the designof formulations for different applications in the consumer sector.Specifically, the combination SAF-12/SDS has the potential to exhibithigh cleaning ability due its low CMC and the low surface tensionof water–oil which is provided by SAF-12. In order to furtheranalyze the sustainability of SAF, ecotoxicity was evaluated throughthe inhibition of Our studies on SAF have demonstrated that this surfactant can exhibitsuperior properties compared with previously reported furan-basedsurfactants, further demonstrating the importance of the linkage betweenthe alkyl chain and the furan ring. The properties reported by Dauenhauerand co-workers were retained while using an ester linkage insteadof a C–C chain (2). The usage of ester linkage makes the scale-upof the alkylation viable since it can be carried out in one step withoutthe need of a purification step; however, the usage is limited tothose applications where pH is neutral due to risk of hydrolysis ofthe ester group. SAF’s properties are superior compared withthe ketone linker (3), which proved to be unsatisfactory in termsof CMC and resistance to hard water and proved to be stable underlight. The carboxylate surfactant has been reported in the literaturewith ether (5), (6) and ester linkers, but the synthesis route reportedrequires HMF or FDCA, which reduces the economic viability of thesesurfactants. Moreover, carboxylate surfactants are known to exhibitpoor resistance to hard water, which limits their performance. In Further improvements of the overall performance canbe achievedby mixing SAF with SDS with improvements in all properties, retaininghigh resistance to hard water, an area where SDS is weak.Thesynthesis procedure of SAF has multiple advantages over previouslyreported furan surfactants since the intermediate can be producedat high purity and yield without the need of any purification step,with the further advantages of a reduction in the reaction steps andimproved secondary properties (color), which are important for obtaininga final surfactant with good specification.FA can be producedfrom furfural with multiple reported oxidationtechniques which already are efficient and provide high yield. Currently,this product is not commercialized at large scale since there areno applications in commodity chemicals and further studies are neededto implement these processes. The implementation of the productionof AF directly from furfural through oxidative esterification canlead to further improvements of the process economics, but a suitablecatalyst needs to be developed to perform this reaction under stoichiometricconditions which can avoid the implementation of a distillation columnto remove the fatty alcohol, which would increase the cost.The implementation of an efficient process for the production ofFA has the potential to establish SAF as a new economically competitivecommodity chemical with higher performance and green credentials comparedwith traditional surfactants and potential in emulsification applicationssuch as cleaning and oil recovery.2 emissions related to the processing, which gives the prospectiveof a competitive market price. Ultimately, the low-cost productionof FA and the compatibility of the sulfonation step in the fallingfilm reactor will define the success of this technology.Inthis study, we explored SAF as a potential replacement of LASin formulation development. The advantages in using SAF lie on theefficient formation of the hydrophobic part of the molecule at highyield simply using sulfuric acid as a catalyst and without the requirementof a purification step, reducing the energy requirements of the plant.This can significantly reduce the operating costs of a scale-up whichcan guarantee an acceptable MSP in association with low COThebasic proprieties of SAF suggest that it can guarantee similaror better performances than LAS in terms of emulsification proprieties,detergency, and personal care thanks to the low surface tension ofwater–oil, CMC, and skin irritation. Moreover, the high resistancein hard water and the improvement of CMC and solubility when SAF ismixed with SDS open the prospective of a new cheaper formulation designwith high performances.Preliminary results on the ecotoxicitysuggest that this surfactantdoes not exhibit any environmental concern compared to LAS. Furtherstudies need to be addressed on the anaerobic and aerobic biodegradabilityof this surfactant.


