Efficient Bioconversion of High Concentration Phytosterol Microdispersion to 4-Androstene-3,17-Dione (AD) by <Emphasis Type="Italic">Mycobacterium</Emphasis> sp. B3805

Low solubility of sterols in aqueous media limits efficient steroid production mediated by biocatalytic microorganisms such as Mycobacterium. Sterol emulsion technologies have been developed with low success rates, largely due to the complexity of generating stable and bioavailable particles. In this study, several aqueous dispersions of sterols in-water of different particle sizes were bioconverted to 4-androstene-3,17-dione (AD) in a solvent-free environment, using a classic microorganism Mycobacterium sp. B3805 as a model system. According to our results, the high concentration (20 g/L) phytosterol dispersions with the smallest particle size tested (370 nm) achieved up to 54% (7.4 g/L) AD production yield in 11 days. Moreover, the use of 0.1 biomass/sterols ratio in a complex bioconversion media containing yeast extract, and a 1:1 glucose/microdispersion ratio in the presence of the surfactant DK-Ester P-160 (HLB16), allowed homogenization and increased microdispersion stability, thus achieving the best results using emulsion technologies to date.     

Microbial Side-Chain Cleavage of Phytosterols by Mycobacteria in Vegetable Oil/Aqueous Two-Phase System

Microbial side-chain cleavage of natural sterols to 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) by Mycobacteria has received much attention in pharmaceutical industry, while low yield of the reaction owing to the strong hydrophobicity of sterols is a tough problem to be solved urgently. Eight kinds of vegetable oils, i.e., sunflower, peanut, corn, olive, linseed, walnut, grape seed, and rice oil, were used to construct oil/aqueous biphasic systems in the biotransformation of phytosterols by Mycobacterium sp. MB 3683 cells. The results indicated that vegetable oils are suitable for phytosterol biotransformation. Specially, the yield of AD carried out in sunflower biphasic system (phase ratio of 1:9, oil to aqueous) was greatly increased to 84.8 % with 10 g/L feeding concentration after 120-h transformation at 30 °C and 200 rpm. Distribution coefficients of AD in different oil/aqueous systems were also determined. Because vegetable oils are of low cost and because of their eco-friendly characters, there is a great potential for the application of oil/aqueous two-phase systems in bacteria whole cell biocatalysis.     

Efficient Biotransformation of Phytosterols to Dehydroepiandrosterone by <Emphasis Type="Italic">Mycobacterium</Emphasis> sp.

In this study, a method for the efficient production of dehydroepiandrosterone (DHEA) from phytosterols in a vegetable oil/aqueous two-phase system by Mycobacterium sp. was developed. After the 3-hydroxyl group of phytosterols was protected, they could be converted into DHEA with high yield and productivity by Mycobacterium sp. NRRL B-3683. In a shake flask biotransformation, 15.05 g l^?1 of DHEA and a DHEA yield of 85.39% (mol mol^?1) were attained after 7 days with an initial substrate concentration of 25 g l^?1. When biotransformation was carried out in a 30-l stirred bioreactor with 25 g l^?1 substrate, the DHEA concentration and yield was 16.33 g l^?1 and 92.65% (mol mol^?1) after 7 days, respectively. The results of this study suggest that inexpensive phytosterols could be utilized for the efficient production of DHEA.     

Nanodimer cyclodextrin ligands with high affinity to steroids

Molecular-imprinting by cross-linking of ligands of ??-cyclodextrin (CD) complex with steroids has been developed for the synthesis of tailor-made CD dimer. Steroids of androstane (9??-hydroxy-androst-4-en-3,17-dione, androst-4-en-3,17-dione, androsta-1,4-dien-3,17-dione (ADD)) and pregnane (hydrocortisone, 6-methyl-hydrocortisone, 20-hydroxymethylpregna-1,4-diene-3-one (HMPD)) series were used as template molecules. For imprinting procedure, crystalline ??-CD complexes of exact stoichiometry (??-CD:steroid template = 2:1) were synthesized following by toluene 2,4-diisocyanate (TDI) cross-linking. The attempts to produce CD dimer for steroid without hydrophobic side chain failed, while tailor-made CD dimer has been obtained using HMPD as a template. The dimer was characterized by ¹H NMR and mass-spectrometry. The complex stability constant (K_S) towards HMPD template exceeded 10⁷ M^?1. The K_S of CD dimer with ADD exceeded the corresponded value of TDI-modified CD monomer by more than an order of magnitude. The dimer was applied for quantitative extraction of ADD from aqueous solution using dialysis membranes impermeable for CD. The value of K_S for ADD estimated from balanced concentrations of dialysis data corresponded to that calculated by nonlinear spectrometric method.     

