Continuous ethanol production by flocculating yeast in the fluidized bed bioreactor

Abstract: Continuous fermentation by a highly flocculant strain of the yeast Saccharomyces cerevisiae was carried out in a tower fluidized-bed bioreactor. The synthetic and molasses media with a total sugar concentration of 17% (w/v) were used for fermentation. Different dilution rates were tested. Stable cell densities of 50 kg m^-3(dry weight) were maintained for all dilution rates. The ethanol productivity was increasing linearly with dilution rates up to 15—20 kg m^-3 h^-1. Aeration of the culture stabilized flocculating activity and viability of yeast and also permitted long-term operation of the bioreactor.     

Relationships between hydrodynamics and rheology of flocculating yeast suspensions in a high-cell-density airlift bioreactor

In this article a hydrodynamic and rheological analysis of a continuous airlift bioreactor with high-cell-density system is presented. A highly flocculating recombinant strain of Sacharomyces cerevisiae containing genes for lactose transport (lactose permease) and hydrolysis (beta-galactosidase) was exploited to ferment lactose from cheese whey to ethanol. The magnetic particle-tracer method was used to assess the effect of operational conditions (air-flow rate, biomass concentration) on hydrodynamic behavior of an airlift bioreactor during the fermentation process. Measurements of liquid circulation velocity showed the existence of a critical value of biomass concentration at which a dramatic deceleration of net liquid flow appeared with increasing biomass quantity. Rheological analysis revealed exponential increase of viscosity of the yeast floc suspension at the same biomass concentration of about 73 g/dm3 corresponding to 42.8% v/v of solid fraction. These facts have a particular importance for the successful processing of a high-cell-density airlift bioreactor as only a circulated flow regime will be favorable to keep the solid particles in suspension state and evenly distributed throughout the bioreactor.     

Diffusivity measurement in a flocculating yeast layer

Summary The diffusion of small molecules (sugars and alcohols) through a dense layer of flocculating yeast has been studied and compared to the theorical value calculated from parameters such as porosity and yeast density. Lactose, ethanol and methanol were chosen as model molecules. In comparison with water, the diffusion rate of lactose is divided by three. Alcohols which cross the cell envelope diffuse relatively faster than lactose which was assumed not to penetrate the cells. An increase of the lactose diffusion rate is observed when the yeast are heat killed.     

Construction of a flocculating yeast for fuel ethanol production

The expression vector pYX212 harboring FLO1 gene and kanMX gene was transformed into Saccharomyces. cerevisiae ZWA46. The transformant, ZWA46-F2, showed strong and stable flocculation ability during 20 serial batch cultivations. The flocculation onset of the strain is in the early stationary growth phase, not coincident with the glucose depletion in the culture medium. The flocculation ability of the transformant showed no difference with the initial pH ranging from 3.5 to 6.0. Furthermore, the ethanol concentration and other properties of the transformant strain ZWA46-F2 were similar to those of the wild-type strain ZWA46.     

Inulin enrichment by fermentation in a flocculating yeast reactor

Summary Simultaneous production of ethanol and fructose enriched syrups was obtained from Jerusalem artichoke extract using a Saccharomyces diastaticus flocculating yeast in a continuous gas-lift reactor with internal biomass recycle. This allowed the production of 42 g/L of ethanol and 70 g/L of inulin containing up to 92% fructose (fructose/glucose ratio of 11). These results can be compared to the batch and chemostat fermentations which gave a higher ethanol concentration but a lower fructose enrichment. Mass transfert limitations can explain both the productivity decrease and the selectivity improvement in the gas-lift reactor.     

Construction of a flocculating yeast for fructose production from inulin

Construction of flocculating yeast lacking for fructose utilisation was realised by integration of the FLO1 flocculation gene in the ribosomal DNA of an hexokinase deficient (hxk1, hxk2) Saccharomyces cerevisiae strain (ATCC36859). Simultaneous production of ethanol and fructose was obtained from glucose/fructose mixtures or from hydrolysed Jerusalem artichoke extracts using the transformed yeast in batch fermentations and in a continuous reactor with internal biomass recycle. This allowed the production of 5 g ethanol/L and 48 g sugars/L containing up to 99 % fructose from diluted hydrolysed Jerusalem artichoke extracts containing 60 g sugars/L. © Rapid Science Ltd. 1998     

