High-Efficiency Carbohydrate Fermentation to Ethanol at Temperatures above 40 degrees C by Kluyveromyces marxianus var. marxianus Isolated from Sugar Mills

A number of yeast strains, isolated from sugar cane mills and identified as strains of Kluyveromyces marxianus var. marxianus, were examined for their ability to ferment glucose and cane syrup to ethanol at high temperatures. Several strains were capable of rapid fermentation at temperatures up to 47 degrees C. At 43 degrees C, >6% (wt/vol) ethanol was produced after 12 to 14 h of fermentation, concurrent with retention of high cell viability (>80%). Although the type strain (CBS 712) of K. marxianus var. marxianus produced up to 6% (wt/vol) ethanol at 43 degrees C, cell viability was low, 30 to 50%, and the fermentation time was 24 to 30 h. On the basis of currently available strains, we suggest that it may be possible by genetic engineering to construct yeasts capable of fermenting carbohydrates at temperatures close to 50 degrees C to produce 10 to 15% (wt/vol) ethanol in 12 to 18 h with retention of cell viability.     

Thermotolerant single cell protein production byKluyveromyces marxianus var.marxianus

Summary Amino acid analyses were undertaken on single cell protein (SCP) produced by thermotolerant strains ofKluyveromyces marxianus var.marxianus grown on sugar cane molasses at 40°C. The maximum conversion of available sugars to biomass at 45°C was only 10.8% (g dry wt.·g^–1 total sugars). The amino acid composition of the SCP did not differ markedly from that reported for other yeast species.     

Ethanol Production by Thermophilic Bacteria: Physiological Comparison of Solvent Effects on Parent and Alcohol-Tolerant Strains of Clostridium thermohydrosulfuricum

The effects of temperature, solvents, and cultural conditions on the fermentative physiology of an ethanol-tolerant (56 g/liter at 60°C) and parent strain of Clostridium thermohydrosulfuricum were compared. An ethanol-tolerant mutant was selected by successive transfer of the parent strain into media with progressively higher ethanol concentrations. Physiological differences noted in the mutant included enhanced growth, tolerance to various solvents, and alterations in the substrate range and the fermentation end product ratio. Ethanol tolerance was temperature dependent in the mutant but not in the parent strain. The mutant grew with ethanol concentrations up to 8.0% (wt/vol) at 45°C, but only up to 3.3% (wt/vol) at 68°C. Low ethanol concentration (0.2 to 1.6% [wt/vol]) progressively inhibited the parent strain to where glucose was not fermented at 2.0% (wt/vol) ethanol. Both strains grew and produced alcohols on glucose complex medium at 60°C in the presence of either 5% methanol or acetone, and these solvents when added at low concentration stimulated fermentative metabolism. The mutant produced ethanol at high concentrations and displayed an ethanol/glucose ratio (mole/mole) of 1.0 in media where initial ethanol concentrations were ≤4.0% (wt/vol), whereas when ethanol concentration was changed from 0.1% to 1.6% (wt/vol), the ethanol/glucose ratio for the parent strain changed from 1.6 to 0.6. These data indicate that C. thermohydrosulfuricum strains are tolerant of solvents and that low ethanol tolerance is not a result of disruption of membrane fluidity or glycolytic enzyme activity.     

Ethanol Inhibition Kinetics of Kluyveromyces marxianus Grown on Jerusalem Artichoke Juice

The kinetics of ethanol inhibition on cell growth and ethanol production by Kluyveromyces marxianus UCD (FST) 55-82 were studied during batch growth. The liquid medium contained 10% (wt/vol) inulin-type sugars derived from an extract of Jerusalem artichoke (Helianthus tuberosus) tubers, supplemented with small amounts of Tween 80, oleic acid, and corn steep liquor. Initial ethanol concentrations ranging from 0 to 80 g/liter in the liquid medium were used to study the inhibitory effect of ethanol on the following parameters: maximum specific growth rate (μ_max), cell and ethanol yields, and sugar utilization. It was found that as the initial ethanol concentration increased from 0 to 80 g/liter, and maximum specific growth rate of K. marxianus cells decreased from 0.42 to 0.09 h⁻¹, whereas the ethanol and cell yields and sugar utilization remained almost constant. A simple kinetic model was used to correlate the μ_max results and the rates of cell and ethanol production, and the appropriate constants were evaluated.     