A New Steroid-Transforming Strain of Mycobacterium neoaurum and Cloning of 3-Ketosteroid 9α-Hydroxylase in NwIB-01

Using enrichment procedures, five strains that can utilize soybean phytosterols as the sole carbon source were isolated from steroids-contaminated soil samples. Among the isolated strains, the strain NwIB-01 with the highest steroid degradation ability was identified as Mycobacterium neoaurum by morphological, physiological, biochemical tests and 16S rRNA sequence analysis. Meanwhile, the key enzyme gene, which was involved in steroid metabolism and encoding 395-amino acid 3-ketosteroid 9α-hydroxylase (KSH), was obtained from M. neoaurum NwIB-01 with the assistance of homology analysis and chromosome walking. To our best knowledge, this is the first report to the gene of key enzyme KSH from M. neoaurum. Strain NwIB-01 exhibited powerful ability of cleaving the side chain specifically from soybean phytosterols to accumulate 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD). It was showed that when cultured in 15 g/l phytosterols, the yield of ADD reached 4.23 g/l while accompanied by 1.76 g/l AD in 96-h-old culture (the molar yield of AD + ADD is 64.7%). The strain NwIB-01 can be applied as excellent phytosterols-transformation strains in potential industrial applications.     

Plantaricin LD1: A Bacteriocin Produced by Food Isolate of Lactobacillus plantarum LD1

Plantaricin LD1, a bacteriocin produced by Lactobacillus plantarum LD1, was characterized for biochemical and antimicrobial properties. Bacteriocin showed stability at high temperatures (100 °C for 20 min and 121 °C for 15 min under 15 psi pressure), in a pH range of 2.0–8.0 and also in the presence of organic solvents, surfactants and detergents. The crude preparation was not affected by catalase, amylase and lipase but activity was reduced in the presence of pepsin, trypsin and proteinase K showing proteinaceous nature of the compound. The molecular weight of bacteriocin was found to be ～6.5 kDa, and antimicrobial activity was confirmed by bioassay. It inhibited not only related strains but also other Gram-positive and Gram-negative bacteria such as Lactobacillus curvatus NRRL B-4562, Lactococcus lactis subsp. lactis NRRL B-1821, Enterococcus faecium NRRL B-2354, Enterobacter cloacae NRRL B-14298, Micrococcus luteus, Staphylococcus aureus, urogenic Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Shigella flexneri and Vibrio sp. These properties of plantaricin LD1 suggest its applications not only in food safety but in therapeutics as well.     

Development of a rapid normal-phase LC-positive ion APCI-MS method for simultaneous detection and quantitation of cholesterol, androst-4-ene-3,1 7-dione, and androsta-1,4-diene-3,17-dione

This paper describes the development of a normal-phase liquid chromatograph-UV-diode array detection-positive ion atmospheric pressure chemical ionization-mass spectrometry method for the simultaneous identification and quantitation of cholesterol, androst-4-ene-3,17-dione (AD), and androsta-1,4-diene-3,17-dione (ADD) in fermentation broths. The compounds detected under positive ion atmospheric pressure chemical ionization on a mass spectrometer by selected ion monitoring are separated by normal-phase high-performance liquid chromatography. [M+H]+ ions are taken into consideration for quantitation of AD and ADD, and [M-H2O+H]+ ions are considered for quantitation of cholesterol. The compounds are analyzed on a Si60 silica (5 microm, 125 x 4-mm i.d.) Merck column using a 2:3 isocratic mixture of isopropyl alcohol and hexane. The calibration curves resulting from the reference compounds in the concentration range of 100-5000 pg on column exhibit a good linear correlation (r2 > or = 0.996). The method is validated by analyzing six replicates of broth samples fortified with three compounds, namely, cholesterol, AD, and ADD, at 0.050 and 0.5 microg/g levels. The mean recoveries for the fortifications range from 90% to 98% with relative standard deviations in the range of 3.36% to 9.78%. The method is developed to study the qualitative as well as quantitative conversion of cholesterol to AD and ADD by a microorganism identified as Nocardia sp. These studies helped the investigation of the reaction kinetics, which showed that the molar biotransformation of cholesterol into AD and ADD was 21%, even when the reaction was prolonged for 96 h.     