Continuous ethanol production by a flocculating strain of Kluyveromyces marxianus: bioreactor performance

A flocculating strain of Kluyveromyces marxianus was used for alcoholic fermentation in a continuous bioreactor working with zero residual concentration in effluent. Specific kinetic parameters were improved by increasing dilution rate, which is similar to results obtained with ultrafiltration systems. Specific biomass accumulation rate had always a value greater than 92.5% of specific biomass growth rate and was independent of the dilution rate. Productivity is shown to be 12.5 times greater than in conventional continuous operation and is directly proportional to dilution rate. Maximum biomass concentration also presents a linear relationship with dilution rate. The largest obtained biomass concentration is 8 times greater than in a conventional continuous fermentor.     

Continuous high-ethanol fermentation from cane molasses by a flocculating yeast

Repeated-batch fermentation by a flocculating fusant, Saccharomyces cerevisiae HA 2, was done in a molasses medium that contained 20% (w/v) total sugar, at 30°C in an automatically controlled fermentor, and the effects of ethanol concentration on the specific growth rate and the specific production rate of ethanol were studied. Both the specific growth rate and the specific production rate of ethanol fell with increase of ethanol concentration, and there was a linear correlation between each rate and the concentration of thanol. The maximum specific growth rate (μ_max) and the maximum specific production rate of ethanol (q_max) were 0.12 h⁻¹ and 0.1 g ethanol/10⁹ cells·h, respectively. The specific growth rate and the specific production rate of ethanol fell to zero at ethanol concentration of 89 g/l and 95 g/l, respectively. The number of viable cells, calculated from the linear inhibition equation, was 1.3 × 10⁹ cells/ml for production of 85 g/l ethanol at a dilution rate (D₁) of 0.2 h⁻¹. Based on this estimation, a laboratory-scale continuous fermentation, using two fermentors in series, was done. In the second fermentor, 85 g/l ethanol was produced at a dilution rate (D₁) of 0.2 h⁻¹ by the active feedig of the fermented mash from the first fermentor into the second fermentor by pumping (hereafter called active feeding). To maintain the number of viable cells above 10⁹ cells/ml in the second fermentor, a active feeding ratio of more than 23% was required. Under these conditions, 81 g/l ethanol was produced in the second fermentor at a dilution rate (D_t) of 0.25 h⁻¹, and the high ethanol productivity of 20.3 g/l·h could be achieved. A bench-scale continuous fermentation, using two fermentors in series, with a active feeding ratio of 25% was done. An ethanol concentration of 84 g/l in the second fermentor at a dilution rate (D_t) of 0.25 h⁻¹ was achieved, just as it was in the laboratory-scale fermentation test.     

Utilization of an external loop bioreactor for the isolation of a flocculating strain ofKluyveromyces marxianus

A continuous open loop bioreactor was used to induce flocculation in an originally nonflocculent strain ofKluyveromyces marxianus. The sedimentation capacity of the isolated strain was of such a magnitude that the cell concentration inside the fermentor was 50 times larger than in the effluent. Also, a batch system was used with the same objective, but no flocculation was obtained.The kinetic parameters of the flocculent strain were compared with those of the mother strain. It was shown that both maximum specific growth rate and maximum specific ethanol production rate were lower in the flocculent strain. Ethanol had a larger inhibitory effect on the kinetic parameters of the isolated strain. Also, the batch fermentations with this strain presented a larger final biomass concentration and a reduced ethanol yield.     

Optimization of two-stage continuous ethanol fermentation using flocculating yeast

Summary Two-stage two-stream continuous ethanol fermentation using flocculating yeast was studied to produce high ethanol concentration from blackstrap cane-molasses. Optimizing pH, RQ, cell concentration, and medium molasses concentration of the first stage to give a good cell yield without a significant decrease in ethanol yield, the final ethanol concentration of as high as 92.0–93.5 g/l at an overall dilution rate of 0.10 l/h was obtained. This process could work stably as long as 3 weeks without any decreases in the efficiency.     