Fungal Invertase as an Aid for Fermentation of Cane Molasses into Ethanol

Comparative studies of the fermentation of cane molasses into ethanol by Saccharomyces cerevisiae in the presence or absence of fungal invertase were performed. When cane molasses was fermented by the yeast at 30°C and pH 5.0, the presence of the enzyme had no effect on ethanol production. At pH 3.5, ethanol production was increased by the addition of invertase. At 40°C, the addition of invertase increased ethanol production by 5.5% at pH 5.0 and by 20.9% at pH 3.5.     

Fermentation Temperature Modulates Phosphatidylethanolamine and Phosphatidylinositol Levels in the Cell Membrane of Saccharomyces cerevisiae

During alcoholic fermentation, Saccharomyces cerevisiae is exposed to a host of environmental and physiological stresses. Extremes of fermentation temperature have previously been demonstrated to induce fermentation arrest under growth conditions that would otherwise result in complete sugar utilization at “normal” temperatures and nutrient levels. Fermentations were carried out at 15°C, 25°C, and 35°C in a defined high-sugar medium using three Saccharomyces cerevisiae strains with diverse fermentation characteristics. The lipid composition of these strains was analyzed at two fermentation stages, when ethanol levels were low early in stationary phase and in late stationary phase at high ethanol concentrations. Several lipids exhibited dramatic differences in membrane concentration in a temperature-dependent manner. Principal component analysis (PCA) was used as a tool to elucidate correlations between specific lipid species and fermentation temperature for each yeast strain. Fermentations carried out at 35°C exhibited very high concentrations of several phosphatidylinositol species, whereas at 15°C these yeast strains exhibited higher levels of phosphatidylethanolamine and phosphatidylcholine species with medium-chain fatty acids. Furthermore, membrane concentrations of ergosterol were highest in the yeast strain that experienced stuck fermentations at all three temperatures. Fluorescence anisotropy measurements of yeast cell membrane fluidity during fermentation were carried out using the lipophilic fluorophore diphenylhexatriene. These measurements demonstrate that the changes in the lipid composition of these yeast strains across the range of fermentation temperatures used in this study did not significantly affect cell membrane fluidity. However, the results from this study indicate that fermenting S. cerevisiae modulates its membrane lipid composition in a temperature-dependent manner.     

High-Temperature Ethanol Fermentation and Transformation with Linear DNA in the Thermotolerant Yeast Kluyveromyces marxianus DMKU3-1042

We demonstrate herein the ability of Kluyveromyces marxianus to be an efficient ethanol producer and host for expressing heterologous proteins as an alternative to Saccharomyces cerevisiae. Growth and ethanol production by strains of K. marxianus and S. cerevisiae were compared under the same conditions. K. marxianus DMKU3-1042 was found to be the most suitable strain for high-temperature growth and ethanol production at 45°C. This strain, but not S. cerevisiae, utilized cellobiose, xylose, xylitol, arabinose, glycerol, and lactose. To develop a K. marxianus DMKU3-1042 derivative strain suitable for genetic engineering, a uracil auxotroph was isolated and transformed with a linear DNA of the S. cerevisiae ScURA3 gene. Surprisingly, Ura⁺ transformants were easily obtained. By Southern blot hybridization, the linear ScURA3 DNA was found to have inserted randomly into the K. marxianus genome. Sequencing of one Lys⁻ transformant confirmed the disruption of the KmLYS1 gene by the ScURA3 insertion. A PCR-amplified linear DNA lacking K. marxianus sequences but containing an Aspergillus α-amylase gene under the control of the ScTDH3 promoter together with an ScURA3 marker was subsequently used to transform K. marxianus DMKU3-1042 in order to obtain transformants expressing Aspergillus α-amylase. Our results demonstrate that K. marxianus DMKU3-1042 can be an alternative cost-effective bioethanol producer and a host for transformation with linear DNA by use of S. cerevisiae-based molecular genetic tools.     

Inhibition of Yeast Growth by Octanoic and Decanoic Acids Produced during Ethanolic Fermentation

The inhibition of growth by octanoic or decanoic acids, two subproducts of ethanolic fermentation, was evaluated in Saccharomyces cerevisiae and Kluyveromyces marxianus in association with ethanol, the main product of fermentation. In both strains, octanoic and decanoic acids, at concentrations up to 16 and 8 mg/liter, respectively, decreased the maximum specific growth rate and the biomass yield at 30°C as an exponential function of the fatty acid concentration and increased the duration of growth latency. These toxic effects increased with a decrease in pH in the range of 5.4 to 3.0, indicating that the undissociated form is the toxic molecule. Decanoic acid was more toxic than octanoic acid. The concentrations of octanoic and decanoic acids were determined during the ethanolic fermentation (30°C) of two laboratory media (mineral and complex) by S. cerevisiae and of Jerusalem artichoke juice by K. marxianus. Based on the concentrations detected (0.7 to 23 mg/liter) and the kinetics of growth inhibition, the presence of octanoic and decanoic acids cannot be ignored in the evaluation of the overall inhibition of ethanolic fermentation.     

