Effects of furfural on ethanol fermentation bySaccharomyces cerevisiae: Mathematical models

Different inocula with high yeast concentration were investigated as a means of overcoming the inhibitory effect of furfural in ethanol fermentation. In order to verify the toxicity of the furfural, a series of fermentation runs were made with 0.25, 5.50, and 9.00 g/L (dry weight) ofSaccharomyces cerevisiae inoculum and 1, 3, and 5 g/L of furfural. The extent of cell death occurring in the early phase of fermentation was dependent on the initial cell concentration. With high initial yeast concentration, the effect of furfural is canceled, because it is depleted at an early stage of fermentation. The ethanol weight yield averaged 0.45 on the basis of sugar consumed. The ethanol productivity and specific growth rate decreased with the increase of furfural concentration, and the inhibitory effect almost disappeared with high cell concentration (9 g/L). Mathematical models were developed that relate productivity and growth rate with furfural and cell concentration.     

Influence of growth factor supplements on butyric acid production from sucrose byClostridium butyricum

During batch fermentation of sucrose to butyric acid byClostridium butyricum the effect of growth factor supplementation was determined: addition of yeast extract (5 g/L) stimulated most. Using biotin as the sole growth factor, average productivity was definitely lower. Beet molasses as a combined source of carbon and growth factor were effective only at a high concentration (150 g/L). The optimal butyric acid production (45 g/L, yield 45%) was achieved with sucrose concentration of 100 g/L in a medium supplemented with yeast extract (5 g/L). It represents an average productivity of 0.90 gL⁻¹ h⁻¹ and relative butyric acid concentration of 91%.     

高糖和氮源对鼠李糖乳杆菌(Lactobacillus rhamnosus)L-乳酸发酵的影响

鼠李糖乳杆菌经实验室耐高糖高酸选育,能够在高糖浓度下高效高产L-乳酸。以酵母粉为氮源和生长因子,葡萄糖初始浓度分别为120 g/L和146 g/L,摇瓶培养120h,L-乳酸产量分别为104g/L和117.5g/L,L-乳酸得率分别为86.7%和80.5%。高葡萄糖浓度对菌的生长和乳酸发酵有一定的抑制。增加接种量,在高糖浓度发酵条件下,可以缩短发酵时间,但对增加乳酸产量效果不明显。乳酸浓度对鼠李糖乳杆菌生长和产酸有显著的影响。初始乳酸浓度到达70g/L以上时,鼠李糖乳杆菌基本不生长和产酸,葡萄糖消耗也被抑制。酵母粉是鼠李糖乳杆菌的优良氮源,使用其它被测试的氮源菌体生长和产酸都有一定程度的下降。用廉价的黄豆粉并补充微量维生素液,替代培养基中的酵母粉,可以使产酸浓度和碳源得率得以基本维持。     

Enhancement of ribitol production during fermentation of <Emphasis Type="Italic">Trichosporonoides oedocephalis</Emphasis> ATCC 16958 by optimizing the medium and altering agitation strategies

To maximize the productivity of ribitol, which is an important starting material for the production of one expensive rare sugar, L-ribose, the effects of culture medium and agitation speed on cell growth as well as on the productivity of ribitol were thoroughly investigated in a 7 L fermentor. The maximum volumetric productivity, 0.322 g/L/h of ribitol, were obtained at an initial glucose concentration of 200 g/L in a batch culture. Based on the optimum glucose concentration, the ribitol yield conversed from glucose was up to 0.193 g/g when 1% yeast extract was used as a nitrogen source. When the agitation speed was maintained at 200 rpm, the ribitol concentration of 38.60 g/L was collected after 120 h of cultivation time. Additionally, the scheme of two-phase agitation and glucose infusion was employed. To begin, in the first 24 h of fermentation, a high agitation rate at 350 rpm and the initial glucose concentration of 50 g/L were applied, and the biomass concentration of 25.50 g/L was achieved at 36 h of incubation; whereas this value was observed until 60 h in the former batch fermentation methods. Then, in the second phase, with the agitation speed reduced to 150 rpm and the infusion amount of glucose controlled at 150 g/L, the yield of ribitol reached to 65.00 g/L in two-phase agitation fermentation and was 1.68 fold of that obtained in one-stage batch fermentation. To our knowledge, this study first demonstrates its significant effectiveness in improving ribitol production with the application of Trichosporonoides oedocephalis ATCC 16958.     

