Continuous fermentation of feed streams containing D-glucose and D-xylose in a two-stage process utilizing immobilized Saccharomyces cerevisiae and Pachysolen tannophilus

In the U.S., forest and crop residues contain enough glucose and xylose to supply 10 times the country's usage of ethanol and ethylene, but an efficient fermentation scheme is lacking,(1,2,3) To develop a strategy for process design, specific ethanol productivities and yields of Pachysolen tannophilus NRRL Y-2460 and Saccharomyces cerevisiae NRRL Y-2235 were compared. Batch cultures and continuous stirred reactors (CSTR) loaded with immobilized cells were fed glucose and xylose. As expected from previous reports, Y-2235 fermented glucose but not xylose. Y-2460 consumed both sugars but fermented glucose inefficiently relative to Y-2235, and it suffered a diauxic lag lasting 10-20 h when given a sugar mixture. Immobilized Y-2235 exhibited increasing productivity but constant yield with in creasing glucose concentration. In contrast, Y-2460 exhibited an optimum productivity at 30-40 g/L xylose and a declining yield with increasing xylose concentration. Immobilized Y-2235 tolerated more than 100 g/L ethanol while the productivity and yield of Y-2460 fell by 80 and 58%, respectively, as ethanol reached 50 g/L. A 38.8-g/L ethanol stream could be produced as 103 g/L xylose was continuously fed to Y-2460. If it was blended with a 274 g/L glucose stream to give a composite of 23.7 g/L ethanol and 107 g/L glucose, Y-2235 could en rich the ethanol to 75 g/L. Taken together these results suggest use of a two-stage continuous reactor for pro cessing xylose and glucose from lignocellulose. An immobilized Y-2460 CSTR (or cascade) would convert the hemicellulose hydrolyzate. Then downstream, an immobilized Y-2235 plug flow reactor would enrich the hemicellulose-derived ethanol to more than 70 g/L upon addition of cellulose hydrolyzate.     

Ethanol production from eucalyptus wood hemicellulose hydrolysate by Pichia stipitis

Ethanol production was evaluated from eucalyptus wood hemicellulose acid hydrolysate using Pichia stipitis NRRL Y-7124. An initial lag phase characterized by flocculation and viability loss of the yeast inoculated was observed. Subsequently, cell regrowth occurred with sequential consumption of sugars and production of ethanol. Polyol formation was detected. Acetic acid present in the hydrolysate was an important inhibitor of the fermentation, reducing the rate and the yield. Its toxic effect was due essentially to its undissociated form. The fermentation was more effective at an oxygen transfer rate between 1.2 and 2.4 mmol/L h and an initial pH of 6.5. The hydrolysate used in the experiences had the following composition (expressed in grams per liter): xylose 30, arabinose 2.8, glucose 1.5, galactose 3.7, mannose 1.0, cellobiose 0.5, acetic acid 10, glucuronic acid 1.5, and galacturonic acid 1.0. The best values obtained were maximum ethanol concentration 12.6 g/L, fermentation time 75 h, fermentable sugar consumption 99% ethanol yield 0.35 g/g sugars consumed, and volumetric ethanol productivity 4 g/L day. (c) 1992 John Wiley & Sons, Inc.     

Optimum pH and temperature conditions for xylose fermentation by Pichia stipitis

Pichia stipitis NRRL Y-7124 is a xylose-fermenting yeast able to accumulate ca. 57 g/L ethanol. Because optimum process conditions are important, data were collected to determine the effects of temperature and pH on growth and fermentation rates and product accumulations. Temperatures (26-35 degrees C) providing optimum biomass and ethanol productivities did not necessarily provide maximum ethanol accumulation. Xylitol and residual xylose concentrations increased with temperature. Maximum ethanol selectivity was achieved at 25-26 degrees C with minimal sacrifice to production rates. The temperature optimum for xylose could not be generalized to glucose fermentations, in which ethanol productivity and accumulation were optimum at 34 degrees C. The optimum pH range for growth and fermentation on xylose was 4-7 at 25 degrees C.     

