Direct conversion of inulin and extract of tubers of Jerusalem artichoke into single cell oil by co-cultures of Rhodotorula mucilaginosa TJY15a and immobilized inulinase-producing yeast cells

In this study, it was found that the immobilized inulinase-producing cells of Pichia guilliermondii M-30 could produce 169.3 U/ml of inulinase activity while the free cells of the same yeast strain only produced 124.3 U/ml of inulinase activity within 48 h. When the immobilized inulinase-producing yeast cells were co-cultivated with the free cells of Rhodotorula mucilaginosa TJY15a, R. mucilaginosa TJY15a could accumulate 53.2% oil from inulin in its cells and cell dry weight reached 12.2 g/l. Under the similar conditions, R. mucilaginosa TJY15a could accumulate 55.4% (w/w) oil from the extract of Jerusalem artichoke tubers in its cells and cell dry weight reached 12.8 g/l within 48 h. When the co-cultures were grown in 2 l fermentor, R. mucilaginosa TJY15a could accumulate 56.6% (w/w) oil from the extract of Jerusalem artichoke tubers in its cells and cell dry weight reached 19.6 g/l within 48 h. Over 90.0% of the fatty acids from the yeast strain TJY15a grown in the extract of Jerusalem artichoke tubers was C_16:0, C_18:1 and C_18:2, especially C_18:1 (50.6%).     

Single cell oil production from hydrolysates of inulin and extract of tubers of Jerusalem artichoke by Rhodotorula mucilaginosa TJY15a

In this study, we found that Rhodotorula mucilaginosa TJY15a could accumulate 48.8% (w/w) oil from hydrolysate of inulin and its cell dry weight reached 14.8 g/l during the batch cultivation while it could accumulate 48.6% (w/w) oil and 52.2% (w/w) oil from hydrolysate of extract of Jerusalem artichoke tubers and its cell dry weight reached 14.4 g/l and 19.5 g/l during the batch and fed-batch cultivations, respectively. At the end of the fed-batch cultivation, only 0.04% of reducing sugar and 0.08% of total sugar were left in the fermented medium. Over 87.6% of the fatty acids from the yeast strain TJY15a cultivated in the hydrolysate of extract of Jerusalem artichoke tubers was C_16:0, C_18:1 and C_18:2, especially C_18:1 (54.7%). Therefore, the results show that hydrolysates of inulin and extract of Jerusalem artichoke tubers were also the good materials for single cell oil production.     

圆红冬孢酵母发酵菊芋块茎产油脂的研究

研究了圆红冬孢酵母Y4发酵菊芋块茎,菊芋品种及其处理方法对发酵产油的影响。结果表明,菊芋浸提汁、酸水解液或菊芋浆均可直接被圆红冬孢酵母Y4利用,发酵积累油脂,但白皮菊芋比紫皮菊芋更有利于油脂发酵。发酵菊芋浸提汁或酸水解液时,无需添加外源营养物,干菌体油脂含量可达到40%（w/w）;发酵菊芋浆时,白皮菊芋转化率达到12.1 g油/100 g去皮干菊芋。菊芋油脂发酵产品主要以16碳和18碳系脂肪酸为主,与常规植物油的脂肪酸组成相似,可作为制备生物柴油的新型替代原料。     

Considerazioni su Gelsi Ingialliti per Fame

Jerusalem artichoke (Helianthus tuberosus L.), an important crop, containing over 50% inulin in its tubers on a dry weight basis is an agricultural and industrial crop with a great potential for production of ethanol and industrial products. Inulin is a good substrate for bioethanol production. Saccharomyces cerevisiae 6525 can produce high concentrations of ethanol, but it cannot synthesize inulinase. In this study, a new integration vector carrying inuA1 gene encoding exoinulinase was constructed and transformed into 18SrDNA site of industrial strain S. cerevisiae 6525. The obtained transformant, BR8, produced 1.1 U mL^? 1 inulinase activity within 72 h and the dry cell weight reached 12.3 g L^? 1 within 48 h. In a small-scale fermentation, BR8 produced 9.5% (v/v) ethanol, with a productivity rate of 0.385 g ethanol per gram inulin, while wild-type S. cerevisiae 6525 produced only 3.3% (v/v) ethanol in the same conditions. In a 5-L fermentation, BR8 produced 14.0% (v/v) ethanol in fermentation medium containing inulin and 1% (w/v) (NH4)₂SO₄. The engineered S. cerevisiae 6525 carrying inuA1 converted pure nonhydrolyzed inulin directly into high concentrations of ethanol.     

