Ethanol production from Jerusalem artichoke juice using flocculent cells of Kluyveromyces marxianus

Summary Flocculent cells ofKluyveromyces marxianus SM 16-10 were used for batch production of ethanol from the inulin sugars derived from Jerusalem artichoke tubers. Using 20% initial sugar concentration, a maximum ethanol concentration of 92 g/l was achieved in 7 h, when the flocculent cell concentration was 30 g dry wt./l bioreactor volume. The same flocculent cells were used repeatedly for 7 batch runs starting with fresh medium at the beginning of each run. The ethanol yield was found to be almost constant at about 94% of the theoretical for all the 7 batch cycles, while the maximum ethanol production rate increased from 17.21 g ethanol/1/h during the first batch run to 21 g ethanol/1/h during the last batch run.     

Kinetics of ethanol production from Jerusalem artichoke juice with some Kluyveromyces species

Summary The kinetics of ethanol production by Kluyveromyces marxianus ATCC 12708 and ATCC 10606, K. cicerisporus ATCC 22295 and K. fragilis 105 have been studied using raw juice of the Jerusalem artichoke in which the carbohydrates were not hydrolysed prior to fermentation. The experiments revealed that this juice contains enough nutrients and can serve as a complete medium without additional nutrients both for growth of the yeasts and for ethanol production. It was found that both specific ethanol productivity and specific glucose uptake rates were the highest with K. marxianus ATCC 12708 (1.68 gg^-1h^-1 and 3.78 gg^-1h^-1 respectively). This microorganism produced an ethanol yield of 87.5% of the theoretical value in 25 hours.     

Continuous ethanol production from Jerusalem artichoke tubers. II. Use of immobilized cells of Kluyveromyces marxianus

Kluyveromyces marxianus UCD (FST) 55-82 cells were immobilized in Na alginate beads and used in a packed-bed bioreactor system for the continuous production of ethanol from the extract of Jerusalem artichoke tubers. Volumetric ethanol productivities of 104 and 80 g ethanol/ L/h were obtained at 80 and 92% sugar utilization, respectively. The maximum volumetric ethanol productivity of the immobilized cell bioreactor system was found to be 15 times higher than that of an ordinary-stirred-tank (CST) bioreactor using cells of K. marxianus. The immobilized cell bioreactor system was operated continuously at a constant dilution rate of 0.66 h(-1) for 12 days resulting in only an 8% loss of the original immobilized cell activity, which corresponds to an estimated half-life of ca. 72 days. The maximum specific ethanol productivity and maximum specific sugar uptake rate of the immobilized cells were found to be 0.55 g ethanol/g/biomass/h and 1.21 g sugars/g biomass/h, respectively.     

The effect of temperature and pH on ethanol production by free and immobilized cells of Kluyveromyces marxianus grown on Jerusalem artichoke extract

The effect of temperature and pH on the kinetics of ethanol production by free and calcium alginate immobilized cells of Kluyveromyces marxianus grown on Jerusalem artichoke extract was investigated. With the free cells, the ethanol and biomass yields were relatively constant over the temperature range 25-35 degrees C, but dropped sharply beyond 35 degrees C. Other kinetic parameters, specific growth rate, specific ethanol production rate, and specific total sugar uptake rate were maximum at 35 degrees C. However, with the immobilized cells, ethanol yield remained almost constant in the temperature range 25-45 degrees C, and the specific ethanol production rate and specific total sugar uptake rate attained their maximum values at 40 degrees C. For the pH range between 3 and 7, the free-cell optimum for growth and product formation was found to be ca. pH 5. At this pH, the specific growth rate was 0.35 h(-1) and specific ethanol production rate was 2.83 g/g/h. At values higher or lower than pH 5, a sharp decrease in specific ethanol production rate as well as specific growth rate was observed. In comparison, the immobilized cells showed a broad optimum pH profile. The best ethanol production rates were observed between pH 4 and 6.     