Random UV-C mutagenesis of Scheffersomyces (formerly Pichia) stipitis NRRL Y-7124 to improve anaerobic growth on lignocellulosic sugars

Scheffersomyces (formerly Pichia) stipitis NRRL Y-7124 was mutagenized using UV-C irradiation to produce yeast strains for anaerobic conversion of lignocellulosic sugars to ethanol. UV-C irradiation potentially produces large numbers of random mutations broadly and uniformly over the whole genome to generate unique strains. Wild-type cultures of S. stipitis NRRL Y-7124 were subjected to UV-C (234 nm) irradiation targeted at approximately 40% cell survival. When surviving cells were selected in sufficient numbers via automated plating strategies and cultured anaerobically on xylose medium for 5 months at 28°C, five novel mutagenized S. stipitis strains were obtained. Variable number tandem repeat analysis revealed that mutations had occurred in the genome, which may have produced genes that allowed the anaerobic utilization of xylose. The mutagenized strains were capable of growing anaerobically on xylose/glucose substrate with higher ethanol production during 250- to 500-h growth than a Saccharomyces cerevisiae yeast strain that is the standard for industrial fuel ethanol production. The S. stipitis strains resulting from this intense multigene mutagenesis strategy have potential application in industrial fuel ethanol production from lignocellulosic hydrolysates.     

Growth, death, and oxygen uptake kinetics of Pichia stipitis on xylose

Pichia stipitis NRRL Y-7124 has potential application in the fermentation of xylose-rich waste streams, produced by wood hydrolysis. Kinetic models of cell growth, death, and oxygen uptake were investigated in batch and oxygen-limited continuous cultures fed a rich synthetic medium. Variables included rates of dilution (D) and oxygen transfer (K(1)a) and concentrations of xylose (X), ethanol (E), and dissolved oxygen (C(ox)). Sustained cell growth required the presence of oxygen. Given excess xylose, specific growth rate (micro) was a Monod function of C(ox). Specific oxygen uptake rate was proportional to mu by a yield coefficient relating biomass production to oxygen consumption; but oxygen uptake for maintenance was negligible. Thus steady-state C(OX) depended only on D, while steady-state biomass concentration was controlled by both D and K(1)a. Given excess oxygen, cells grew subject to Monod limitation by xylose, which became inhibitory above 40 g/L. Ethanol inhibition was consistent with Luong's model, and 64. 3 g/L was the maximum ethanol concentration allowing growth. Actively growing cells died at a rate that was 20% of micro. The dying portion increased with E and X.     

休哈塔假丝酵母乙醇发酵性能的研究

木糖的高效发酵是制约纤维素燃料乙醇生产的技术瓶颈之一,高性能发酵菌种的开发是本领域研究的重点。以木糖发酵的典型菌株休哈塔假丝酵母为材料,研究氮源配比、葡萄糖和木糖初始浓度、葡萄糖添加及典型抑制物等因素对其木糖利用和乙醇发酵性能的影响规律。结果表明,硫酸铵更适宜于木糖和葡萄糖发酵产乙醇。在摇瓶振荡发酵条件下,该酵母可发酵164.0 g/L葡萄糖生成61.9 g/L乙醇,糖利用率和乙醇得率分别为99.8%和74.0%;受酵母细胞膜上转运体系的限制,对木糖的最高发酵浓度为120.0 g/L,可生成45.7 g/L乙醇,糖利用率和乙醇得率分别达到94.8%和87.0%。休哈塔假丝酵母发酵木糖的主要产物为乙醇,仅生成微量的木糖醇;添加葡萄糖可促进木糖的利用;休哈塔假丝酵母在葡萄糖发酵时的乙酸和甲酸的耐受浓度分别为8.32和2.55 g/L,木糖发酵时的乙酸和甲酸的耐受浓度分别为6.28和1.15 g/L。     

A parametric study on ethanol production from xylose byPichia stipitis

Characteristics of ethanol production by a xylose-fermenting yeast,Pichia stipitis Y-7124, were studied. The sugar consumption rate and specific growth rate were higher in the glucose-containing medium than in the xylose-containing medium. Specific activities of xylose reductase and xylitol dehydrogenase were higher in the medium with xylose than glucose, suggesting their induction by xylose. Maximum specific growth rate and ethanol yield were achieved at 30 g xylose/L concentration without formation of by-products such as xylitol and acetic acid whereas a maximum ethanol concentration was obtained at 130 g/L xylose. Adding a respiratory inhibitor, rotenone, increased a maximum ethanol concentration by 10% compared with the control experiment. In order to evaluate the pattern of ethanol inhibition on specific growth rate, a kinetic model based on Luong’s equations was applied. The relationship between ethanol concentration and specific growth rate was hyperbolic for glucose and parabolic for xylose. A maximum ethanol concentration at which cells did not grow was 33.6 g/L for glucose and 44.7 g/L for xylose.     