Proline enhances <Emphasis Type="Italic">Torulopsis glabrata</Emphasis> growth during hyperosmotic stress

This study investigated that the importing of compatible solute proline could enhance the growth of the yeast Torulopsis glabrata under hyperosmotic stress. Osmolarity progressively increased from 860 to 2,603 mOsmol/kg by accumulation of sodium pyruvate in the culture broth, leading to a significant decrease in cell growth. When 1.0 g/L of proline as a compatible solute was added to the culture medium, it was imported and enhanced cell growth by 59.0% at 2,603 mOsmol/kg. By addition of proline during pyruvate production, the concentration, productivity, and yield of pyruvate increased 22.1, 38.4, and 14.3%, respectively. These results suggested that T. glabrata can import proline as an osmoprotectant against high osmotic stress, thus enhance pyruvate productivity. The improvement of yeast growth and viability under hyperosmotic stress by the addition of proline provided an alternative approach to enhance the organic acids production by yeast.     

发酵产丁二酸过程中废弃细胞的循环利用

对厌氧发酵产丁二酸后的废弃细胞进行破壁处理,考察了以细胞水解液作为有机氮源重新用于丁二酸发酵的可行性。比较了超声破碎、盐溶、酶解3种方法破碎细胞获得的水解液作为氮源发酵产丁二酸的效果,结果表明酶解制得的细胞水解液效果最佳。以总氮含量为1.11g/L的酶解液(相当于10g/L酵母膏)作为氮源发酵,丁二酸产量可达42.0g/L,继续增大酶解液用量对耗糖、产酸能力没有显著提高。将细胞酶解液与5g/L酵母膏联用发酵36h后,丁二酸产量达75.5g/L,且丁二酸生产强度为2.10g/(L·h),比使用10g/L酵母膏时提高了66.7%。因此,厌氧发酵产丁二酸结束后的废弃细胞酶解液可以替代原培养基中50%的酵母膏用于发酵。     

Expanded bed adsorption of human serum albumin from very dense Saccharomyces cerevesiae suspensions on fluoride-modified zirconia.

The adsorption of proteins from high cell density yeast suspensions on mixed-mode fluoride-modified zirconia (FmZr) particles (38 to 75 microm, surface area of 29 m(2)/g and density of 2.8 g/cm(3)) was investigated using human serum albumin (HSA) added to Saccharomyces cerevesiae as the model expression host. Because of the high density of the porous zirconia particles, HSA (4 mg/mL) can be adsorbed from a 100 g dry cell weight (DCW)/L yeast suspension in a threefold-expanded bed of FmZr. The expanded bed adsorption of any protein from a suspension containing >50 g DCW/L cells has not been previously reported. The FmZr bed expansion characteristics were well represented by the Richardson-Zaki correlation with a particle terminal velocity of 3.1 mm/s and a bed expansion index of 5.4. Expanded bed hydrodynamics were investigated as a function of bed expansion using residence time distribution studies with sodium nitrite as the tracer. The adsorption of HSA on FmZr exhibited features of multicomponent adsorption due to the presence of dimers. The protein binding capacity at 5% breakthrough decreased from 22 mg HSA/mL settled bed void volume for 20 g DCW/L yeast to 15 mg HSA/mL settled bed void volume for 40 g DCW/L yeast and remained unchanged for the higher yeast concentrations (60 to 100 g DCW/L). However, the batch (or equilibrium) binding capacity decreased monotonically as a function of yeast concentration (20 to 100 g DCW/L) and the binding capacity at 100 g DCW/L yeast was fivefold lower compared with that at 20 g DCW/L yeast. The lower batch binding capacity at high cell concentrations resulted from the adsorption of cells at the surface of the particles restricting access of HSA to the intraparticle surface area. Batch (or equilibrium) and column HSA adsorption results indicated that the adsorption of HSA on FmZr occurred at a time scale that may be much faster than that of yeast cells. The zirconia particles were cleaned of adsorbed HSA and yeast with a total of 1500 to 2000 column volumes (over many cycles) of 0. 25 M NaOH, without any significant effect on the chromatographic performance.     