Improved ethanol production in Jerusalem artichoke tubers by overexpression of inulinase gene in Kluyveromyces marxianus

Ethanol production from Jerusalem artichoke tubers through a consolidated bioprocessing (CBP) strategy using the inulinase-producing yeast Kluyveromyces marxianus is an economical and competitive than that from a grainbased feedstock. However, poor inulinase production under ethanol fermentation conditions significantly prolongs the fermentation time and compromises ethanol productivity. Improvement of inulinase activity appears to be promising for increasing ethanol production from Jerusalem artichoke tubers by CBP. In the present study, expression of the inulinase gene INU with its own promoter in K. marxianus (K/INU2) was explored using the integrative cassette. Overexpression of INU was explored using chromosome integration via the HO locus of the yeast. Inulinase activity and ethanol were determined from inulin and Jerusalem artichoke tubers under fed-batch operation. Inulinase activity was 114.9 U/mL under aerobic conditions for K/INU2, compared with 52.3 U/mL produced by the wild type strain. Importantly, inulinase production was enhanced in K/INU2 under ethanol fermentation conditions. When using 230 g/L inulin and 220 g/L Jerusalem artichoke tubers as substrates, inulinase activities of 3.7 and 6.8 U/mL, respectively, were measured using K/INU2, comparing favorably with 2.4 and 3.1 U/mL, respectively, using the wide type strain. Ethanol concentration and productivity for inulin were improved by the recombinant yeast to 96.2 g/L and 1.34 g/L/h, respectively, vs 93.7 g/L and 1.12 g/L/h, respectively, by the wild type strain. Ethanol concentration and productivity improvements for Jerusalem artichoke tubers were 69 g/L and 1.44 g/L/h, respectively, from the recombinant strain vs 62 g/L and 1.29 g/L/h, respectively, from the wild type strain.     

Continuous ethanol production from Jerusalem artichoke tubers. I. Use of free cells of Kluyveromyces marxianus

The Continuous fermentation of Jerusalem artichoke juice to ethanol by free cells of Kluyveromyces marxianus UCD (FST) 55-82 has been studied in a continuous-stirred-tank bioreactor at 35 degrees C and pH 4.6. A maximum yield of 90% of the theoretical was obtained at a dilution rate of 0.05 h(-1). About 95% of the sugars were utilized at dilution rates lower than 0.15 h(-1). Volumetric ethanol productivity and volumetric biomass productivity reached maximum values of 7 g ETOH/L/h and 0.6 g dry wt/L/h, respectively, at a dilution rate of 0.2 h(-1). The maintenance energy coefficient for K. marxianus culture was found to be 0.46 g sugar/g biomass/h/ Oscillatory behavior was following a change in dilution rate from a previous steady state and from batch to continuous culture. Values of specific ethanol production rate and specific sugar uptake were found to increase almost linearly with the increase of the dilution rate. The maximum specific ethanol production rate and maximum specific sugar uptake rate were found to be 2.6 g ethanol/g/ cell/h and 7.9 sugars/g cell/h, respectively. Washout occurred at a dilution rate of 0.41 h(-1).     

Kinetics of ethanol production by immobilized cells of Zymomonas mobilis at varying d-glucose concentrations

Ethanol fermentation by cells of Zymomonas mobilis immobilized in calcium alginate gel has been studied using 5 to 30 wt% initial d-glucose in the medium. Up to 27% d-glucose was completely fermented and the maximum ethanol concentration of 12.6% (w/v) was obtained using an immobilized cell concentration of 58 g dry wt l^?1 of bead volume. The ethanol yield coefficient was almost unaffected by initial d-glucose concentration and its value was >95% of theoretical. The rates of ethanol production and d-glucose utilization first increased, with an increase in initial d-glucose concentration up to 13.6%, and then started to decrease upon a further increase in initial d-glucose concentration. Cell leakage from the calcium alginate beads was very low.     

Repeated batch production of ethanol from Jerusalem artichoke tubers using recycled immobilized cells of Kluyveromyces fragilis

Summary Recycled immobilized cells of Kluyveromyces fragilis ATCC 28244 were used for repeated batch production of ethanol from the inulin sugars derived from Jerusalem artichoke tubers. Using 10% initial sugar concentration, a maximum ethanol concentration of 48 g/l was achieved in 7 h when the immobilized cell concentration in the Ca alginate beads was 72 g dry wt. immobilized cell/l bead volume. The maximum ethanol production rate was 13.5 g ethanol/l bioreactor volume/h. The same Ca alginate beads containing the cells were used repeatedly for 11 batch runs starting with fresh medium at the beginning of each run. The ethanol yield was found to be almost constant at 96% of the theoretical for all 11 batch runs, while the maximum ethanol production rate during the last batch run was found to be 70% of the original ethanol rate obtained in the first batch run.     