Comparative evaluation of ethanol production by xylose-fermenting yeasts presented high xylose concentrations

Summary Three strains ofPichia stipitis and three ofCandida shehatae were compared withPachysolen tannophilus in their abilities to ferment xylose at concentrations as high as 200 g/L when subjected to both aerobic and microaerophilic conditions. Evaluations based on accumulated ethanol concentrations, ethanol productivities, xylose consumption, and ethanol and xylitol yields were determined from batch culture time courses. Of the strains considered,P.stipitis NRRL Y-7124 seemed most promising since it was able to utilize all but 7 g/L of 150 g/L xylose supplied aerobically to produce 52 g/L ethanol at a yield of 0.39 g per gram xylose (76% of theoretical yield) and at a rate comparable to the fastest shown byC.shehatae NRRL Y-12878. For all strains tested, fermentation results from aerobic cultures were more favorable than those from microaerophilic cultures.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

发酵木糖生产乙醇的野生菌和转基因菌的研究进展

木糖发酵是利用植物纤维原料生物转化制取乙醇工业化生产的技术基础和关键。野生酵母中有些种属菌株可以高效利用木糖产生乙醇,其中毕赤酵母（Pichiastipim）的乙醇转化速度最高达到0．99g／L／h,转化率几乎接近理论值0．5g／g,发酵液中最高乙醇浓度可迭到（61±9）g／L。但工业生产中要达到毕赤酵母所要求的微氧最佳发酵条件比较困难。近十几年来许多研究尝试根据代谢工程原理,利用基因工程技术对酿酒酵母进行改造。从而提高其发酵木糖产生乙醇的能力。这些研究大多是将毕赤酵母的一些木糖发酵关键酶基因（XYL1、XYL2、XYL3以及ADHl、ADH2等）转入酿酒酵母细胞内,并试图得到正常转录和表达。但到目前为止,大部分的重组菌株的乙醇发酵性能还没有达到工业生产的要求。     

纤维素降解产物对Kluyveromyces marxianus 1727共发酵葡萄糖和木糖的影响

研究纤维素酸水解产生的4种副产物乙酸、甲酸、糠醛、5-羟甲基糠醛及发酵产物乙醇对Kluyveromyces marxianus 1727共发酵葡萄糖和木糖的影响。结果表明:5.0 g/L乙酸和1.0 g/L甲酸对葡萄糖和木糖共发酵具有明显的抑制作用;1.0 g/L糠醛和5-羟甲基糠醛基本不影响K.marxianus 1727发酵葡萄糖,且能够被K.marxianus1727转化为毒性相对较低的物质。由于5-羟甲基糠醛的转化速率慢,对K.marxianus 1727发酵木糖的抑制程度大于糠醛。乙醇对K.marxianus 1727发酵木糖具有抑制作用,当乙醇质量浓度大于20 g/L时,生物量及木糖利用率约是对照的44%和70%。     

Culture nutrition and physiology impact the inhibitor tolerance of the yeast Pichia stipitis NRRL Y-7124

Pichia stipitis NRRL Y-7124 is one of the natural yeasts best able to utilize biomass because it is able to ferment hexoses and the pentose, xylose, to economically recoverable concentrations of ethanol. To test the impact of culture conditions on inhibitor tolerance, inhibitors were spiked to growing or stationary-phase P. stipitis supplied either glucose or xylose and varying nitrogen and mineral compositions; then the ensuing specific death rate response was measured. Resistance of glucose- or xylose-grown cells to inhibitors was generally greater in stationary-phase cells than log-phase cells, despite a greater exposure of stationary cells to ethanol. Consistent with this, the specific productivity of detoxification products, furan methanol or furan-2,5-dimethanol, from respective spikes of furfural or HMF increased as cultures progressed into stationary phase. However, when xylose was the substrate, ethanol resistance behaved uniquely and was greater for log- than stationary-phase cells. Amino acid enrichment of the growth medium significantly enhanced ethanol tolerance if xylose was the carbon source, but had no impact if glucose supplied carbon. Regardless of the carbon source, amino acid enrichment of the culture medium enhanced the ability of cells to resist furfural and HMF exposure. Mineral compositions tested had little impact on inhibitor resistance except stationary-phase xylose-grown cells were more susceptible to inhibitor exposure when magnesium sulfate was excessive. Observed tolerance optimization based on specific death rate as a function of culture physiological state, carbon source, nitrogen source and mineral composition provides new knowledge supporting process designs to convert biomass to ethanol using P. stipitis.     