In contrast, cultivar ‘Southern Glen’ showed no response in bulb weight or allicin. This was likely due to high soil background S concentrations masking treatment effects. Subsequently, a solution culture experiment with cv. ‘Glenlarge’ evaluated six S application rates . In solution culture, bulb weight and allicin concentration increased with S rate. Highest bulb weight (~53 g bulb−1) and allicin concentration (~11 mg g−1 DW) were recorded at an S application of 1504 mg S plant−1. This is the first report to conclusively demonstrate the effect of S on yield and allicin in garlic grown in solution culture.Improving bulb yield and allicin content of garlic is important in meeting fresh and pharmaceutical market demands. Garlic plants have a high demand for sulfur (S) since allicin contains S atoms. Two experiments were conducted to identify the effect of S application rate on garlic yield and quality. In a field trial assessing six S application rates (0–150 kg S ha Allicin is the main medicinal compound in garlic and a target concentration of >4.5 mg allicin gindustry  and healindustry  antioxid6H11NO3S) was first determined by Stoll and Seebeck [6H10OS2), which contains two S atoms, suggesting that crop S nutrition plays a critical role in contributing to the allicin content of garlic bulbs. The S concentration in garlic bulbs ranges from ~7.4 to 16 g kg−1 (dry weight basis (DW) [−1) and onion (~5–10 g kg−1) [−1) [−1) and lettuce (3.0 g kg−1) [−1) [Sulfur (S) is a structural element of the allicin molecule and its precursor alliin . The che g kg−1)  and cons−1) [−1) , beetroo g kg−1) . Althoug−1, bulb weight and allicin concentration was either unaffected [−1 [−1 DW [−1 DW [−1 DW) [A number of studies have evaluated the effect of S application rate on garlic bulb weight, alliin and allicin concentration, under either field or glasshouse conditions ,12,13,14affected ,12 or inaffected . In thescted [−1 ,14,15. T [−1 DW) ,17. The In soil, S availability and supply can be increased by S fertilizer application . Furthermore, S may also be derived from atmospheric inputs including air pollution and sea-born sulfate aerosols , and soiThis research aimed to determine how S supply impacts yield and quality of two Australian subtropical garlic cultivars grown in the field and to identify whether solution culture technique can discern S limitations in garlic.−1 (p > 0.05) of S supply on bulb weight and yield for Southern Glen (−1) and fresh yield (~12 t ha−1) than Southern Glen (~65 g bulb−1 and ~9 t ha−1 (fresh yield)) at the optimum S rate of 75 k ha−1   and time constraints.In support of the field trial results, in solution culture the bulb fresh weight of Glenlarge also increased in a curvilinear manner with S application rate ( < 0.05) B. Bulb wp > 0.05) when S application rate increased from 0–150 kg ha−1, and the DM% was 34% in both garlic cultivars (data not shown). In the solution culture experiment, the DM% of Glenlarge bulbs ranged from ~25–32%, but was not significantly different between S application rates (p > 0.05) (data not shown).The percentage dry matter (DM%) of bulbs grown in the field trial showed no significant difference (p > 0.05) in the field trial when S application rates increased from 0 to 150 kg ha−1 (data not shown). However, the concentration of S (~6.2 g kg−1) in bulbs of Glenlarge was lower (p < 0.05) than that of Southern Glen (~8.5 g kg−1).The bulb S concentrations showed no significant differences   B.p < 0.05) in response to the six S rates (−1 (DW), when the S application rate increased from 0 to 50 kg ha−1. With a further increase in application from 75 to 150 kg S ha−1, allicin concentration fluctuated between 13.8 and 14.3 mg g−1 DW but the effect was not significant. Bulb allicin content in Glenlarge increased by ~30% when the S application rate increased from 0 to 75 kg ha−1 (−1 DW) or bulb allicin content (~375 mg bulb−1)  .−1 DW) was substantially lower (p < 0.05) than that of Southern Glen (~16.8 mg g−1 DW). However, despite this difference, the net allicin content in bulbs of Glenlarge (396 mg bulb−1) was significantly greater than that of Southern Glen (375 mg bulb−1), across the S application rates, with the exception of the 0 kg S ha−1 treatment (Allicin concentration in Glenlarge (~13.6 mg greatment  due to t−1 DW at an application rate of 188 mg S plant−1 and increased by ~5 times to ~11.0 mg g−1 DW at an application rate of 1502 mg S plant−1 (−1 (at 188 mg S plant−1) to ~180 mg bulb−1 (at 1504 mg S plant−1)  B.