Consolidated bioprocessing strategy for ethanol production from Jerusalem artichoke tubers by Kluyveromyces marxianus under high gravity conditions

Aims: Developing an innovative process for ethanol fermentation from Jerusalem artichoke tubers under very high gravity (VHG) conditions. Methods and Results: A consolidated bioprocessing (CBP) strategy that integrated inulinase production, saccharification of inulin contained in Jerusalem artichoke tubers and ethanol production from sugars released from inulin by the enzyme was developed with the inulinase‐producing yeast Kluyveromyces marxianus Y179 and fed‐batch operation. The impact of inoculum age, aeration, the supplementation of pectinase and nutrients on the ethanol fermentation performance of the CBP system was studied. Although inulinase activities increased with the extension of the seed incubation time, its contribution to ethanol production was negligible because vigorously growing yeast cells harvested earlier carried out ethanol fermentation more efficiently. Thus, the overnight incubation that has been practised in ethanol production from starch‐based feedstocks is recommended. Aeration facilitated the fermentation process, but compromised ethanol yield because of the negative Crabtree effect of the species, and increases the risk of contamination under industrial conditions. Therefore, nonaeration conditions are preferred for the CBP system. Pectinase supplementation reduced viscosity of the fermentation broth and improved ethanol production performance, particularly under high gravity conditions, but the enzyme cost should be carefully balanced. Medium optimization was performed, and ethanol concentration as high as 94·2 g l^?1 was achieved when 0·15 g l^?1 K₂HPO₄ was supplemented, which presents a significant progress in ethanol production from Jerusalem artichoke tubers. Conclusions: A CBP system using K. marxianus is suitable for efficient ethanol production from Jerusalem artichoke tubers under VHG conditions. Significance and Impact of the Study: Jerusalem artichoke tubers are an alternative to grain‐based feedstocks for ethanol production. The high ethanol concentration achieved using K. marxianus with the CBP system not only saves energy consumption for ethanol distillation, but also significantly reduces the amount of waste distillage discharged from the distillation system.     

Production of fuel ethanol at high temperature from sugar cane juice by a newly isolated Kluyveromyces marxianus

Kluyveromyces marxianus DMKU 3-1042, isolated by an enrichment technique in a sugar cane juice medium supplemented with 4% (w/v) ethanol at 35 degrees C, produced high concentrations of ethanol at both 40 and 45 degrees C. Ethanol production by this strain in shaking flask cultivation in sugar cane juice media at 37 degrees C was highest in a medium containing 22% total sugars, 0.05% (NH(4))(2)SO(4), 0.05% KH(2)PO(4), and 0.15% MgSO(4).7H(2)O and having a pH of 5.0; the ethanol concentration reached 8.7% (w/v), productivity 1.45 g/l/h and yield 77.5% of theoretical yield. At 40 degrees C, a maximal ethanol concentration of 6.78% (w/v), a productivity of 1.13 and a yield 60.4% of theoretical yield were obtained from the same medium, except that the pH was adjusted to 5.5. In a study on ethanol production in a 5l jar fermenter with an agitation speed of 300 rpm and an aeration rate of 0.2 vvm throughout the fermentation, K. marxianus DMKU 3-1042 yielded a final ethanol concentration of 6.43% (w/v), a productivity of 1.3g/l/h and a yield of 57.1% of theoretical yield.     

一步法发酵菊芋生产乙醇

利用马克斯克鲁维酵母(Kluyveromyces marxianus)YX01具有菊粉酶生产能力且乙醇发酵性能良好的特点,直接发酵菊粉生成乙醇.在摇瓶中考察了该菌株最适发酵温度,进而在2.5L发酵罐中考察了通气量和底物浓度的影响.实验结果表明:该菌株最适发酵温度为35℃;在通气量为50 mL/min和100 mL/min时菌体生长加快,发酵时间缩短,但在不通气条件下糖醇转化率明显提高;在菊粉浓度235 g/L时,发酵终点乙醇浓度达到92.2 g/L,乙醇对糖的得率为0.436,为理论值的85.5%.在此基础上,使用近海滩涂种植海水灌溉收获的菊芋为底物,以批式补料方式直接发酵菊芋干粉浓度为280 g/L的底物,发酵终点乙醇浓度为84.0 g/L,乙醇对糖的得率为0.405,为理论值的80.0%.这些研究工作,为以菊芋为原料的燃料乙醇技术开发奠定了基础.     

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.     