Single-cell protein production from spent sulfite liquor utilizing cell-recycle and computer monitoring

Spent sulfite liquor (SSL), a waste product of the paper pulping industry, is produced at a rate of 1 ton (dry basis) per ton of pulp. The sugar content of SSL is about 30 g/L. To reduce the biological oxygen demand (BOD) of SSL before disposal, torula yeast (Candida utilis) is produced by a continuous culture process, the productivity of which is limited by sugar concentration and cell growth rate. To increase productivity, a recycle system has been designed and tested. Cells were sedimented continuously with a flocculating agent (bentonite) before being recycled to the fermentor. A bentonite concentration of 0.02 g/g cell was required. A computer monitoring system based on material balancing techniques was developed to monitor and control the recycle system. With this computer system, productivity was raised to 6.1 g/L h, with cell concentration up to 65 g/L in the recycle stream and 24 g/L in the fermentor. This represents a productivity increase of 150% over continuous culture with no recycle.     

Preparation and performance of immobilized yeast cells in columns containing no inert carrier

Schizosaccharomyces pombe was cultivated in a medium of glucose (10 g/L) malt extract (3 g/L), yeast extract (3 g/L), and bactopeptone (5 g/L) to form flocs. More than 95% of the cell population were flocculated. Variation in glucose concentration (from 10 to 100 g/L) did not affect flocculation. Yeast extract helped induce flocculation. Application of the immobilized yeast for the continuous production of ethanol was tested in a column reactor. Soft yeast flocs (50-200 mesh) underwent morphological changes to heavy particles (0.1-0.3 cm diameter) after continuously being fed with fresh substrates in the column. Productivity as high as 87 g EtOH L(-1) h(-1) was obtained when a 150 g/L glucose medium was fed. The performance of this yeast reactor was stable over a two-month period. The ethanol yield was 97% of the theoretical maximum based upon glucose consumed.     

Application of medium optimization tools for improving recombinant human interferon gamma production from Kluyveromyces lactis

The present study is focused upon improving biomass of Kluyveromyces lactis cells expressing recombinant human interferon gamma (hIFN-γ), with the aim of augmenting hIFN-γ concentration using statistical and artificial intelligence approach. Optimization of medium components viz., lactose, yeast extract, and trace elements were performed with Box–Behnken design (BBD) and artificial neural network linked genetic algorithm (ANN-GA) for maximizing biomass of recombinant K. lactis (objective function). The studies resulted over 1.5-fold improvement in the biomass concentration in a medium composed of 80?g/L lactose, 10.353?g/L yeast extract, and 15?mL/L trace elements as compared with initial biomass value. In the same study hIFN-γ concentration reached 881?µg/L which was 2.28-fold higher as compared with initial hIFN-γ concentration obtained in unoptimized medium. Further the batch fermentation study displayed mixed growth associated kinetics with the maximum hIFN-γ production rate of 1.1?mg/L. BBD and ANN-GA, both optimization techniques predicted a higher lactose concentration was clearly beneficial for augmenting K. lactis biomass which in turn increased hIFN-γ concentration.     

Kinetic modeling of Candida shehatae ATCC 22984 on xylose and glucose for ethanol production

Candida shehatae ATCC 22984, a xylose-fermenting yeast, showed an ability to produce ethanol in both glucose and xylose medium. Maximum ethanol produced by the yeast was 48.8?g/L in xylose and 52.6?g/L in glucose medium with ethanol yields that varied between 0.3 and 0.4?g/g depended on initial sugar concentrations. Xylitol was a coproduct of ethanol production using xylose as substrate, and glycerol was detected in both glucose and xylose media. Kinetic model equations indicated that growth, substrate consumption, and product formation of C. shehatae were governed by substrate limitation and inhibition by ethanol. The model suggested that cell growth was totally inhibited at 40?g/L of ethanol and ethanol production capacity of the yeast was 52?g/L, which were in good agreement with experimental results. The developed model could be used to explain C. shehatae fermentation in glucose and xylose media from 20 to 170?g/L sugar concentrations.     