−1 (p < 0.05) when S application increased from 0 to 75 kg ha−1  on garlic bulb weight of Southern Glen under field conditions. Arunachalam et al. [−1 in three consecutive years and showed that bulb weight increased by ~30%, from ~13 to 17 g bulb−1, but the maximum bulb weight was substantially lower than that of the two garlic cultivars grown under field conditions in the present study.In the field trial, across six S application rates, bulb weight of Glenlarge increased by ~20%, from 75.7 to 89.7 g bulb kg ha−1 A, but bu kg ha−1 B. Despitha−1   B. In the-Kielian  showed t−1) (−1)  A was sub−1) (−1) B. The lo−1) (−1)  which mamilation . through−1) than Glenlarge (6.2 mg kg−1) (p < 0.05). The non-significant effect of S application rate on bulb tissue S concentration is perhaps due to the high S supply in the irrigation water, which was estimated at ~69 kg ha−1. This supply is greater than the requirement for S in both cultivars and was in excess of S uptake for Glenlarge and Southern Glen in the 0 kg S ha−1 treatment. Similarly, the study of Arunachalam et al. [−1) on bulb S concentration (~7.0–8.0 mg kg−1).In the field trial, there were no significant differences in bulb S concentrations across the six S application rates for both Glenlarge and Southern Glen (data not shown). However, Southern Glen bulbs had a higher S concentration  between S uptake and S application rate for Glenlarge, increasing to ~26 kg ha−1 at an application of 150 kg S ha−1. However, there was no significant difference in S uptake (~25 kg S ha−1) across S application rates for Southern Glen  when S application increased from 188 to 1504  (p < 0.05). This suggests that genetic limitations in S uptake capacity exist in the tested variety Glenlarge.In the solution culture experiment, S uptake increased from 97 to 416 mg plant S ha−1) B. In the S ha−1)   showed no S response  in allicin content (~30%), from ~315 to ~420 mg bulb−1, when S application increased from 0 to 75 kg ha−1 (−1) was not significantly different across the six S application rates (p > 0.05)  .In the solution culture experiment, allicin concentration of Glenlarge increased with S application rate , confirm-c kg−1 and 7 mg kg−1 available sulfur  to evaluate the effect of S application rate on garlic bulb yield and allicin concentration. The soil was a classified as a Brown Dermosol according to the Australian Soil Classification and the soil had pH 7.9, a cation exchange capacity of 27.7 cmole sulfur . −1  and Southern Glen . Vegetative seed-cloves of these cultivars were selected and planted in a split-plot randomised complete block factorial with 4 replications. The first factor (main plot) was S rate, and the sub-plots were cultivars. The main plots were 4.0 m × 3.0 m, with a 1 m buffer between them. The main plots consisted of two beds of 1.5 m width, with each bed containing 2 rows of plants at 0.55 m spacing. The within-row plant spacing was 10 cm, giving a population density of ~133,000 plants ha−1 .−1) in the form of magnesium sulfate heptahydrate . Hence, we do not consider that the added Mg confounded the S response. Other nutrients were applied at sufficient rates to optimise garlic growth (rates as kg per ha): 180 N, 120 K, 0.7 B, 0.7 Zn, 0.5 Mn, 0.3 Cu, and 0.11 Mo. No P, Ca and Fe was applied because the soil was inherently very high in these nutrients.Sulfur was added at six rates  were collected and a mineral nutrient analysis was conducted , K, Na, Ca, Zn, Mg, Mn, Cu, Fe, P, S and B , a composite reference leaf sample (youngest fully expanded leaf) was collected from each replicate of each treatment. The leaf samples were dried at 70 °C for 24 h and analysed for mineral nutrients. At 185 days after planting, the bulbs in each plot were harvested and cured for 28 days in an open-sided well-ventilated shed. Five bulbs (~45–65 mm diameter) of each treatment were selected and used for mineral nutrient and allicin analysis. The samples for allicin analysis included 4 cloves removed from the outer clove-ring of each bulb. The remaining cloves from the bulb were sliced into 3 cm pieces, weighed, dried at 70 °C for 7 days. The dried leaf and bulb samples were sent to Incitec-Pivot  for mineral nutrient analysis . The quantification of allicin was using the method described by Nguyen et al. .The solution culture experiment was conducted in a glasshouse at the University of Queensland Gatton Campus  in 2020 to evaluate the effect of S supply on the growth of garlic and its allicin concentration.Twenty-four 13 L white polythene buckets were filled with 12 L of