Ethanol from Sugar Cane: Flask Experiments Using the EX-FERM Technique

Alcohol production at the laboratory scale from sugar cane pieces by the EX-FERM technique was studied with 37 strains of Saccharomyces spp. The EX-FERM process is novel in that it employs the simultaneous extraction and fermentation of the sucrose in a cane-water suspension. Two types of cane treatments were used: chips and shredded pith, either fresh or dried. A mother culture of the yeast was prepared in enriched cane juice and then added to the cane-water mixture. After static fermentation for 40 h at 30°C, the cane was removed, and fresh cane was added to the yeast-alcohol broth. After an additional 24 h, the cane was again removed and the liquor was analyzed. After the first 40-h cycle, sugar consumption was above 99% with 10 of the 37 yeast strains tested, and ethanol reached levels of 1.29 to 4.00 g per 100 ml, depending on the yeast strain. The final ethanol concentration reached 4.27 to 5.37 g per 100 ml, and sugar consumption was above 98% in three cases during a second EX-FERM cycle employing previously air-dried chips and pith. Product yields were within accepted values. Cane treatment did not appear to affect the results at this level.     

Ethanol Production from Colpomenia sinuosa by an Alginate Fermentation Strain Meyerozyma guilliermondii

With the consumption of energy and the spread of COVID-19, the demand for ethanol production is increasing in the world. The industrial ethanol fermentation microbes cannot metabolize the alginate component of macro algae, which affects the ethanol yield. In this research, the ethanol production process from macro algae by an alginate fermentation yeast Meyerozyma guilliermondii, especially the pretreatment process of Colpomenia sinuosa, was studied. At the same time, the experimental design of Box-Behnken was carried out to achieve the optimum fermentation performance. The concentration of KH₂PO₄ (A: 2–6 g.L⁻¹), pH (B: 4–7), reaction time (C: 60–120 h) and temperature (D: 24–34 °C) were variable input parameters. During the ethanol production process, the algae powder was firstly mixed with water at 90 °C for 0.5 h. Later the fermentation culture medium was prepared and then it was fermented by the yeast Meyerozyma guilliermondii to produce ethanol. And the optimal fermentation parameters were as follows: fermentation temperature of 28 °C, KH₂PO₄ dosage of 4.7 g.L⁻¹, initial pH of 6, and fermentation time of 99 h. The ethanol yield reached 0.268 g.g⁻¹ (ethanol to algae), close to the predicted value of model. The generation of alginate lyase during the fermentation of algae was also examined. The highest alginate lyase activity reached 46.42 U.mL⁻¹.     

Production of ethanol at high temperatures in the fermentation of Jerusalem artichoke juice and a simple medium byKluyveromyces marxianus

Summary Temperatures as high as 36°C and 40°C did not negatively affect the ethanol productivity of Jerusalem artichoke (J.a.) juice batch fermentation and the final concentrations of ethanol were close to those produced at lower temperatures. At higher process temperatures (36–40°C), ethanol toxicity inKluyveromyces marxianus was less important during the fermentation of J.a. juice as compared with a simple medium. In simple medium, the heat-sticking of fermentation was observed and the percentage of unfermented sugars steeply increased from 28°C up to 40°C.     

Production of Aseptic Spores of Vesicular-Arbuscular Endophytes and Their Viability After Chemical and Physical Stress