Biotransformation of Phytosterols into Androstenedione—A Technological Prospecting Study

Androstenedione (AD) is a key intermediate in the body’s steroid metabolism, used as a precursor for several steroid substances, such as testosterone, estradiol, ethinyl estradiol, testolactone, progesterone, cortisone, cortisol, prednisone, and prednisolone. The world market for AD and ADD (androstadienedione) exceeds 1000 tons per year, which stimulates the pharmaceutical industry’s search for newer and cheaper raw materials to produce steroidal compounds. In light of this interest, we aimed to investigate the progress of AD biosynthesis from phytosterols by prospecting scientific articles (Scopus, Web of Science, and Google Scholar databases) and patents (USPTO database). A wide variety of articles and patents involving AD and phytosterol were found in the last few decades, resulting in 108 relevant articles (from January 2000 to December 2021) and 23 patents of interest (from January 1976 to December 2021). The separation of these documents into macro, meso, and micro categories revealed that most studies (articles) are performed in China (54.8%) and in universities (76%), while patents are mostly granted to United States companies. It also highlights the fact that AD production studies are focused on “process improvement” techniques and on possible modifications of the “microorganism” involved in biosynthesis (64 and 62 documents, respectively). The most-reported “process improvement” technique is “chemical addition” (40%), which means that the addition of solvents, surfactants, cofactors, inducers, ionic liquids, etc., can significantly increase AD production. Microbial genetic modifications stand out in the “microorganism” category because this strategy improves AD yield considerably. These documents also revealed the main aspects of AD and ADD biosynthesis: Mycolicibacterium sp. (basonym: Mycobacterium sp.) (40%) and Mycolicibacterium neoaurum (known previously as Mycobacterium neoaurum) (32%) are the most recurrent species studied. Microbial incubation temperatures can vary from 29 °C to 37 °C; incubation can last from 72 h to 14 days; the mixture is agitated at 140 to 220 rpm; vegetable oils, mainly soybean, can be used as the source of a mixture of phytosterols. In general, the results obtained in the present technological prospecting study are fundamental to mapping the possibilities of AD biosynthesis process optimization, as well as to identifying emerging technologies and methodologies in this scenario.     

A streamlined high throughput screening method for the Mycobacterium neoaurum mutants with expected yield of biotransformation derivatives from sterols

Mycobacterium neoaurum is ideal strain for bioconversion of sterol into steroid drugs. 96-well plate high throughput screen was validated to be able to discriminate the mimic mixtures of 4-androstene-3,17-dione (AD), androsta-1,4-diene-3,17-dione (ADD) and bisnoraldehyde (BA) optimized by uniform design, which is more rapid and higher throughput than the HPLC-based method.     

Production Optimization and Characterization of Recombinant Cutinases from Thermobifida fusca sp. NRRL B-8184

Two genes, cut1 and cut2, of Thermobifida fusca NRRL B-8184 with cutin-hydrolyzing activity were cloned and expressed in Escherichia coli BL21 (DE3) separately. Enhanced expression was achieved after screening of six different media, optimization of the culture conditions and medium components. Among the screened media, modified Terrific Broth was found to be the best for maximum production of recombinant cutinases in E. coli BL21 (DE3). Under optimal conditions, the production of recombinant Cut1 and Cut2 (cutinases) were found to be 318?±?0.73 and 316?±?0.90 U/ml, respectively. The production of recombinant cutinases was increased by 11-fold as compared with T. fusca NRRL B-8184 wild-type strain. Both the recombinant cutinases were purified to homogeneity. They were found to be thermostable, organic solvent, and surfactant tolerant. Both the cutinase were active in a broad range of temperature (40–80 °C) and pH (6.8–9) with optimum activity at pH 8.0 and 55 °C.     