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.     

Consolidated ethanol production from Jerusalem artichoke tubers at elevated temperature by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> engineered with inulinase expression through cell surface display

Ethanol fermentation from Jerusalem artichoke tubers was performed at elevated temperatures by the consolidated bioprocessing strategy using Saccharomyces cerevisiae MK01 expressing inulinase through cell surface display. No significant difference was observed in yeast growth when temperature was controlled at 38 and 40 °C, respectively, but inulinase activity with yeast cells was substantially enhanced at 40 °C. As a result, enzymatic hydrolysis of inulin was facilitated and ethanol production was improved with 89.3 g/L ethanol produced within 72 h from 198.2 g/L total inulin sugars consumed. Similar results were also observed in ethanol production from Jerusalem artichoke tubers with 85.2 g/L ethanol produced within 72 h from 185.7 g/L total sugars consumed. On the other hand, capital investment on cooling facilities and energy consumption for running the facilities would be saved, since regular cooling water instead of chill water could be used to cool down the fermentation system.     

Enzymatic hydrolysis of inulin to fructose by glutaraldehyde fixed yeast cells

Inulin, a polyfruction, is found as the reserve carbohydrate in the roots and tubers of various plants (i.e. Jerusalem artichoke, chicory, and dahlia tubers). The beta-fructofuranosidase (inulase) from the yeast Kluyveromyces fragilis is of interest because of its industrial potential in fructose syrup and alcohol production from inulin containing plants. We have found that the inulase of K. fragilis can be immobilized in the yeast cells by glutaraldehyde treatment. These cells are resistant to physical and enzymatic destruction. Although the exact nature of the immobilization is not fully understood, the kinetic parameters of the immobilized enzyme are similar to those of the soluble enzyme. No reduction of enzyme activity was observed after glutaraldehyde treatment and glutaraldehyde concentration did not affect enzyme activity. A 96% hydrolysis of dahlia inulin was achieved in 10.5 h with a 9.5% (w/v) fixed enzyme suspension. A Jerusalem artichoke extract containing 16.8%polyfructan was completely hydrolyzed in 3.5 h with a 0.24% (w/v)fixed enzyme suspension. This is a time frame feasible for industrial consideration.     

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.     

COMPARATIVE STUDIES ON VARIOUS CALLUSES AND CROWN GALL OF JERUSALEM ARTICHOKE II. SOME BIOCHEMICAL PROPERTIES

A callus isolated from Jerusalem artichoke tuber was found tohave much more cell wall material and tyrosinase activity thanthe original tuber, and its dry weight was dependent on thesucrose concentration in the culture medium. All calluses anda crown gall tested contained no detectable inulin. Even thoughdifferent cultures were widely different in their dry weight,this was closely related to their total hexose content, andthe latter in turn was proportional to their free hexose content. (Received January 18, 1967; )     

鹰嘴豆孢克鲁维酵母利用菊芋原料同步糖化与发酵生产乙醇

菊芋含有大量的菊粉多糖,且种植简单、产量高,是极具开发价值的替代玉米等粮食作物生产燃料乙醇的原料。文中研究了鹰嘴豆孢克鲁维酵母Y179利用菊芋原料同步糖化与发酵生产乙醇。鹰嘴豆孢克鲁维酵母Y179具有高效分泌菊粉酶的能力,摇瓶试验显示Y179酵母能够利用完全由菊芋原料配制而成的培养基良好生长并发酵产生乙醇。通气及温度对乙醇产量影响明显,相对厌氧环境对Y179酵母发酵产乙醇具有促进作用,30℃发酵温度相对37℃和42℃更有利于乙醇产量提高。种子液培养时间及接种量对乙醇产量影响较小。在5 L发酵罐中以10%(V/V)量接入预培养36 h的Y179种子液,发酵液完全由菊芋干粉配制而成,总糖含量22%(W/V),30℃不通气,300 r/min搅拌,发酵144 h时,乙醇浓度达到12.3%(V/V),糖醇转化效率86.9%,糖利用率大于93.6%。初步研究结果显示鹰嘴豆孢克鲁维酵母Y179在利用菊芋原料生产乙醇方面具有良好应用前景。     