nutrient solution prepared from aliquots of stock solutions . A sleev−1) which were broken into cloves and uniform cloves with a weight of 6.5 ± 0.5 g selected.The experiment was conducted on garlic cv. Glenlarge, which is high yielding and with an intermediate allicin concentration, and is the dominant subtropical variety in Australia. Due to logistical and time constraints, cv Southern Glen could not be tested. Seed-cloves for planting were selected from large bulbs (~130 g fresh weight bulbThe seed-cloves were imbibed in wet towels for ~7 days to initiate root development and then one clove was transplanted into each of the three cups in each bucket . The clo−1) on plant growth, and alliin and allicin concentration. The S was applied as (NH4)2SO4 and MgSO4 with rates equivalent to 25, 50, 75, 100, 150 and 200 kg S ha−1 based on a plant population of 133,000 plants ha−1. To achieve optimum garlic growth potential, the amount of nutrient per plant was applied at rates of 2114 mg N, 928 mg K, 514 mg P, 487 mg Ca, 159 mg Mg, 6.7 mg Fe, 1.76 mg B, 2.58 mg Zn, 1.81 mg Mn, 0.42 mg Cu, 0.06 mg Mo, 0.30 mg Co and 0.30 mg Ni  in the glasshouse were monitored using digital recorders .At maturity , the bulbs were harvested and cured (air dried under cover) for 4 weeks. After curing, the bulbs were weighed, and the number of cloves determined in each bulb. Whole bulbs (three per replicate of each treatment) were taken and allicin was determined using the method described by Nguyen et al. . The remp < 0.05. The graphs were presented as mean and standard errors, and appropriate regression analyses conducted using SigmaPlot 14.0 .Data were analysed using Minitab 18.1 software for Windows , using one-way and two-way ANOVA. The differences in measured parameters  between treatments were determined with Tukey’s HSD at −1 whereas Southern Glen showed no optimum S response. However, mineralisation of organic S in soil and supply of S in irrigation water likely confounded the S rate response. Hence, a solution culture method was developed to grow garlic plants under controlled S supply. This showed that Glenlarge bulb weight and allicin content were maximal at an equivalent S rate of 1504 mg g−1 nominally equivalent to ~200 kg ha−1. In solution culture, garlic had only 50% S uptake efficiency and further study is required to understand the factors determining S uptake.In the field trial, Glenlarge showed maximal bulb weight and allicin content at a S application of 75 kg ha


Fervidobacterium pennivorans subsp. keratinolyticus subsp. nov. strain T was isolated from a terrestrial, high-altitude hot spring in Tajikistan. This strain is an obligate anaerobic rod and their cells occur singly, in pairs, or as short chains under the optimal growth conditions of a temperature of 65 °C and pH 6.5, with peptone, glucose, and galactose as the preferred substrates. The minimum generation time of this strain is 150 min. Strain T can efficiently degrade feather keratin at 65–75 °C; this unusual feature is also exhibited by a few other members of the Fervidobacterium genus. The total genome size of this bacterial strain is 2,002,515 base pairs, with a C + G content of 39.0%. The maximum digital DNA–DNA hybridization (dDDH) value of 76.9% was observed on comparing the genome of this strain with that of Fervidobacterium pennivorans type strain DSM9078. This study describes the physiological and genomic properties of strain T, with an emphasis on its keratinolytic power and differences from other members of the genus Fervidobacterium. Fervidobacterium [Fervidobacterium can grow on a range of complex substrates such as cellulose, starch, or proteinaceous compounds; however, only a few members have been described as being capable of breaking down keratin, including Fervidobacterium islandicum strain AW-1, Fervidobacterium thailandense, and Fervidobacterium pennivorans [https://www.ncbi.nlm.nih.gov/data-hub/taxonomy/200918/; accessed on 2 August 2022), thereby representing a huge resource of hyper-stable enzymes with potential biotechnological applications.The phylum Thermotogae includes extremely thermophilic anaerobic bacteria, with optimum growth temperatures up to 80 °C . They reacterium , six moracterium ,10,11,12nivorans ,10,11. AFervidobacterium group, their thermophilic features, and the increasing availability of thermostable proteases for the degradation of proteinaceous biowaste from agriculture and fisheries have immense potential for biotechnological applications [Fervidobacterium pennivorans subspecies