The anaerobic degradation of deoxycholic acid by PSEUDOMONAS SP. NCIB 10590

The anaerobic metabolism of deoxycholic acid by Pseudomonas sp. NCIB 10590 was studied. The metabolic pathway was similar to that operating under aerobic conditions with 12β-hydroxyandrosta-1,4-dien-3,17-dione as the major neutral product an metabolites which are not produced during aerobic metabolism were isolated and evidence is presented for the following structures: 9α-hydroxyandrost-1-en-3,17-dione, 12α,17)β-dihydroxyandrosta-1,4-dien-3-one; 3β,12β-dihydroxy-5β-androstan-17-one an formation and significance of the phenolic secosteroid is discussed.     

Excretion profile of boldenone and its metabolites after oral administration to veal calves

The residue profiles of boldenone (17β-Bol), its epimer (17α-Bol) and the related compound androsta-1,4-diene-3,17-dione (ADD), were investigated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in urine of male calves orally treated with boldenone, boldenone esters, and/or ADD. In all the experiments with the administered steroids residues of 17α-Bol decreased rapidly after end of treatment; detectable amounts of 17α-Bol were however noticed along the withdrawal observation period after end of treatment. Differently, residues of 17β-Bol were detectable only shortly after administration. This in vivo research concerning oral treatments of cattle with boldenone related substances proves ADD to be a very active boldenone precursor in bovine animals.     

Urine stability and steroid profile: towards a screening index of urine sample degradation for anti-doping purpose

The presence of microorganisms in urine samples, under favourable conditions of storage and transportation, may alter the concentration of steroid hormones, thus altering the correct evaluation of the urinary steroid profile in doping control analysis. According to the rules of the World Anti-Doping Agency (WADA technical document TD2004 EAAS), a testosterone deconjugation higher than 5% and the presence of 5α-androstane-3,17-dione and 5β-androstane-3,17-dione in the deconjugated fraction, are reliable indicators of urine degradation. The determination of these markers would require an additional quantitative analysis since the steroids screening analysis, in anti-doping laboratories, is performed in the total (free + conjugated) fraction. The aim of this work is therefore to establish reliable threshold values for some representative compounds (namely 5α-androstane-3,17-dione and 5β-androstane-3,17-dione) in the total fraction in order to predict directly at the screening stage the potential microbial degradation of the urine samples. Preliminary evidence on the most suitable degradation indexes has been obtained by measuring the urinary concentration of testosterone, epitestosterone, 5α-androstane-3,17-dione and 5β-androstane-3,17-dione by gas chromatography–mass spectrometric every day for 15 days in the deconjugated, glucuronide and total fraction of 10 pools of urines from 60 healthy subjects, stored under different pH and temperature conditions, and isolating the samples with one or more markers of degradation according to the WADA technical document TD2004EAAS. The threshold values for 5α-androstane-3,17-dione and 5β-androstane-3,17-dione were therefore obtained correlating the testosterone deconjugation rate with the urinary concentrations of 5α-androstane-3,17-dione and 5β-androstane-3,17-dione in the total fraction. The threshold values suggested as indexes of urine degradation in the total fraction were: 10 ng mL⁻¹ for 5α-androstane-3,17-dione and 20 ng mL⁻¹ for 5β-androstane-3,17-dione. The validity of this approach was confirmed by the analysis of routine samples for more than five months (i.e. on a total of more than 4000 urine samples): samples with a concentration of total 5α-androstane-3,17-dione and 5β-androstane-3,17-dione higher than the threshold values showed a percentage of free testosterone higher than 5 of its total amount; whereas free testosterone in a percentage higher than 5 of its total amount was not detected in urines with a concentration of total 5α-androstane-3,17-dione and 5β-androstane-3,17-dione lower than the threshold values.     