Enhancement of tannase production by Lactobacillus plantarum CIR1: validation in gas-lift bioreactor

The optimization of tannase production by Lactobacillus plantarum CIR1 was carried out following the Taguchi methodology. The orthogonal array employed was L18 (2¹ × 3⁵) considering six important factors (pH and temperature, also phosphate, nitrogen, magnesium, and carbon sources) for tannase biosynthesis. The experimental results obtained from 18 trials were processed using the software Statistical version 7.1 using the character higher the better. Optimal culture conditions were pH, 6; temperature, 40 °C; tannic acid, 15.0 g/L; KH₂PO₄, 1.5 g/L; NH₄Cl, 7.0 g/L; and MgSO₄, 1.5 g/L which were obtained and further validated resulting in an enhance tannase yield of 2.52-fold compared with unoptimized conditions. Tannase production was further carried out in a 1-L gas-lift bioreactor where two nitrogen flows (0.5 and 1.0 vvm) were used to provide anaerobic conditions. Taguchi methodology allowed obtaining the optimal culture conditions for the production of tannase by L. plantarum CIR1. At the gas-lift bioreactor the tannase productivity yields increase 5.17 and 8.08-fold for the flow rates of 0.5 and 1.0 vvm, respectively. Lactobacillus plantarum CIR1 has the capability to produce tannase at laboratory-scale. This is the first report for bacterial tannase production using a gas-lift bioreactor.     

Increase of ethanol productivity by cell-recycle fermentation of flocculating yeast

Using the recombinant flocculating Angel yeast F6, long-term repeated batch fermentation for ethanol production was performed and a high volumetric productivity resulted from half cells not washed and the optimum opportunity of residual glucose 20 g l⁻¹ of last medium. The obtained highest productivity was 2.07 g l⁻¹ h⁻¹, which was improved by 75.4% compared with that of 1.18 g l⁻¹ h⁻¹ in the first batch fermentation. The ethanol concentration reached 8.4% corresponding to the yield of 0.46 g g⁻¹. These results will contribute greatly to the industrial production of fuel ethanol using the commercial method with the flocculating yeast.     

絮凝酵母吸附Cr(VI) 的机理

絮凝酵母SPSC01为酿酒酵母Saccharomyces cerevisiae和粟酒裂殖酵母Schizosaccharomyces pombe的融合菌株,用其吸附水溶液中的重金属Cr(VI),可以大大降低生物吸附的固液分离成本。为了探讨SPSC01菌体絮凝蛋白对Cr(VI) 还原吸附的影响,对SPSC01与其亲本菌株的吸附行为进行了比较。结果表明,SPSC01和其具有絮凝性状的亲本S. pombe的Cr(VI) 去除速率基本同步,远优于无絮凝性状的亲本S. cerevisiae;达到吸附平衡时,S. pombe、SPSC01和S. cerevisiae对总Cr去除率分别达68.8％、48.6％和37.5％;从而证明了絮凝有利于Cr(VI) 的还原、吸附,絮凝蛋白在Cr(VI) 的还原吸附过程中起促进作用。通过化学屏蔽方法和傅立叶变换红外光谱 (FTIR) 分析,对SPSC01菌体表面吸附Cr(VI) 的机理进行了研究,结果表明SPSC01菌体表面吸附Cr(VI) 起主要作用的基团是氨基、羧基和酰胺基。     

Simulated scale-up of a yeast fermentation using a loop bioreactor

A tubular loop reactor was used to carry out the simulated scale-up of aSaccharomyces cerevisiae fermentation. Performance of the microorganism was assessed in terms of biomass, ethanol concentration respiratory quotient and yield. The behaviour of the culture was shown to be similar in loop reactor (t_c, 10s) and STR.     

絮凝酵母吸附Cr(VI)的机理

絮凝酵母SPSC01为酿酒酵母Saccharomyces cerevisiae和粟酒裂殖酵母Schizosaccharomyces pombe的融合菌株,用其吸附水溶液中的重金属Cr(VI),可以大大降低生物吸附的固液分离成本。为了探讨SPSC01菌体絮凝蛋白对Cr(VI)还原吸附的影响,对SPSC01与其亲本菌株的吸附行为进行了比较。结果表明,SPSC01和其具有絮凝性状的亲本S.pombe的Cr(VI)去除速率基本同步,远优于无絮凝性状的亲本S.cerevisiae;达到吸附平衡时,S.pombe、SPSC01和S.cerevisiae对总Cr去除率分别达68.8%、48.6%和37.5%;从而证明了絮凝有利于Cr(VI)的还原、吸附,絮凝蛋白在Cr(VI)的还原吸附过程中起促进作用。通过化学屏蔽方法和傅立叶变换红外光谱(FTIR)分析,对SPSC01菌体表面吸附Cr(VI)的机理进行了研究,结果表明SPSC01菌体表面吸附Cr(VI)起主要作用的基团是氨基、羧基和酰胺基。     