The survival of germinating spores of vesicular-arbuscular endophytes after treatments with oxidizing agents, antibiotics, moist heat, ultrasonic radiation, and ultraviolet radiation was compared with that of their contaminating microbes. Spores of three species were rapidly decontaminated by treatment with 0.42% (wt/vol) chlorine available from 5.0% (wt/vol) chloramine-T at 30°C for 20 to 40 min depending on the species and the soil from which they were extracted. This treatment did not change spore viability. The survival of spores was reduced by exposure for 20 min to 1.11% chlorine at 30°C for Glomus caledonius or at 35°C for Acaulospora laevis. Growth of any bacteria surviving treatment with oxidizing agents was inhibited by 100 μg of chloramphenicol per ml in agar; however, spore germination and germ tube growth were reduced only by concentrations greater than 200 μg/ml in agar. Spore germination was decreased by concentration of pimaracin, which controlled fungal growth. The spores survived moist heat at 40°C for 80 min, 55°C for 10 min, and 60°C for less than 1 min. The viability of spores was unaffected by ultrasonic irradiation for up to 4 min. Spores of G. caledonius and A. laevis were extremely resistant to ultraviolet radiation. Their viability was unaffected by exposure to 5 × 10⁸ ergs cm⁻² from an ultraviolet source of 253.7nm. The spores had very thick, pigmented walls, and the possibility that these provided some protection against the physical and chemical treatments is discussed. The degree of physiological damage to the spores caused by the treatments demonstrated some adverse effects of basic laboratory procedures. This information, together with that on the comparative sensitivity of contaminating microbes to the treatments, was used in the development of protocol for producing large numbers of uncontaminated spores.     

Effects of Temperature on Bacterial Communities and Metabolites during Fermentation of Myeolchi-Aekjeot,a Traditional Korean Fermented Anchovy Sauce

Myeolchi-aekjeot (MA) in Korea is produced outdoors without temperature controls, which is a major obstacle to produce commercial MA products with uniform quality. To investigate the effects of temperature on MA fermentation, pH, bacterial abundance and community, and metabolites were monitored during fermentation at 15°C, 20°C, 25°C, and 30°C. Initial pH values were approximately 6.0, and pH values increased after approximately 42 days, with faster increases at higher temperatures. Bacterial abundances increased rapidly in all MA samples after quick initial decreases during early fermentation and then they again steadily decreased after reaching their maxima, which were significantly greater at higher temperatures. Bacterial community analysis revealed that Proteobacteria and Tenericutes were predominant in all initial MA samples, but they were rapidly displaced by Firmicutes as fermentation progressed. Photobacterium and Mycoplasma belonging to Proteobacteria and Tenericutes, respectively, which may include potentially pathogenic strains, were dominant in initial MA, but decreased with the growth of Chromohalobacter, which occurred faster at higher temperatures––they were dominant until 273 and 100 days at 15°C and 20°C, respectively, but not detected after 30 days at 25°C and 30°C. Chromohalobacter also decreased with the appearance of subsequent genera belonging to Firmicutes in all MA samples. Tetragenococcus, halophilic lactic acid bacteria, appeared predominantly at 20°C, 25°C, and 30°C; they were most abundant at 30°C, but not detected at 15°C. Alkalibacillus and Lentibacillus appeared as dominant genera with the decrease of Tetragenococcus at 25°C and 30°C, but only Lentibacillus was dominant at 15°C and 20°C. Metabolite analysis showed that amino acids related to tastes were major metabolites and their concentrations were relatively higher at high temperatures. This study suggests that high temperatures (approximately 30°C) may be appropriate in MA fermentation, in the light of faster disappearance of potentially pathogenic genera, higher amino acids, growth of Tetragenococcus, and faster fermentation.     

Intermediate-Scale, Semicontinuous Solid-Phase Fermentation Process for Production of Fuel Ethanol from Sweet Sorghum

A novel, semicontinuous solid-phase fermentation system was used to produce fuel ethanol from sweet sorghum. The process was at an intermediate scale. In the process, dried and shredded sweet sorghum was rehydrated to 70% moisture, acidified to pH 2.0 to 3.0, and either pasteurized (12 h at 70 to 80°C) or not pasteurized before spray inoculation with a broth culture of Saccharomyces cerevisiae. Fermented pulp exited the semicontinuous fermentor after a retention time of 72 h and contained approximately 6% (vol/vol) ethanol. Ethanol yields from dry sweet sorghum were 176 to 179 liters/10³ kg (85% of theoretical). Production costs for a greatly scaled-up (×1,400) conceptual version of this system were projected by calculation to average $0.47/liter for 95% ethanol. The calculated energy balance (energy output/energy input ratio) was estimated to be 1.05 when pasteurization was included and 1.31 when pasteurization was omitted. In calculating the energy balances, the output energy of the protein feed byproduct and the input energy for growing the sweet sorghum were not considered. A design for the scaled-up plant (farm scale) is provided.     