Formation of boldenone and boldenone-analogues by maggots of Lucilia sericata

Current evidence suggests that neo formation of the anabolic steroid boldenone (androsta-1,4-diene-17-ol-3-one) occurs in calves' faecal material, making it difficult to distinguish between illegally administered boldenone and its potential endogenous presence. This strengthens the urgent need to elucidate the pathway leading to boldenone formation. In our laboratory, the invertebrate Neomysis integer (Crustacea, Mysidacea) was used since 2004 as an alternative model for the partial replacement of vertebrate animals in metabolisation studies with illegal growth promotors and veterinary drugs, e.g. boldenone. The present study evaluates the metabolic capacity of other invertebrates, the brine shrimp Artemia franciscana and maggots of the greenbottle fly Lucilia sericata. The first results indicate that maggots of L. sericata are able to convert phytosterols and -stanols, nowadays in substantial amounts added to animal feed, into androsta-1,4-diene-3,17-dione (ADD), the precursor of boldenone, at a yield of 0.10-0.14% (p<0.001, significance compared to endogenous excretion of maggots) but not to boldenone itself. Furthermore, beta-testosterone, an endogenous hormone, was transformed into androst-4-ene-3,17-dione (AED), ADD and beta-boldenone at a significant (p<0.001, significance compared to endogenous excretion of maggots) yield of circa 13%, 0.80% and 2.2%, respectively. In future studies these results are of value to further evaluate the use of maggots of L. sericata as an invertebrate model in metabolisation studies.     

Quantitative Analysis and Separation of Chiral (S)-Ethyl 3-Hydroxyglutarate in Bioconversion Mixtures by LC and TLC

A convenient ion-pair LC procedure was firstly established for rapid analysis of ethyl 3-hydroxyglutarate (3-EHG) in an enzymatic-hydrolysis mixture, and the detection limit was as low as 0.45 μmol L^?1; high repeatability was achieved with intra-day (n = 5) and inter-day (n = 5) relative standard deviation (RSD) values of 1.56 and 2.38%, respectively. The good linearity was established for 3-EHG concentration in the broad range from 0.005 to 0.30 mol L^?1, with a coefficient (r) of 0.9992. (S)- and (R)-3-EHG were separated by normal-phase LC after simple derivatization with (R)-(+)-phenylethanamine, ee value (≥95%) of 3-EHG prepared by Lipase B catalyzed hydrolysis of diethyl 3-hydroxyglutatate (3-DHG) was determined after optimization of the mobile phase, and the RSD was 0.75% (n = 9) for repeatability. The results showed that the above methods were highly reproducible and reliable for analysis and separation of (S)-3-EHG from bioconversion mixture.     

Studies related to the origin of C18 neutral steroids isolated from extracts of urine from the male horse: the identification of urinary 19-oic acids and their decarboxylation to produce estr-4-en-17beta-ol-3-one (19-nortestosterone) and estr-4-ene-3,17-dione (19-norandrost-4-ene-3,17-dione) during sample processing

For almost two decades we have known that enzymatic hydrolysis of "normal" urine samples from the entire male horse using Escherichia coli (E. coli) followed by solvolysis (ethyl acetate:methanol:sulphuric acid) results in the detection of significant amounts of estr-4-ene-3,17-dione (19-norandrost-4-ene-3,17-dione) along with estr-4-en-17beta-ol-3-one (19-nortestosterone, nandrolone) in extracts of the hydrolysed urine and that both steroids are isolated from the solvolysis fraction. This solvolysis process is targeted at the steroid sulphates. Also we have shown that 19-norandrost-4-ene-3,17-dione and 19-nortestosterone are isolated from testicular tissue extracts. Subsequently, evidence was obtained that 19-nortestosterone detected in extracts of "normal" urine from male horses may not be derived from the 17beta-sulphate conjugate. However, following administration of 19-nortestosterone based proprietary anabolic steroids to all horses (males, females and castrates), the urinary 19-nortestosterone arising from the administration is excreted primarily as the 17beta-sulphate conjugate. Thus, if the 19-nortestosterone-17beta-sulphate conjugate arises only following administration this has interesting implications for drug surveillance programmes to control administration of 19-nortestosterone based anabolic preparations to male horses. These results have led us to consider that the precursors to 19-nortestosterone and 19-norandrost-4-ene-3,17-dione, present in the urine prior to the hydrolysis steps, have the same basic structure except for the functionality at the 17-position. We have used preparative high pressure liquid chromatography (LC) and LC fractionation to separate these precursors from the high amounts of oestrogenic sulphates present in "normal" urine from the entire male horse. Purified fractions have then been studied by liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) to identify the precursors.     

Degradation of deoxycholic acid by Pseudomonas Sp. NCIB 10590

The microbial degradation of deoxycholic acid 1 by Pseudomonas NCIB 10590 has been studied and two major products have been isolated and identified as 12β-hydroxyandrosta-1,4-dien-3,17-dione 2 and 12α-hydroxypregna-1,4-dien-3-one-20-carboxylic acid 9. Three minor products were isolated and evidence is given for the following structures: 12α-hydroxyandrosta-1,4-dien-3,17-dione 4, 12β-hydroxyandrosta-4-en-3,17-dione 7 and 12?, 17?-dihydroxyandrosta-1,4-dien-3-one 8.     