Single-cell protein production from Jerusalem artichoke extract by a recently isolated marine yeast <Emphasis Type="Italic">Cryptococcus aureus</Emphasis> G7a and its nutritive analysis

After crude protein of the marine yeast strains maintained in this laboratory was estimated by the method of Kjehldahl, we found that the G7a strain which was identified to be a strain of Cryptococcus aureus according to the routine identification and molecular methods contained high level of protein and could grow on a wide range of carbon sources. The optimal medium for single-cell protein production was seawater containing 6.0 g of wet weight of Jerusalem artichoke extract per 100 ml of medium and 4.0 g of the hydrolysate of soybean meal per 100 ml of medium, while the optimal conditions for single-cell protein production were pH 5.0 and 28.0°C. After fermentation for 56 h, 10.1 g of cell dry weight per liter of medium and 53.0 g of crude protein per 100 g of cell dry weight (5.4 g/l of medium) were achieved, leaving 0.05 g of reducing sugar per 100 ml of medium and 0.072 g of total sugar per 100 ml of medium total sugar in the fermented medium. The yeast strain only contained 2.1 g of nucleic acid per 100 g of cell dry weight, but its cells contained a large amount of C_16:0 (19.0%), C_18:0 (46.3%), and C_18:1 (33.3%) fatty acids and had a large amount of essential amino acids, especially lysine (12.6%) and leucine (9.1%), and vitamin C (2.2 mg per 100 g of cell dry weight). These results show that the new marine yeast strain was suitable for single-cell protein production.     

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.     

发酵菊芋汁生产果糖糖浆研究

分析了来自不同地区的菊芋成分,干物质含量在23％～26％,菊粉多糖含量为17％～18％（鲜重）。制备菊芋汁中的主要固形物成分是菊粉多糖,游离的还原糖和可溶性蛋白质含量很低,菊芋用于果糖或果糖糖浆生产具有较高的经济价值;通过摇瓶发酵试验确定了利用菊芋汁生产果糖糖浆的工艺,用自动模拟发酵罐进行了生产模拟实验。产品总糖含量为61％,其中果糖95％,葡萄糖5％,通过发酵法生产果糖糖浆总糖得率为90％。     

Astaxanthin from Jerusalem artichoke: Production by fed-batch fermentation using Phaffia rhodozyma and application in cosmetics

Jerusalem artichoke extract or powder was used for astaxanthin production using Phaffia rhodozyma without acidic or enzymatic inulin hydrolysis. The culture medium containing Jerusalem artichoke as carbon source was optimized, and feeding strategies, including constant, exponential, pH-stat, and substrate feedback fed-batch fermentations, were also compared for enhancing the cell biomass and astaxanthin synthesis by P. rhodozyma. Substrate-feedback fed-batch fermentation resulted in the highest dry cell weight of 83.60 g/L, with a carotenoid concentration and yield of 982.50 mg/L and 13.30 mg/g, respectively, under optimized medium components using Jerusalem artichoke extract as carbon source in a 3-L stirred-tank bioreactor. Moreover, 482.50 mg/L of carotenoids and 253.10 mg/L of astaxanthin were obtained by continuous feeding of Jerusalem artichoke powder, which was used as carbon source. Astaxanthin essence with high DPPH-scavenging activity was obtained from the extracted astaxanthin, and the DPPH free radical scavenging rate of 40 ppm astaxanthin essence reached 76.29%. When stored at 4 °C, astaxanthin essence showed the highest stability, with a minimum k value of 0.0099 week⁻¹ and maximum half-life (t_1/2) value of 70 weeks.     