Kinetics of ethanol fermentations in membrane cell recycle fermentors

Ethanol fermentation by yeast was carried out in a cell filtration recycle system with a hollow-fiber membrane filter. Maximum biomass concentrations up to 210 g dry wt/L were obtained, but in normal operation concentrations they were between 100 and 150 g/L. The ethanol productivity using 14% glucose feed was 85 g/L h, with an ethanol concentration of 65 g/L and an ethanol yield of over 90%. The ethanol productivity and yeast growth rate decreased as the cell concentration increased beyond a certain level. The cell mass in the reactor was maintained by a proper manipulation of diluticn rate and bleed ratio depending on the growth rate.     

Fermentative production of succinic acid from glucose and corn steep liquor byAnaerobiospirillum succiniciproducens

Anaerobiospirillum succiniciproducens requires expensive complex nitrogen sources such as yeast extract and polypeptone for its growth and succinic acid production. It was found thatA. succiniciproducens was able to grow in a minimal medium containing glucose when supplemented with corn steep liquor (CSL) as the sole complex nitrogen source. The concentration of CSL had a significant effect on the glucose consumption byA. succiniciproducens. When 10–15 g/L of CSL was supplemented, cells were grown to an OD₆₆₀ of 3.5 and produced 17.8 g/L succinic acid with 20 g/L glucose. These results are similar to those obtained by supplementing yeast extract and polypeptone, thereby suggesting that succinic acid can be produced more economically using glucose and CSL.     

Effect of air supplement on the performance of continuous ethanol fermentation system

For the purpose of improving ethanol productivity, the effect of air supplement on the performance of continuous ethanol fermentation system was studied. The effect of oxygen supplement on yeast concentration, cell yield, cell viability, extracellular ethanol concentration, ethanol yield, maintenance coefficient, specific rates of glucose assimilation, ethanol production, and ethanol productivity have been evaluated, using a high alcohol tolerant Saccharomyces cerevisiae STV89 strain and employing a continuous fermentor equipped with an accurate air metering system in the flow rate range 0-11 mL air/L/h. It was found that, when a small amount of oxygen up to about 80mu mol oxygen/L/h was supplied, the ethanol productivity was significantly enhanced as compared to the productivity of the culture without any air supplement. It was also found that the oxygen supplement improved cell viability considerably as well as the ethanol tolerance level of yeast. As the air supply rate was increased, from 0 to 11 mL air/L/h while maintaining a constant dilution rate at about 0.06 h(-1), the cell concentration increased from 2.3 to 8.2 g/L and the ethanol productivity increased from 1.7 to 4.1 g ethanol/L/h, although the specific ethanol production rate decreased slightly from 0.75 to 0.5 g ethanol/g cell/h. The ethanol yield was slightly improved also with an increase in air supply rate, from about 0.37 to 0.45 ethanol/g glucose. The maintenance coefficient increased by only a small amount with the air supplement. This kind of air supplement technique may very well prove to be of practical importance to a development of a highly productive ethanol fermentation process system especially as a combined system with a high density cell culture technique.     

戊糖乳杆菌发酵培养基氮源条件的优化

对戊糖乳杆菌发酵培养基的氮源条件进行了优化。通过单因素实验及响应面分析优化利用木糖高产乳酸的戊糖乳杆菌发酵培养基的不同氮源组合。优化得到的牛肉膏与柠檬酸氢二铵复合的最佳组成为牛肉膏17.72 g/L,柠檬酸氢二铵1.91 g/L,得到乳酸实际最大产量42.37 g/L。添加玉米浆与酵母粉和无机氮源复合的最佳组成为玉米浆46.54 g/L,酵母粉21.95 g/L,柠檬酸氢二铵9.95 g/L,可得到乳酸最大产量41.06 g/L。通过响应面优化减少了有机氮源的种类。牛肉膏与柠檬酸氢二铵的复合得到了更高的乳酸产量,且减少了有机氮源用量,节约了成本。玉米浆与酵母粉的复合解决了单一玉米浆造成的木糖利用速率过低的问题,同样得到较高浓度的乳酸。     

