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.     

Continuous ethanol production from pineapple cannery waste using immobilized yeast cells

The cells of Saccharomyces cerevisiae ATCC 24553, were immobilized in k-carrageenan and packed in a tapered glass column reactor for ethanol production from pineapple cannery waste at temperature 30 degrees C and pH 4.5. The maximum productivity was 42.8 g ethanol 1(-1) h(-1) at a dilution rate of 1.5 h(-1). The volumetric ethanol productivity of the immobilized cells was ca. 11.5 times higher than the free cells. The immobilized cell reactor was operated over a period of 87 days at a dilution rate of 1.0 h(-1), without any loss in the immobilized cell activity. The maximum specific ethanol productivity and specific sugar uptake rate of the immobilized cells were 1.2 g ethanol g(-1) dry wt. cell h(-1) and 2.6 g sugar g(-1) dry wt. cell h(-1), respectively, at a dilution rate of 1.5 h(-1).     

Alcoholic fermentation by agar-immobilized yeast cells

Immobilized yeast cells in agar gel beads were used in a packed bed reactor for the production of ethanol from cane molasses at 30°C, pH 4.5. The maximum productivity, 79.5g ethanol/l.h was obtained with 195g/l reducing sugar as feed. Substrate (64.2%) was utilized at a dilution of 1.33h^-1. The immobilized cell reactor was operated continuously at a constant dilution rate of 0.67h^-1 for 100 days. The maximum specific ethanol productivity and specific sugar uptake rate were 0.610g ethanol/g cell.h and 1.275g sugar/g cell.h, respectively.     

Continuous growth kinetics of Candida utilis in pineapple cannery effluent

Candida utilis was grown on a pineapple cannery effluent as the sole carbon and energy source in a chemostat at dilution rates between 0.10 and 0.62 h(-1) to determine the growth kinetics. The principal sugars in the effluent were sucrose, glucose, and fructose. The cell yield coefficient on carbohydrate varied with dilution rate and a maximum value of 0.63 was observed at a dilution rate of 0.33 h(-1). The steady-state concentrations of carbohydrate, reducing sugar, and chemical oxygen demand (COD) appeared to follow Monod saturation kinetics with increasing dilution rate, although none of the measured parameters represented a pure substrate. The maximum specific growth rate and reducing sugar saturation constant were 0.64 h(-1) and 0.060 g/L, respectively. A maximum cell mass productivity of 2.3 g/L h was observed at a dilution rate of 0.51 h(-1). At this dilution rate, only 68% of the COD was removed. A 95% COD removal was attained at a dilution rate of 0.10 h(-1). Optimal yeast productivity and COD reduction occurred at a dilution rate of 0.33 h(-1).     

Ethanol from whey: Continuous fermentation with cell recycle

The production of ethanol from cheese whey lactose has been demonstrated using a single-stage continuous culture fermentation with 100% cell recycle. In a two-step process, an aerobic fed batch operation was used initially to allow biomass buildup in the absence of inhibitory ethanol concentrations. In the anaerobic ethanol-producing second step, a strain of Kluyveromyces fragilis selected on the basis of batch fermentation data had a maximum productivity of 7.1 g ethanol/L/h at a dilution rate of 0.15 h(-1), while achieving the goal of zero residual sugar concentration. The fermentation productivity diminished when the feed sugar concentation exceeded 120 g/L despite the inclusion of a lipid mixture previous shown to enhance batch fermentation productivities.     