Continuous ethanol fermentation in a tower reactor with flocculating yeast recycle: scale-up effects on process performance, kinetic parameters and model predictions

An unsegregated and unstructured model developed for a small-scale process of ethanol production in a tower reactor with cell recycle was applied to describe the experimental data obtained in a large-scale process. The model was developed considering the following points: reactor hydrodynamic behavior analogous to that of ideal CSTR, substrate limitation, inhibition phenomena linked both to ethanol and to biomass, absence of fermentation in the settler, and no loss of cell viability. The scale-up criterion consisted in maintaining an identical relation settler volume/fermentor volume on the two scales. All large-scale experiments were carried out using a flocculating yeast strain IR-2, isolated from fermented food, and identified as Saccharomyces cerevisiae. Sugarcane juice was used as the substrate source with sugar concentrations of 150?g/l. Different values of dilution rate and recycle ratio were employed (D?=?0.11–0.33?h^?1, α?=?5.4–18.0) and the temperature was of 32?°C. The kinetic parameters were similar on both scales and the model predictions agreed well with the large-scale experimental data.     

Fermentation of lactose to ethanol with recombinant yeast in an immobilized yeast membrane bioreactor

A novel concept of membrane bioreactor in which living cells are sandwiched between ultrafiltration (UF) and reverse osmosis (RO) membranes was applied for lactose fermentation to ethanol by genetically engineered yeast cells. The productivity of the Lactophile 13B strains was higher than that of the Lactophile 13D strains. In both cases performance data similar to those for glucose fermentation to ethanol by Saccharomyces cerevisiae were obtained. However, the operational stability of recombinant yeast cells was improved in the new bioreactor in comparison to the stability of these cells in a shake flask.     

发酵抑制物对絮凝酵母戊糖发酵的影响

将絮凝剂加入酵母溶液中,使酵母絮凝成颗粒以此作为固定化酵母进行戊糖发酵。研究了常见发酵抑制物(甲酸、乙酸、糠醛和乳酸等)对絮凝酵母发酵木糖的影响。结果表明:在60.0g/L木糖发酵液中,经过24h发酵,木糖利用率达94.6%,当分别添加抑制物甲酸、乙酸、糠醛、乙醇和乳酸时,聚氧乙烯絮凝酵母分别对其的耐受浓度为0.5、0.5、1.0、30.0和8.0g/L。当抑制物添加量超过各自的耐受浓度后,对絮凝酵母发酵会产生明显的抑制作用。     

Exploration of a natural reservoir of flocculating genes from various Saccharomyces cerevisiae strains and improved ethanol fermentation using stable genetically engineered flocculating yeast strains

Flocculating yeast strains with good fermentation ability are desirable for brewing industry as well as for fuel ethanol production, however, the genetic diversity of the flocculating genes from natural yeast strains is largely unexplored. In this study, FLO1, FLO5, FLO9, FLO10 and FLO11 PCR products were obtained from 16 yeast strains from various sources, and the PCR product amplified from FLO1 of the self-flocculating yeast strain SPSC01 was used for the construction of expression cassette flanked by homologous fragments of the endonuclease gene HO for chromosome integration. A genetically engineered flocculating yeast BHL01 with good fermentation performance was obtained by transforming an industrial strain Saccharomyces cerevisiae 4126 with the expression cassette. The fermentation performances of SPSC01 and BHL01 in flask fermentation were compared using 208 g/L glucose. BHL01 completed the fermentation 8 h earlier than SPSC01, while no significant difference between BHL01 and S. cerevisiae 4126 was observed. In very high gravity repeated batch ethanol fermentation using 255 g/L glucose, BHL01 maintained stable flocculation for at least over 24 batches, while SPSC01 displayed severe deflocculation under the same conditions. The natural reservoir of flocculating genes from yeast strains may represent an unexplored gene source for the construction of new flocculating yeast strains for improved ethanol production.