Production of Thermostable α-Amylase, Pullulanase, and α-Glucosidase in Continuous Culture by a New Clostridium Isolate

The production of α-amylase, pullulanase, and α-glucosidase and the formation of fermentation products by the newly isolated thermophilic Clostridium sp. strain EM1 were investigated in continuous culture with a defined medium and an incubation temperature of 60°C. Enzyme production and excretion were greatly influenced by the dilution rate and the pH of the medium. The optimal values for the formation of starch-hydrolyzing enzymes were a pH of 5.9 and a dilution rate of 0.075 to 0.10 per h. Increase of the dilution rate from 0.1 to 0.3 per h caused a drastic drop in enzyme production. The ethanol concentration and optical density of the culture, however, remained almost constant. Growth limitation in the chemostat with 1% (wt/vol) starch was found optimal for enzyme production. Under these conditions 2,800 U of pullulanase per liter and 1,450 U of α-amylase per liter were produced; the amounts excreted were 70 and 55%, respectively.     

Ethanol-Induced Leakage in Saccharomyces cerevisiae: Kinetics and Relationship to Yeast Ethanol Tolerance and Alcohol Fermentation Productivity

Ethanol stimulated the leakage of amino acids and 260-nm-light-absorbing compounds from cells of Saccharomyces cerevisiae. The efflux followed first-order kinetics over an initial period. In the presence of lethal concentrations of ethanol, the efflux rates at 30 and 36°C were an exponential function of ethanol concentration: k_e^X = k_e^Xme^{E (X-X_m)}, where k_e^X and k_e^Xm are the efflux rate constants, respectively, in the presence of a concentration X of ethanol or the minimal concentration of ethanol, X_m, above which the equation was applicable, coincident with the minimal lethal concentration of ethanol. E is the enhancement constant. At 36°C, as compared with the corresponding values at 30°C, the efflux rates were higher and the minimal concentration of ethanol (X_m) was lower. The exponential constants for the enhancement of the rate of leakage (E) had similar values at 30 or 36°C and were of the same order of magnitude as the corresponding exponential constants for ethanol-induced death. Under isothermic conditions (30°C) and up to 22% (vol/vol) ethanol, the resistance to ethanol-induced leakage of 260-nm-light-absorbing compounds was found to be closely related with the ethanol tolerance of three strains of yeasts, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Saccharomyces bayanus. The resistance to ethanol-induced leakage indicates the possible adoption of the present method for the rapid screening of ethanol-tolerant strains. The addition to a fermentation medium of the intracellular material obtained by ethanol permeabilization of yeast cells led to improvements in alcohol fermentation by S. cerevisiae and S. bayanus. The action of the intracellular material, by improving yeast ethanol tolerance, and the advantages of partially recycling the fermented medium after distillation were discussed.     

Reduction of seminal plasma concentration can decrease detrimental effects of seminal plasma on chilled ram spermatozoa

This study was conducted to investigate the effect of different levels of seminal plasma (SP) and cold-shock on ram spermatozoa during 36 h storage at 5°C. In both ejaculated spermatozoa coated with egg yolk (second ejaculate; coated spermatozoa) and epididymal spermatozoa, samples were treated with 0, 50 and 100% seminal plasma. Different levels of seminal plasma were added on the basis of ram spermatocrit (32%). Then half of aliquots were suddenly put on ice water (cold-shock) and other half were gradually (0.25°C/min) chilled (non- cold shock). Sperm motility, viability and functional membrane integrity were determined in both aliquots at 0, 12, 24 and 36 h storage at 5°C. Under non- cold shock and cold-shock conditions, coated spermatozoa treated with 0% SP showed the highest motility compared to ejaculated spermatozoa (first ejaculate; uncoated spermatozoa) after 12, 24 and 36 h of storage at 5°C (P<0.05). Under non- cold shock and cold-shock conditions, viability and functional membrane integrity was higher in the coated spermatozoa treated with 0% SP than in the uncoated spermatozoa during 36 h storage (P<0.05). There was no significant difference between coated spermatozoa treated with 0 and 50% SP in the percentage of motility and viability after 24 and 36 h of storage (P>0.05). Under non- cold shock and cold-shock conditions, the percentage of motility of epididymal spermatozoa treated with 0% SP was significantly (P<0.05) higher than those treated with 100% SP after 36 h of storage at 5°C. In conclusion, removal of seminal plasma and/or reduction (up to 50%) of its concentration can decrease detrimental effects of seminal plasma on chilled ram spermatozoa.     