自絮凝酵母SPSC01在组合反应器系统中酒精连续发酵的研究

建立了一套由四级磁力搅拌发酵罐串联组成、总有效容积4000mL的小型组合生物反应器系统 ,其中一级罐作为种子培养罐。以脱胚脱皮玉米粉双酶法制备的糖化液为种子培养基和发酵底物 ,进行了自絮凝颗粒酵母酒精连续发酵的研究。种子罐培养基还原糖浓度为100g L ,添加 (NH₄)₂HPO₄ 和KH₂PO₄ 各 20g L ,以0.017h^-1 的恒定稀释速率流加 ,并溢流至后续酒精发酵系统。发酵底物初始还原糖浓度 220g/L ,添加 (NH₄)₂HPO₄ 15g/L和KH₂PO₄2 5g/L ,流加至第一级发酵罐 ,稀释速率分别为 0.017、0.025、0.033、0.040和0.05 0h^-1。实验数据表明 ,自絮凝颗粒酵母在各发酵罐中呈部分固定化状态 ,在稀释速率0.040h^-1 条件下 ,发酵系统呈一定的振荡行为 ,其他四个稀释速率实验组均能够达拟稳态。当稀释速率不超过 0 0 33h^-1 ,流出末级发酵罐的发酵液中酒精浓度可以达到 12 % (V/V)以上 ,残还原糖和残总糖分别在 0 11%和 0 35 % h^-1,流出末级发酵罐的发酵液中酒精浓度可以达到12%（V/V）以上,残还原糖和残总糖分别在0.11%和0.35%（W/V）以下。在稀释速率为0.033h^-1时,计算发酵系统酒精的设备生产强度指标为3.32（g·L^-1·h^-1）,与游离酵母细胞传统酒精发酵工艺相比,增加约1倍。     

Physiology of the yeast Kluyveromyces marxianus during batch and chemostat cultures with glucose as the sole carbon source

Growth, substrate consumption, metabolite formation, biomass composition and respiratory parameters of Kluyveromyces marxianus ATCC 26548 were determined during aerobic batch and chemostat cultivations, using mineral medium with glucose as the sole carbon source, at 30 degrees C and pH 5.0. Carbon balances closed within 95-101% in all experiments. A maximum specific growth rate of 0.56 h(-1), a biomass yield on glucose of 0.51 g g(-1), and a maximum specific consumption of oxygen of 11.1 mmol g(-1) h(-1) were obtained during batch cultures. The concentration of excreted metabolites was very low at the culture conditions applied, representing 6% of the consumed carbon at most. Acetate and pyruvate were excreted to a larger extent than ethanol under the batch conditions, and the protein content accounted for 54.6% of the biomass dry weight. Steady states were obtained during chemostats at dilution rates of 0.1, 0.25 and 0.5 h(-1). At the two former dilution rates, cells grew at carbon limitation and the biomass yield on glucose was similar to that obtained under the batch conditions. Metabolite formation was rather low, accounting for a total of 0.005 C-mol C-mol(-1) substrate. At 0.5 h(-1), although the biomass yield on glucose was similar to the value obtained under the above-mentioned conditions, the cultivation was not under carbon limitation. Under this condition, 2-oxoglutarate, acetate, pyruvate and ethanol were the prevalent metabolites excreted. Total metabolite formation only accounted to 0.056 C-mol C-mol(-1) of substrate. A very high protein and a low carbohydrate content (71.9% and 9.6% of biomass dry weight, respectively) were measured in cells under this condition. It is concluded that K. marxianus aligns with the so-called aerobic-respiring or Crabtree-negative yeasts. Furthermore, it has one of the highest growth rates among yeasts, and a high capacity of converting sugar into biomass, even when carbon is not the limiting nutrient. These results provide useful data regarding the future application of K. marxianus in processes aimed at the production of biomass-linked compounds, with high yields and productivities.     

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.     

Production of ethanol by Clostridium thermosaccharolyticum: I. Effect of cell recycle and environmental parameters

The direct microbial conversion (DMC) process for the production of ethanol from lignocellulosic biomass is limited by low volumetric ethanol production rates due to the low cell densities of Clostridium thermosaccharolyticum which is a key organism for ethanol production in this process. Hence, this study focuses on the use of a continuous- culture cell recycle system to improve the volumetric ethanol productivity and yield of the fermentation of xylose by C. thermosaccharolyticum. Early experiments with the continuous-culture cell recycle system showed a two-fold improvement in volumetric ethanol productivity. However, the ethanol yield at the higher dilution rates suffered because of the large amount of lactate produced. The manipulation of two environmental parameters-iron concentration in the nutrient medium and the N(2) purge rate of the fermentor headspace-allowed a dramatic reduction in the lactate production and a simultaneous improvement in the ethanol titer and yield. Under the improved conditions of increased iron concentration (12.5 mg/L FeSO(4) . 7H(2)O) and decreased N(2) purge rate (0.1 L/min), a continuous culture of C. thermosaccharolyticum operating at a dilution rate of 0.24 h(-1) and 50% cell recycle produced 8.6 g/L ethanol and less than 1 g/L each of acetate and lactate. The volumetric ethanol productivity was 2.2 g/L/h, which is 8 times larger than obtained for a continuous culture operated with no cell recycle and the same specific growth rate.     