recovered from a hot spring in Tajikistan, which effectively breaks down native chicken feathers under anaerobic conditions at temperatures up to 75 °C.Keratin is a structural protein present in feathers, hair, skin, wool, horns, etc. and is one of the most abundant polymers on Earth after cellulose and chitin . The preications ,24. In tThe sample was collected from the Khodja-Obi-Garm geothermal field, located on the Bank of the Varzob River, 50 km North of Dushanbe, Tajikistan, at 38°53′41.45″ N, 68°47′15.64″ E, at an altitude of 1800 m. The temperature and pH of the spring water were 93 °C and 8.5, respectively, with a conductivity of 4378.3 µS/cm . Water s4·7H2O, 0.3 g; KCl, 0.3 g; NH4Cl, 0.5 g; CaCl2·2H2O, 0.1 g; and KH2PO4, 0.3 g. A trace element solution (1 mL) was also added, containing (per liter): HCl (25%), 10 mL; FeCl2·4H2O, 1.5 g; CoCl2·6H2O, 190 mg; MnCl2·H2O, 100 mg; ZnCl2, 70 mg; Na2MoO4·2H2O, 36 mg; NiCl2·6H2O, 24 mg; H3BO3, 6 mg; and CuCl2·H2O, 2 mg. Finally, 0.5 mL of resazurin (0.2%) was added to the medium as a redox indicator. After sterilization at 121 °C for 20 min and cooling to approximately 60 °C under continuous flushing with nitrogen gas, 10 mL of the vitamin solution was added, which contained the following ingredients: 4-aminobenzoic acid, 8 mg/L; D(+) biotin, 2 mg/L; nicotinic acid, 20 mg/L; Ca-D(+) pantothenic acid, 10 mg/L; pyridoxamine·2HCl, 30 mg/L; thiamine dichloride, 20 mg/L; and vitamin B12, 10 mg/L. Finally, 25% HCl-cysteine (2 mL) was added, and the pH was adjusted to 7.0 with 1 M HCl. The medium was transferred to 50 or 100 mL serum flasks using the Hungate technique, closed with butyl rubber stoppers, crimped with metal seals [Anaerobic enrichment cultures were prepared according to a modified Hungate technique . The minal seals , and sub−1 dilution; from this one, a ten-fold dilution series up to 10−9 was prepared. All the flasks were incubated at 65 °C for 48 h and the process was repeated using inoculum from the highest dilution that yielded growth. The most diluted growth-yielding culture was examined by phase contrast microscopy and appeared to be pure, and this was used as a stock culture for all the following experiments. The strain was stored at 4 °C, and fresh cultures were prepared by inoculating 1 mL of inoculum from the stock culture into 30 mL of an anoxic MMF-based medium as described above. The standard incubation temperature was 65 °C. Additional yeast extract (0.1%) was used for determining the temperature and pH-dependency and when denser cultures were needed.To obtain pure cultures, ten-fold serial dilutions for extinction experiments were carried out, yielding a bacterial isolate termed strain T. A battery of eight flasks filled with 9 mL of MMF medium supplemented with 0.5% peptone and 0.05% yeast extract was prepared. One mL of the original culture was transferred to one of these new flasks to make a 10https://www.uib.no/ en/rg/mic) (accessed on 11 October 2022) and the Electron Microscopy Laboratory (ELMILab) of the University of Bergen (https://www.uib.no/en/geo/111662/scanning-electron-microscope) (accessed on 11 October 2022) using a JEOL JSM-7400F  and Zeiss Supra 55VP scanning electron microscope , respectively. The samples were fixed in 2.5% glutaraldehyde (diluted in the culture media) and stored at 4 °C for 24 h until further processed, as described previously [A phase-contrast microscope  equipped with an oil immersion lens (100×) and a Nikon DS-Fi3 camera was used to observe the bacterial cells and obtain photomicrographs. Scanning electron microscopy (SEM) was performed at the Molecular Imaging Center (MIC)  containing (per liter) Na2HPO4 (2.5 g), NaCl (8 g), KCl (0.2 g), and KH2PO4 (0.2 g) prior to an OD measurement. PBS was used as the blank. All the measurements were performed in triplicates. The generation time (g) was calculated from the exponential part of the growth curve using the equation: r is the growth rate of the organism, calculated using the equation: OD2 and OD1 are the optical densities measured at times t2 and t1, respectively.The strain was tested for growth under strict anoxic conditions on the following substrates: sucrose, lactose, arabinose, galactose, mannose, sorbitol, mannitol, starch, CM-cellulose, glucose, and peptone using an MMF medium supplemented with 0.05% yeast extract. After 24 and 48 h of incubation at 65 °C, the growth was determined using phase-contrast microscopy. Doubling of the cell density after 24 or 48 h was scored as positive or slow growth, respectively. To determine