Respiratory capacity of the Kluyveromyces marxianus yeast isolated from the mezcal process during oxidative stress

During the mezcal fermentation process, yeasts are affected by several stresses that can affect their fermentation capability. These stresses, such as thermal shock, ethanol, osmotic and growth inhibitors are common during fermentation. Cells have improved metabolic systems and they express stress response genes in order to decrease the damage caused during the stress, but to the best of our knowledge, there are no published works exploring the effect of oxidants and prooxidants, such as H₂O₂ and menadione, during growth. In this article, we describe the behavior of Kluyveromyces marxianus isolated from spontaneous mezcal fermentation during oxidative stress, and compared it with that of Saccharomyces cerevisiae strains that were also obtained from mezcal, using the W303-1A strain as a reference. S. cerevisiae strains showed greater viability after oxidative stress compared with K. marxianus strains. However, when the yeast strains were grown in the presence of oxidants in the media, K. marxianus exhibited a greater ability to grow in menadione than it did in H₂O₂. Moreover, when K. marxianus SLP1 was grown in a minibioreactor, its behavior when exposed to menadione was different from its behavior with H₂O₂. The yeast maintained the ability to consume dissolved oxygen during the 4 h subsequent to the addition of menadione, and then stopped respiration. When exposed to H₂O₂, the yeast stopped consuming oxygen for the following 8 h, but began to consume oxygen when stressors were no longer applied. In conclusion, yeast isolated from spontaneous mezcal fermentation was able to resist oxidative stress for a long period of time.     

Direct ethanol production from cellulosic materials at high temperature using the thermotolerant yeast Kluyveromyces marxianus displaying cellulolytic enzymes

To exploit cellulosic materials for fuel ethanol production, a microorganism capable of high temperature and simultaneous saccharification–fermentation has been required. However, a major drawback is the optimum temperature for the saccharification and fermentation. Most ethanol-fermenting microbes have an optimum temperature for ethanol fermentation ranging between 28 °C and 37 °C, while the activity of cellulolytic enzymes is highest at around 50 °C and significantly decreases with a decrease in temperature. Therefore, in the present study, a thermotolerant yeast, Kluyveromyces marxianus, which has high growth and fermentation at elevated temperatures, was used as a producer of ethanol from cellulose. The strain was genetically engineered to display Trichoderma reesei endoglucanase and Aspergillus aculeatus β-glucosidase on the cell surface, which successfully converts a cellulosic β-glucan to ethanol directly at 48 °C with a yield of 4.24 g/l from 10 g/l within 12 h. The yield (in grams of ethanol produced per gram of β-glucan consumed) was 0.47 g/g, which corresponds to 92.2% of the theoretical yield. This indicates that high-temperature cellulose fermentation to ethanol can be efficiently accomplished using a recombinant K. marxianus strain displaying thermostable cellulolytic enzymes on the cell surface.     

Ethanol fermentation with Kluyveromyces marxianus from Jerusalem artichoke grown in salina and irrigated with a mixture of seawater and freshwater

Aims: To study fuel ethanol fermentation with Kluyveromyces marxianus ATCC8554 from Jerusalem artichoke (Helianthus tuberosus) grown in salina and irrigated with a mixture of seawater and freshwater. Methods and Results: The growth and ethanol fermentation of K. marxianus ATCC8554 were studied using inulin as substrate. The activity of inulinase, which attributes to the hydrolysis of inulin, the main carbohydrate in Jerusalem artichoke, was monitored. The optimum temperatures were 38°C for growth and inulinase production, and 35°C for ethanol fermentation. Aeration was not necessary for ethanol fermentation with the K. marxianus from inulin. Then, the fresh Jerusalem artichoke tubers grown in salina and irrigated with 25% and 50% seawater were further examined for ethanol fermentation with the K. marxianus, and a higher ethanol yield was achieved for the Jerusalem artichoke tuber irrigated with 25% seawater. Furthermore, the dry meal of the Jerusalem artichoke tubers irrigated with 25% seawater was examined for ethanol fermentation at three solid concentrations of 200, 225 and 250 g l^?1, and the highest ethanol yield of 0·467, or 91·5% of the theoretical value of 0·511, was achieved for the slurry with a solid concentration of 200 g l^?1. Conclusions: Halophilic Jerusalem artichoke can be used for fuel ethanol production. Significance and Impact of the Study: Halophilic Jerusalem artichoke, not competing with grain crops for arable land, is a sustainable feedstock for fuel ethanol production.