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.     

Performance assessment of two patent strains ofZymomonas mobilis in batch and continuous fermentations

Summary The performance ofZymomonas mobilis strains ATCC 31821 and ATCC 31823 was assessed in batch and continuous culture. In batch culture using a medium containing 250 g/l glucose, identical maximum specific growth rates of 0.16/h were found, though final biomass concentration and growth yield were significantly lower for ATCC 31 823 than for ATCC 31 821. Final ethanol concentrations in this medium were about 110 g/l vor both organisms. In continuous culture at increasing dilution rates using a medium containing 100 g/l glucose, no significant differences were seen between the two strains with respect to the fermentation parameters studied. For ATCC 31 821, maximum rates of glucose uptake (Q_s) and ethanol produktion (Q_p) of 8.7 g glu/g/h and 4.4 g eth/g/h, respectively, were found. Both strains showed a similar performance at a fixed dilution rate of 0.1/h, where maximum ethanol concentrations of about 68 g/l were reached at a feed glucose concentration of about 139 g/l. At this dilution rate the maximum values of Q_s and Q_p were about 5.8 g glu/g/h and 2.8 g eth/g/h, respectively. Test tube experiments showed that growth, measured as optical density, decreased with increasing concentrations of exogenous ethanol with complete inhibition of growth at ethanol concentrations >8% (v/v). As evidenced by the results presented here, we have been unable to practice the invention as described in U.S. Patent 4,403,034 (Rogers and Tribe 1983).Nomenclature D Dilution rate, 1/h - _max maximum specific growth rate, 1/h - S_R Initial substrate concentration, g glucose/1 - S Residual substrate concentration, g glucose/1 - S₀ Effluent substrate concentration, g glucose/1 - X Blomass concentration; g cells/l - OD₆₂₀ Optical density at 620 nm, dimensionless - [P] Product concentration, g ethanol/1 - Y_x/s Growth yield, g cells/g glucose used - Y_p/s Product yield, g ethanol/g glucose used - %, Yield Percentage yield, Y_p/sx100/Y _p ^s/max =Y_p/sx100/0.51 - Q_s Specific rate of glucose uptake, g glucose/g cells/h - Q_p Specific rate of ethanol formation, g ethanol/g cells/h - m_e Maintenance energy coefficient, g glucose/g cells/h - VP Volumetric productivity, g ethanol/l/h - t Fermentation time, h     

An novel immobilization method of Saccharomyces cerevisiae to sorghum bagasse for ethanol production

Natural sorghum bagasse without any treatment was used to immobilize Saccharomyces cerevisiae at 0.6+/-0.2g dry cell weight (DCW)/g dry sorghum bagasse weight (DSW) through solid-state or semi-solid state incubation. The scanning electron microscopy (SEM) of the carriers revealed the friendship between yeast cells and sorghum bagasse are adsorption and embedding. The ethanol productivity of the immobilized cells was 2.24 times higher than the free cells. In repeated batch fermentation with an initial sugar concentration of 200g/L, nearly 100% total sugar was consumed after 16 h. The ethanol yield and productivity were 4.9 g/g consumed sugar on average and 5.72 g/(Lh), respectively. The immobilized cell reactor was operated over a period of 20 days without breakage of the carriers, while the free cell concentration in the effluent remained less than 5 g/L thoughout the fermentation. The maximum ethanol productivity of 16.68 g/(Lh) appeared at the dilution rate of 0.3h(-1).     

Influence of specific growth rate on biomass yield, productivity, and compostion of Candida utilis in batch and continuous culture.

Candida utilis was grown in batch and continuous culture on prickly pear juice as sole carbon and energy source. In batch culture the maximum specific growth rate (mum) and the substrate yield coefficient (Yps) varied according to sugar concentration. When the fermentation was carried out with 1% sugar, mum and Ys were 0.47/h and 42.6%, respectively. The best yields occurred in a chemostat at the pH range of 3.5 to 4.5 and temperature of 30 C. A beneficial effect on Ys was observed when the dilution rate (D) was increased. At a D of 0.55/h, the productivity was 2.38 g/liter per h. The maintenance coefficient attained a value of 0.09 g of sugar/g of biomass per h. Increases of D produced higher protein contents of the biomass. The information obtained indicates that protein production with Candida utilis, using prickly pear juice, should be carried out a high dilution rates where the Ys and protein content of the cell mass are also higher.     