the temperature range and optimal temperature for growth, the bacterial strain was subjected to 38, 55, 65, 70, 75 and 80 °C and tested in duplicates using the MMF medium supplemented with peptone (0.5%) and yeast extract (0.1%). A growth curve was prepared by measuring the optical density of the strain at 600 nm using a UV MIN 1240 Shimadzu spectrophotometer. The cells were grown in batch cultures in 50 or 100 mL serum flasks containing 30 mL of MMF supplemented with 0.5% peptone and 0.05% yeast extract. One milliliter aliquots from a freshly inoculated culture incubated at 65 °C were taken every two hours and centrifuged at 12,000× TM Bacterial Genomic DNA Kit  according to the manufacturer’s guidelines. The 16S rRNA gene was amplified using the primers 27F (5′-GAGTTTGATCCTGGCTCAG) and 1525R (5′-GAAAGGAGGTGATCCAGCC) [Fervidobacterium including the type strains of the six described species using the ClustalW tool [Thermosipho africanus was used as the outgroup.Genomic DNA was extracted and purified from cell pellets using the GenEluteTCCAGCC) . The PCRTCCAGCC) . The 16SalW tool . A phyloalW tool  algorithalW tool ,33. The alW tool . The trealW tool . The 16STM Bacterial Genomic DNA Kit (Sigma-Aldrich) following the manufacturer’s guidelines. The genomic DNA was sequenced using PacBio technology at Eurofins Genomics, Constance, Baden, Germany (https://eurofinsgenomics.eu/) (accessed on 11 October 2022). Processing of the reads and de novo assembly were performed using CLC Genomic Workbench version 21 , which yielded a complete genome sequence of 2,002,515 bases with a coverage of 254×. The genome sequence was submitted to the NCBI database (accession No. CP050868) and annotated using an automatic NCBI annotation pipeline. A genome-based phylogenetic analysis was conducted using the Type (Strain) Genome Server (TYGS) available at the DSMZ website (https://tygs.dsmz.de) (accessed on 11 October 2022) [Genomic DNA from strain T was extracted using the GenEluteer 2022)  and the er 2022) .The keratinolytic activity was assessed using native chicken breast feathers as the keratin-based substrates. The feathers were washed in a methanol: ethanol solution (1:1) before autoclaving . Then, 15 ± 5 mg of the feathers was added to 30 mL of MMF medium supplemented with yeast extract (0.05%), to which a 1 mL inoculum of the bacterium was added. An uninoculated flask containing only medium with feather was used as the negative control. The cultures were incubated anaerobically at 70 °C. Every 24 h, the flasks were visually inspected to check for changes in the feather integrity.Following two dilution-to-extinction series in the MMF medium supplemented with peptone, an apparently pure culture was obtained, and termed strain T, with a cellular morphology like other members of the Thermotogales: straight to slightly curved cells (around 1.5 µm × 5 µm) with a membranous sheath-like toga. A spheroid extension of the toga was observed at the end of the cells and was visible under a phase-contrast and scanning electron microscope . Strain Fervidobacterium, with a 98.88% sequence identity to the 16S rRNA gene sequence of Fervidobacterium pennivorans DSM 9078T (CP003260.1). A phylogenetic tree based on the 16S rRNA gene sequence was constructed for strain T and other members of the genus Fervidobacterium (F. pennivorans strain T was placed between F. pennivorans type strain (DSM 9078) and F. pennivorans strain DYC. A maximum-likelihood [A Neighbor-Joining phylogenetic analysis of the 16S rRNA gene sequence of strain T showed that it belongs to the genus acterium . Accordikelihood ,39 tree Fervidobacterium. F. pennivorans strain T can degrade native chicken feathers at temperatures up to 70 °C, a characteristic that is also exhibited by other fervidobacteria, except F. nodosum and F. gondwanense.A low concentration of yeast extract (0.05%) was essential for the growth of strain T. The strain clearly possessed a heterotrophic metabolism with a preference for carbohydrates such as lactose, galactose, glucose, and sorbitol, as well as protein-derived substrates such as peptone . It alsoAll the tested strain T cultures were capable of growing on peptone and glucose. Strain T grew within a narrow pH range . Growth was not observed at NaCl concentrations higher than 3%, with an optimal salt concentration of 1%. Strain T grew within a temperature range of 55 to 75 °C, with an optimal temperature of 65 °C. Growth was not observed at 50 or 80 °C.F. pennivorans strain T was measured under conditions of 65 °C in MMF