The ultimate veal calf reference experiment: hormone residue analysis data obtained by gas and liquid chromatography tandem mass spectrometry

A lifetime controlled reference experiment has been performed using 42 veal calves, 21 males and 21 females which were fed and housed according to European regulations and common veterinary practice. During the experiment feed, water, urine and hair were sampled and feed intake and growth were monitored. Thus for the first time residue analysis data were obtained from guaranteed lifetime-untreated animals. The analysis was focused on the natural hormones estradiol and testosterone and their metabolites, on 17beta- and 17alpha-nortestosterone, on 17beta- and 17alpha-boldenone and androsta-1,4-diene-3,17-dione (ADD), and carried out by gas chromatography tandem mass spectrometry (GC/MS/MS), an estrogen bioassay and liquid chromatography (LC) MS/MS. Feed, water and hair samples were negative for the residues tested. Female calf urines showed occasionally low levels of 17alpha-estradiol and 17alpha-testosterone. On one particular sampling day male veal calf urines showed very high levels of 17alpha-testosterone (up to 1000 ng mL(-1)), accompanied by lower levels of estrone and 17beta-testosterone. Despite these extreme levels of natural testosterone, 17beta-boldenone was never detected in the same urine samples; even 17alpha-boldenone and ADD were only occasionally beyond CCalpha (maximum levels 2.7 ng mL(-1)). The data from this unique experiment provide a set of reference values for steroid hormones in calf urine and demonstrate that 17beta-boldenone is not a naturally occurring compound in urine samples.     

Improvement of Strain Penicillium sp. EZ-ZH190 for Tannase Production by Induced Mutation

In the search for an efficient producer of tannase, Penicillium sp. EZ-ZH190 was subjected to mutagenesis using heat treatment and strain EZ-ZH290 was isolated. The maximum tannase in this mutant strain was 4.32 U/mL with an incubation period of 84 h as compared to wild strain EZ-ZH190 where the incubation period was 96 h with a maximum enzyme activity of 4.33 U/mL. Also, the Penicillium sp. EZ-ZH290 tannase had a maximum activity at 40 °C and pH 5.5. Then, the spores of strain EZ-ZH290 were subjected to γ irradiation mutagenesis and strain EZ-ZH390 was isolated. Strain EZ-ZH390 exhibited higher tannase activity (7.66 U/mL) than the parent strain EZ-ZH290. It was also found that Penicillium sp. EZ-ZH390 tannase had an optimum activity at 35 °C and a broad pH profile with an optimum at pH 5.5. The tannase pH stability of Penicillium sp. EZ-ZH390 and its maximum production of tannase followed the same trend for five generations confirming the occurrence of stable mutant. This paper is shown that γ irradiation can mutate the Penicillium sp. leading to increase the tannase production.     

Selection of a Thermotolerant Kluyveromyces marxianus Strain with Potential Application for Cellulosic Ethanol Production by Simultaneous Saccharification and Fermentation

The development of technologies for cellulosic ethanol production by simultaneous saccharification and fermentation (SSF) depends on the use of microorganisms with high fermentative rates and thermotolerance. In this study, the ability of five Kluyveromyces marxianus strains to produce ethanol from glucose at 45 °C was investigated. The highest fermentative parameters were observed with K. marxianus NRRL Y-6860, which was then further studied. An initial evaluation of the oxygen supply on ethanol production by the selected yeast and a comparison of SSF process from acid pretreated rice straw between K. marxianus NRRL Y-6860 and Saccharomyces cerevisiae at 30 and 45 °C were carried out. Under the lowest evaluated conditions of aeration and agitation, K. marxianus NRRL Y-6860 produced 21.5 g/L ethanol from 51.3 g/L glucose corresponding to Y_P/S of 0.44 g/g and Q_P of 3.63 g/L h. In the SSF experiments, K. marxianus NRRL Y-6860 was more efficient than S. cerevisiae at both evaluated temperatures (30 and 45 °C), attained at the highest temperature an ethanol yield of 0.24 g/g and productivity of 1.44 g/L h.