Mutants of Pachysolen tannophilus showing enhanced rates of growth and ethanol formation from d-xylose

Mutants of Pachysolen tannophilus NRRL Y-2460 have been sought that show enhanced rates of d-xylose fermentation. Mutagenesis followed by enrichment in urea-xylitol broth generally resulted in a lower frequency of good ethanol producers than enrichment in nitrate-xylitol broth. Under aerobic conditions, the best xylose-fermenting strains (which were obtained from nitrate-xylitol broth) produced ethanol from xylose twice as fast and in 32% better yield than the parent strain. Under anaerobic conditions, these strains produced ethanol from xylose 50% faster than (but in the same yield as) the parent strain. These findings show that enrichment in nitrate-xylitol broth is a promising method for obtaining mutants of Pachysolen having enhanced fermentation rates.     

Alcoholic fermentation of D-xylose by immobilizedPichia stipitis yeast

Summary Pichia stipitis NRRL Y-7124 yeast cells were for the first time immobilized both in agar gel beads and on fine nylon net for ethanol fermentation on D-xylose, in order to investigate the possibility of using the biocatalyst for improved utilization of the biomass pentose fraction. With free cells the initial xylose level affected little ethanol production, with a maximum of 22 g/l ethanol obtained in 5 days on 5% and of 40 g/l in 8 days on 10% xylose, and an average volumetric productivity of about 0.22 g/lh. The maximum ethanol concentration of 19.5% on 5% xylose with the nylon net attached cells in a continuous packed-bed column reactor was obtained with 35 h residence time. The volumetric productivities of 0.56 g/lh at 19.5 g/l ethanol and 1.0 g/lh at 15.0 g/l ethanol were markedly higher than those obtained with free cells. The stability of the immobilized biocatalyst was excellent. The same reactor could be used for at least 80 days without significant activity loss.     

The expression of a Pichia stipitis xylose reductase mutant with higher K(M) for NADPH increases ethanol production from xylose in recombinant Saccharomyces cerevisiae

Xylose fermentation by Saccharomyces cerevisiae requires the introduction of a xylose pathway, either similar to that found in the natural xylose-utilizing yeasts Pichia stipitis and Candida shehatae or similar to the bacterial pathway. The use of NAD(P)H-dependent XR and NAD(+)-dependent XDH from P. stipitis creates a cofactor imbalance resulting in xylitol formation. The effect of replacing the native P. stipitis XR with a mutated XR with increased K(M) for NADPH was investigated for xylose fermentation to ethanol by recombinant S. cerevisiae strains. Enhanced ethanol yields accompanied by decreased xylitol yields were obtained in strains carrying the mutated XR. Flux analysis showed that strains harboring the mutated XR utilized a larger fraction of NADH for xylose reduction. The overproduction of the mutated XR resulted in an ethanol yield of 0.40 g per gram of sugar and a xylose consumption rate of 0.16 g per gram of biomass per hour in chemostat culture (0.06/h) with 10 g/L glucose and 10 g/L xylose as carbon source.     

Effect of glucose supplements on the fermentation of xylose by Pachysolen tannophilus

Hemicellulosic sugars, predominantly D-xylose, comprise about one-half the total carbohydrate that can be obtained from hardwoods and agricultural residues through dilute acid hydrolysis. Because rates and yields in the xylose fermentation are low, economic utilization of these materials as fermentation feedstocks is difficult. Pachysolen tannophilus formed 5.5% ethanol from 12% glucose but only 2% ethanol from 12% xylcose. Aeration doubled the specific rate of D-glucose fermentation by P. tannophilus, as compared to anaerobic fermentation, but the specific rate of the xylose fermentation remained unchanged. Periodic additions of 0.5% D-glucose to aerobic fermentations of 3% xylose increased the yield of ethanol from 0.28 g/g xylose to greater than 0.41 g/g xylose utilized. The rate of xylose utilization remained unchanged, and radiotracer studies showed that addition of 0.5% glucose did not inhibit xylose utilization under aerobic or anaerobic conditions. No enhancement was observed anaerobically, nor was enhancement observed with acid hydrolysates, apparently because of the presence of acetic acid which inhibited growth and fermentation.     