supplemented with 0.5% peptone and 0.1% yeast extract , 1875 protein-coding sequences were found, 194 of which were assigned to a subsystem according to the annotation conducted by the RAST server. Additionally, 57 RNA genes were identified  and metallopeptidases were annotated. Enzymes involved in the reduction of disulfide bonds were also identified; thus, strain T possesses an arsenal of keratin-degrading enzymes. Moreover, this approach resulted in the identification of known genes predicted to play a role in the resistance to environmental stresses, such as oxidative stress, cold shock (the CSP family), heat shock, and detoxification. In this regard, the organism seemed to possess many defense mechanisms against heavy metals  and general drugs via a multidrug resistance efflux pump. Two complete 16S rRNA operons were identified and aligned, showing a 100% sequence identity. Three CRISPR arrays, including 19 genes encoding CRISPR-associated proteins, were identified. Although motility has not been reported in fervidobacteria, 56 genes involved in chemotaxis and flagellar motility were identified. The organism did not contain plasmids.F. pennivorans strain T against the complete genomes of the members of the genus Fervidobacterium was performed using in silico DNA–DNA hybridization (dDDH) analysis using the TYGS server, average nucleotide identity (ANI) calculation, and phylogenomic tree reconstructions. Based on the TYGS analysis, strain T was most closely affiliated with F. pennivorans type strain DSM9078, sharing a dDDH identity value of 76.9%  [The genomes of representative strains of r (BRIG) , with thr (BRIG)  program r (BRIG) . These pr (BRIG) ,44 can aF. pennivorans strain T and type strain DSM 9078 was performed using Mauve [A full genome alignment of ng Mauve   could be identified, suggesting that these two bacteria may be classified as different subspecies. The strain grew well on substrates such as glucose, peptone, and galactose; however, turbid cultures were obtained only when yeast extract was added as a supplemental nutrient, suggesting the need for a mixture of the auxiliary nutrients for growth. This behavior is not unique to this organism and has also been described for other strains belonging to the F. pennivorans species [F. pennivorans species was established. It showed a lag phase dependent on both physical and physiological conditions: an exponential phase of 11 h with a generation time of 150 min and a long stationary phase. The total genome size of strain T was 2,002,515 base pairs, similar to those of the other members of the genus Fervidobacterium, with a similar number of coding gene sequences. Whole-genome alignment with the type strain showed three large, inverted regions flanked by transposase-encoding genes. Chromosomal rearrangements are not unusual for this group and have also been reported in other Thermotogales species. Chromosomal rearrangements have been suggested to affect species evolution [The new isolate described in this study belongs to the  species  as well volution ,48.Fervidobacterium species. However, F. changbaicum CB5-1 and F. islandicum AW-1 were sufficiently similar to be grouped into the same genome species, with an ANI value of 98.79% , whereas the DYC strain forms a deeper and separate branch and shares less than a 92% ANI value with strain T and DSM9078, suggesting that strain DYC constitutes a separate f 98.79% . AccordiF. pennivorans strain T allowed the identification of several keratinase enzyme candidates. The strain showed strong keratinolytic activity and could completely degrade chicken feathers after 48 h incubation at 75 °C. Furthermore, scanning electronic microscope images revealed that this bacterium physically attaches to the feathers during the degradation process, suggesting that the cell wall or membrane-bound enzymes participate in the process. Nevertheless, the features required for keratin degradation are not fully understood, and most research in this field centers on screening novel microorganisms with keratinolytic activity, as well as their secreted protease arsenal. Furthermore, for other complex substrates, such as cellulose, it has been suggested that the degradation of keratin does not occur by the action of a single enzyme but requires a mixture of different enzymes with hydrolytic activity. F. pennivorans is an anaerobic and thermophilic bacterium, making it especially convenient for industrial processes, as neither oxygen supply nor refrigeration systems are required.Analysis of the genome of