Continuous production of fructose syrup and ethanol from hydrolysed Jerusalem artichoke juice

Summary The results from this study showed that Jerusalem artichoke juice can be used for the production of very enriched fructose syrup by selective conversion of glucose to ethanol in a continuous process using immobilized cells ofSaccharomyces cerevisiae ATCC 36859. The product contained up to 99% of the total carbohydrates as fructose compared to 76% in the feed. Using Jerusalem artichoke juice supplemented with some glucose a product was obtained with 7.5% w/v ethanol which made ethanol recovery economically favourable. It was found that some fructose was consumed in these continuous processes; the glucose/fructose conversion rate ratio was regulated by the glucose concentration in the product stream.     

Thermotolerant Kluyveromyces marxianus and Saccharomyces cerevisiae strains representing potentials for bioethanol production from Jerusalem artichoke by consolidated bioprocessing

Thermotolerant inulin-utilizing yeast strains are desirable for ethanol production from Jerusalem artichoke tubers by consolidated bioprocessing (CBP). To obtain such strains, 21 naturally occurring yeast strains isolated by using an enrichment method and 65 previously isolated Saccharomyces cerevisiae strains were investigated in inulin utilization, extracellular inulinase activity, and ethanol fermentation from inulin and Jerusalem artichoke tuber flour at 40?°C. The strains Kluyveromyces marxianus PT-1 (CGMCC AS2.4515) and S. cerevisiae JZ1C (CGMCC AS2.3878) presented the highest extracellular inulinase activity and ethanol yield in this study. The highest ethanol concentration in Jerusalem artichoke tuber flour fermentation (200?g?L(-1)) at 40?°C achieved by K. marxianus PT-1 and S. cerevisiae JZ1C was 73.6 and 65.2?g?L(-1), which corresponded to the theoretical ethanol yield of 90.0 and 79.7?%, respectively. In the range of 30 to 40?°C, temperature did not have a significant effect on ethanol production for both strains. This study displayed the distinctive superiority of K. marxianus PT-1 and S. cerevisiae JZ1C in the thermotolerance and utilization of inulin-type oligosaccharides reserved in Jerusalem artichoke tubers. It is proposed that both K. marxianus and S. cerevisiae have considerable potential in ethanol production from Jerusalem artichoke tubers by a high temperature CBP.     

Ethanol production from Jerusalem artichoke tubers (Helianthus tuberosus) using Kluyveromyces marxianus and Saccharomyces rosei

This article examines the potential of Jerusalem artichoke as a source for ethanol and single-cell protein SCP. In addition, experimental results are presented on batch fermentation kinetics employing two strains of Kluyveromyces marxianus and one strain of Saccharomyces rosei grown on the extract derived from the tubers of Jerusalem artichoke. Of the three cultures examined, Kluyveromyces marxianus UCD (FST) 55-82 was found to be the best producer of ethanol grown in a simple medium at 35 degrees C. The ethanol production was found to be growth-associated having a mu(max) = 0.41. h(-1) and the ethanol and biomass yields were determined to be Y(p/s) = 0.45 (88% of the theoretical) and Y(x/s) = 0.04 with 92% of the original sugars utilized. On the basis of carbohydrate yields of Jerusalem artichoke reported in the literature and these batch kinetic studies with K. maxxianus, the calculated ethanol yields were found to range from 1400 kg ethanol acre (-1) yr(-1)to a maximum of 2700 kg ethanol acre (-1) yr(-1). The SCP yields for K. marxianus were calculated to range between 130 to 250 kg dry wt cell acre (-1) yr(-1). The potential for developing an integrated process to produce ethanol and SCP is also discussed.     

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.     

Isolation of a respiratory-deficient Kluyveromyces fragilis mutant for the production of ethanol from Jerusalem artichoke

A respiratory-deficient, mutant of Kluyveromyces fragilis was isolated using a ethidium bromide mutagenesis. It was characterized by a loss of cytochromes a + a3 and by an improvement of its inulinase activity. Under anaerobic conditions this mutant was always better than the wild strain for ethanol production especially from Jerusalem artichoke extracts containing large amounts of high polyfructosans ("early" extracts).     

High-yield ethanol production from Jerusalem artichoke tubers

Fermentation conditions were optimized for the production of ethanol from Jerusalem artichoke with a strain of Saccharomyces cerevisiae able to use high-concentration juice and undiluted pulp. Yields (95 to 125 g ethanol/l=85 to 98% of the theoretical value) exceeded those obtained with strain of Kluyveromyces used classically.The authors are with the Laboratoire de Pharmacognosie et Biotechnologie, UFR Pharmacie, 28 Place Henri-Dunant, 63001 Clermont-Ferrand Cédex, France. H. Pourrat is the corresponding author.