Alcoholic Fermentation of d-Xylose by Yeasts

Type strains of 200 species of yeasts able to ferment glucose and grow on xylose were screened for fermentation of d-xylose. In most of the strains tested, ethanol production was negligible. Nineteen were found to produce between 0.1 and 1.0 g of ethanol per liter. Strains of the following species produce more than 1 g of ethanol per liter in the fermentation test with 2% xylose: Brettanomyces naardenensis, Candida shehatae, Candida tenuis, Pachysolen tannophilus, Pichia segobiensis, and Pichia stipitis. Subsequent screening of these yeasts for their capacity to ferment d-cellobiose revealed that only Candida tenuis CBS 4435 was a good fermenter of both xylose and cellobiose under the test conditions used.     

The D-xylose fermenting capacities of immobilizedPichia stipitis andPachysolen tannophilus

Summary The yeastsP. stipitis NRRL Y-7124 andP. tannophilus NRRL Y-2460 were entrapped in -carrageenan beads and used for repeated batch fermentation of D-xylose, in a series of four reactors. The operating conditions finally chosen gave an oxygen coefficient (K_La) of 0.83 min^–1, as measured by the sulphite method. Ethanol yields were 0.40 g/g forP. stipitis and 0.36 g/g forP. tannophilus (respectively 78.4% and 70.5% of the theoretical yields). In spite of its lower retention by the gel,P. stipitis exhibited greater fermenting capacities thanP. tannophilus.     

树干毕赤酵母NLP31发酵产乙醇的研究

研究了树干毕赤酵母NLP31在木糖质量浓度为45 g/L的3种发酵培养基Ⅰ、Ⅱ和Ⅲ上发酵3轮的发酵性能以及在45 g/L木糖或混合糖(葡萄糖30 g/L,木糖15 g/L)的发酵培养基Ⅲ上的代谢历程。结果表明:树干毕赤酵母NLP31在发酵培养基Ⅲ上,乙醇浓度和乙醇得率均达到最高,分别为(17.29±0.15)g/L和(84.65±0.58)%。在45 g/L木糖或混合糖(葡萄糖30 g/L,木糖15 g/L)的发酵培养基Ⅲ上的代谢历程表明:混合糖发酵达到最大乙醇得率的时间仅为12 h,要比单一木糖发酵缩短了8 h。树干毕赤酵母NLP31在以廉价的无机N源为发酵培养基上的乙醇发酵性能高,能够降低燃料乙醇的生产成本。     

分批补料对木糖发酵生产乙醇的影响

以树干毕赤酵母为发酵菌种,纯木糖为发酵底物,通过分批补料来提高糖利用率以及乙醇得率。结果表明,在24h内,最佳初始木糖浓度为80g/L,在28h的发酵周期中,可以将木糖浓度提高至90g/L,在32h发酵周期内可以将木糖浓度提高至100g/L。通过分批补料,乙醇浓度得到明显提高。当总糖浓度分别为80g/L、90g/L时,24h发酵周期内,分批补料次数以1次为宜,乙醇浓度分别达30.95g/L、32.60g/L,相比于不补料即一次性投料,乙醇浓度分别提高了9.36%、9.18%。总糖浓度100g/L,28h发酵周期内,补料2次效果最佳,乙醇浓度达37.49g/L,比一次性投料下提高了10.36%,较一次性投料达到相同发酵效果缩短了4h。     

Detoxification of corn stover prehydrolyzate by trialkylamine extraction to improve the ethanol production with Pichia stipitis CBS 5776

In order to realize the separated ethanol fermentation of glucose and xylose, prehydrolysis of corn stover with sulfuric acid at moderate temperature was applied, while inhibitors were produced inevitably. A complex extraction was adopted to detoxify the prehydrolyzate before fermentation to ethanol with Pichia stipitis CBS 5776. The best proportion of mixed extractant was 30% trialkylamine-50% n-octanol -20% kerosene. Detoxification results indicated that 73.3% of acetic acid, 45.7% of 5-hydroxymethylfurfural and 100% of furfural could be removed. Compared with the undetoxified prehydrolyzate, the fermentability of the detoxified prehydrolyzate was significantly improved. After 48 h fermentation of the detoxified prehydrolyzate containing 7.80 g/l of glucose and 52.8 g/l of xylose, the sugar utilization ratio was 93.2%; the ethanol concentration reached its peak value of 21.8 g/l, which was corresponding to 82.3% of the theoretical value.