菊芋粕菊粉的酶法提取、组成及抗氧化活性研究

本研究拟应用酶法提取技术解决菊芋菊粉工业化生产中的废弃物——菊芋粕再利用程度低的问题,并评价菊芋粕菊粉的抗氧化活性功效。对菊芋粕菊粉的果胶酶酶法提取的最佳条件通过响应面法进行了优化,并对菊芋初次水提菊粉(primary water-extracted inulin,PWI)和二次酶提菊粉(secondary enzymatic-extracted inulin,SEI)的组成成分和抗氧化活性进行了比较分析。响应面法优化确定的菊芋粕菊粉最佳酶法提取条件为:pH4.5、提取温度50 ℃、酶底比7.5 U/g、提取时间2 h,该提取方法所得菊芋粕菊粉的得率为35.30%±0.85%,与传统热水浸提法相比菊粉得率提高38.16%。组成分析结果显示,SEI的总糖和菊粉含量均显著高于PWI( P <0.05)。在菊粉聚合度方面,PWI中蔗果三糖和蔗果四糖含量较高,而SEI中蔗果五糖、蔗果六糖及其以上聚合度菊粉的含量较高。此外,SEI的抗氧化活性优于PWI。因此,果胶酶辅助提取方法有望为菊芋粕菊粉的再利用难题提供新的解决思路。     

Simultaneous saccharification of inulin and ethanol fermentation by recombinantSaccharomyces cerevisiae secreting inulinase

VariousSaccharomyces cerevisiae strains were transformed with a 2 μ-based multicopy expression plasmid, pYIGP, carryingKluyveromyces marxianus inulinase gene under the control ofGAPDH promoter. Among them two strains, SEY2102 and 2805, showed high levels of cell growth and inulinase expression, and were selected to study their fermentation properties on inulin. Jerusalem artichoke inulin was more effective for cell growth (10∼11 g-dry wt./L at 48 hr) and inulinase expression (1.0 units/mL with SEY2102/pYIGP and 2.5 units/mL with 2805/pYIGP) than other inulin sources such as dahlia and chicory. It was also found that maximal ethanol production of 9 g/L was obtained from Jerusalem artichoke inulin at the early stationary phase (around 30 hr), indicating that recombinantS. cerevisiae cells secreting exoinulinase could be used for the simultaneous saccharification of inulin and ethanol fermentation.     

Molecular properties and prebiotic effect of inulin obtained from artichoke (Cynara scolymus L.)

A high molecular weight inulin has been prepared from artichoke (Cynara scolymus L.) agroindustrial wastes using environmentally benign aqueous extraction procedures. Physico-chemical analysis of the properties of artichoke inulin was carried out. Its average degree of polymerization was 46, which is higher than for Jerusalem artichoke, chicory, and dahlia inulins. GC-MS confirmed that the main constituent monosaccharide in artichoke inulin was fructose and its degradation by inulinase indicated that it contained the expected beta-2,1-fructan bonds. The FT-IR spectrum was identical to that of chicory inulin. These data indicate that artichoke inulin will be suitable for use in a wide range of food applications. The health-promoting prebiotic effects of artichoke inulin were demonstrated in an extensive microbiological study showing a long lasting bifidogenic effect on Bifidobacterium bifidum ATCC 29521 cultures and also in mixed cultures of colonic bacteria.     

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.     

Process for biodiesel production from Cryptococcus curvatus

The objective of the current report is process optimization for economical production of lipids by the well known oleaginous yeast Cryptococcus curvatus and conversion of the lipids to biodiesel. A high cell density fed-batch cultivation on low cost substrate viz. crude glycerol resulted in a dry biomass and oil yield of up to 69 g/L and 48% (w/w), respectively. The process was scaled up easily to 26 L. The oil extraction process was also optimized using environmentally safe solvents. The oil profile indicated a high oleic acid content followed by palmitic acid, stearic acid and linoleic acid. The oil was trans-esterified to biodiesel and thoroughly characterized. This is the first end to end report on production of biodiesel from the C. curvatus oil.     

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.