Influence of specific growth rate on biomass yield, productivity, and compostion of Candida utilis in batch and continuous culture.

Candida utilis was grown in batch and continuous culture on prickly pear juice as sole carbon and energy source. In batch culture the maximum specific growth rate (mum) and the substrate yield coefficient (Yps) varied according to sugar concentration. When the fermentation was carried out with 1% sugar, mum and Ys were 0.47/h and 42.6%, respectively. The best yields occurred in a chemostat at the pH range of 3.5 to 4.5 and temperature of 30 C. A beneficial effect on Ys was observed when the dilution rate (D) was increased. At a D of 0.55/h, the productivity was 2.38 g/liter per h. The maintenance coefficient attained a value of 0.09 g of sugar/g of biomass per h. Increases of D produced higher protein contents of the biomass. The information obtained indicates that protein production with Candida utilis, using prickly pear juice, should be carried out a high dilution rates where the Ys and protein content of the cell mass are also higher.     

Continuous ethanol production by thermotolerant Kluyveromyces marxianus IMB3 immobilized on mineral Kissiris at 45°C

Thermotolerant Kluyveromyces marxianus var. marxianus IMB3 yeast strain was immobilized on Kissiris (mineral glass foam derived from lava) in column packed reactors, and used for ethanol production from glucose or molasses under continuous culture conditions at temperatures between 40 and 50°C. Both ethanol yield and fermentation efficiency were highest at 45°C and a dilution rate (D) of 0.15/h. Increasing sugar concentration led to an increase in ethanol yield of up to 68.6 and 55.9 g/l on approx. 200g glucose or molasses, respectively. Optimum fermentation efficiency (experimental yields over theoretical maximum yields) however was at about 15% sugar for both glucose and molasses. Slight aeration (25 ml of air/min) through the medium addition line was found advantageous due to its mixing effect and probable maintenance of activity.     

Kinetics of batch ethanol fermentation of cheese-whey powder (CWP) solution as function of substrate and yeast concentrations

A lactose utilizing yeast strain, Kluyveromyces marxianus DSMZ-7239 was used for ethanol formation from cheese-whey powder (CWP) solution in batch experiments. Effects of initial substrate (CWP) and yeast concentrations on the rate and extent of ethanol formation were investigated. The initial pH and oxidation-reduction potential (ORP) was kept at 5 and -250 mV, respectively. The rate and extent of ethanol formation increased with increasing CWP concentration up to 156 g l(-1) (75 g l(-1) sugar) and then decreased for larger CWP concentrations due to substrate inhibition at high sugar concentrations. The ethanol yield coefficient was also maximum (0.54 g EtOH/g sugar) and equal to the theoretical yield at CWP concentration of 156 g l(-1). The growth yield coefficient was found to be Y(x/s)=1.2+/-0.1g biomass g sugar(-1). The rate of sugar utilization and ethanol formation also increased linearly with increasing initial biomass concentrations. A kinetic model describing the rate of sugar utilization and substrate inhibition as function of the initial substrate and the biomass concentrations was developed. The kinetic constants were determined using the experimental data. Model predictions of sugar utilization rates were in good agreement with the experimental data. The results indicated that the initial sugar concentration should be below 75 g l(-1) (CWP<156 g l(-1)) and the initial biomass should be above 850 mg l(-1) to obtain high rates and yields of ethanol formation and to avoid substrate inhibition.     

Ethanol production from concentrated whey permeate using a fed-batch culture ofKluyveromyces fragilis

Summary Fed-batch fermentation of non-supplemented concentrated whey permeate resulted in high ethanol productivity for feeds of lactose for which batch fermentation had a poor performance. At an initial lactose concentration of 100 g/L and a constant lactose feeding rate of 18 g/h we have obtained: ethanol concentration 64 g/L, ethanol productivity 3.3 g/Lh, lactose consumption 100%, ethanol yield 0.47 g/g, and biomass yield 0.058 g/g.Nomenclature St total lactose fed per medium volume in the bioreactor, g/L - Si initial lactose concentration, g/L - F lactpse feeding rate, g/h - P final ethanol concentration, g/L - Y_p/s ethanol yield, g ethanol/g lactose - Y_x/s biomass yield, g biomass/g lactose - X_S lactose consumption, % - Qp overall ethanol volumetric productivity, g/Lh - m maximum specific growth rate, h - qsm maximum specific lactose consumption rate, g/gh - qpm maximum specific ethanol production rate, g/gh     

Batch fermentation kinetics of ethanol production by Zymomonas mobilis on cellulose hydrolysate

Summary Growth and ethanol production by three strains (MSN77, thermotolerant, SBE15, osmotolerant and wild type ZM4) of the bacterium Zymomonas mobilis were tested in a rich medium containing the hexose fraction from a cellulose hydrolysate (Aspen wood). The variations of yield and kinetic parameters with fermentation time revealed an inhibition of growth by the ethanol produced. This inhibition may result from the increase in medium osmolality due to ethanol formation from glucose.Nomenclature S glucose concentration (g/L) - C conversion of glucose (%) - t fermentation time (h) - q_S specific glucose uptake rate (g/g.h) - q_p specific ethanol productivity (g/g.h) - Q_p volumetric ethanol productivity (g/L.h) - Q_X volumetric biomass productivity (g/L.h) - Y_X/S biomass yield (g/g) - Y_p/S ethanol yield (g/g) - specific growth rate (h^-1)     

Studies on growth and metabolism of Oenococcus oeni on sugars and sugar mixtures

AIMS: To study the effect of sugars and sugar mixtures on the growth kinetics of Oenococcus oeni NCIMB 11648 in batch culture with the aim of producing a high cell productivity system for starter cultures. METHODS AND RESULTS: The growth of O. oeni was investigated on single sugars (glucose, fructose or sucrose) and their mixtures (glucose-fructose, glucose-sucrose or fructose-sucrose). Better growth was obtained on sugar mixtures compared with growth on a single sugar. The production system of O. oeni biomass was investigated in batch culture with or without pH control with respect to kinetics, specific growth rate and biomass yield. The effect of pH and substrate concentration on fermentation balances and ATP yield were determined. The optimal growth of O. oeni was achieved on the glucose-fructose mixture (9 g l(-1), 1 : 1) at pH 4.5 and 25 degrees C with pH control, with highest cell volumetric productivity (7.9 mg cell l(-1) h(-1)), biomass yield (0.041 g cell g(-1) sugar) and specific growth rate (0.066 h(-1)). CONCLUSIONS: The limitations to the growth of O. oeni were pH and inhibition by end product resulting in poor utilization of the medium with low cell yields. The cell productivity of the system can be improved by the appropriate use of mixed sugar growth medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This study uniquely showed that appropriate sugar mixtures with the correct environmental conditions can significantly improve the productivity of O. oeni cultures.     

Ethanol production from wood hydrolysate using genetically engineered Zymomonas mobilis

An ethanologenic microorganism capable of fermenting all of the sugars released from lignocellulosic biomass through a saccharification process is essential for secondary bioethanol production. We therefore genetically engineered the ethanologenic bacterium Zymomonas mobilis such that it efficiently produced bioethanol from the hydrolysate of wood biomass containing glucose, mannose, and xylose as major sugar components. This was accomplished by introducing genes encoding mannose and xylose catabolic enzymes from Escherichia coli. Integration of E. coli manA into Z. mobilis chromosomal DNA conferred the ability to co-ferment mannose and glucose, producing 91 % of the theoretical yield of ethanol within 36 h. Then, by introducing a recombinant plasmid harboring the genes encoding E. coli xylA, xylB, tal, and tktA, we broadened the range of fermentable sugar substrates for Z. mobilis to include mannose and xylose as well as glucose. The resultant strain was able to ferment a mixture of 20 g/l glucose, 20 g/l mannose, and 20 g/l xylose as major sugar components of wood hydrolysate within 72 h, producing 89.8 % of the theoretical yield. The recombinant Z. mobilis also efficiently fermented actual acid hydrolysate prepared from cellulosic feedstock containing glucose, mannose, and xylose. Moreover, a reactor packed with the strain continuously produced ethanol from acid hydrolysate of wood biomass from coniferous trees for 10 days without accumulation of residual sugars. Ethanol productivity was at 10.27 g/l h at a dilution rate of 0.25 h(-1).