Ethanol production from cellulosic materials using cellulase-expressing yeast

We demonstrate direct ethanol fermentation from amorphous cellulose using cellulase-co-expressing yeast. Endoglucanases (EG) and cellobiohydrolases (CBH) from Trichoderma reesei, and β-glucosidases (BGL) from Aspergillus aculeatus were integrated into genomes of the yeast strain Saccharomyces cerevisiae MT8-1. BGL was displayed on the yeast cell surface and both EG and CBH were secreted or displayed on the cell surface. All enzymes were successfully expressed on the cell surface or in culture supernatants in their active forms, and cellulose degradation was increased 3- to 5-fold by co-expressing EG and CBH. Direct ethanol fermentation from 10 g/L phosphoric acid swollen cellulose (PASC) was also carried out using EG-, CBH-, and BGL-co-expressing yeast. The ethanol yield was 2.1 g/L for EG-, CBH-, and BGL-displaying yeast, which was higher than that of EG- and CBH-secreting yeast (1.6 g/L ethanol). Our results show that cell surface display is more suitable for direct ethanol fermentation from cellulose.     

Modeling of Xanthophyllomyces dendrorhous growth on glucose and overflow metabolism in batch and fed-batch cultures for astaxanthin production

An astaxanthin-producing yeast Xanthophyllomyces dendrorhous ENM5 was cultivated in a liquid medium containing 50 g/L glucose as the major carbon source in stirred fermentors (1.5-L working volume) in fully aerobic conditions. Ethanol was produced during the exponential growth phase as a result of overflow metabolism or fermentative catabolism of glucose by yeast cells. After accumulating to a peak of 3.5 g/L, the ethanol was consumed by yeast cells as a carbon source when glucose in the culture was nearly exhausted. High initial glucose concentrations and ethanol accumulation in the culture had inhibitory effects on cell growth. Astaxanthin production was partially associated with cell growth. Based on these culture characteristics, we constructed a modified Monod kinetic model incorporating substrate (glucose) and product (ethanol) inhibition to describe the relationship of cell growth rate with glucose and ethanol concentrations. This kinetic model, coupled with the Luedeking-Piret equation for the astaxanthin production, gave satisfactory prediction of the biomass production, glucose consumption, ethanol formation and consumption, and astaxanthin production in batch cultures over 25-75 g/L glucose concentration ranges. The model was also applied to fed-batch cultures to predict the optimum feeding scheme (feeding glucose and corn steep liquor) for astaxanthin production, leading to a high volumetric yield (28.6 mg/L) and a high productivity (5.36 mg/L/day).     

Biotransformation of benzaldehyde by Saccharomyces cerevisiae: characterization of the fermentation and toxicity effects of substrates and products

Although higher initial rates of phenylacetyl carbinol formation were observed in fermentations containing a high starting benzaldehyde level, a massive reduction in yeast viability was observed resulting in early cessation of production formation. Pulse feeding to maintain lower benzaldehyde concentrations resulted in a lower initial reaction rate, but prolonged yeast viability and the biotransformation. This resulted in higher overall product tilers. As benzaldehyde concentration was increased, yeast growth rate was reduced (0.5 g/L), inhibited (1-2 g/L), or cell viability reduced (3 g/L). Benzaldehyde appeared to alter the cell permeability barrier to substrates and products. Reductions in yeast biomass levels and especially protein and lipid content were observed during the biotransformation. The effects of benzaldehyde and reaction products on yeast pyruvate decarboxylase and alcohol dehydrogenase stability were determined. Homogenized yeast cells produced similar phenylacetyl carbinol levels to whole yeast only if supplemented with thiamine pyrophosphate and magnesium.     

Improved production of curdlan with concentrated cells ofAgrobacterium sp.

The addition of a limited concentration of yeast extract to a minimal salt medium (MSM) enhanced cell growth and increased the production of curdlan whereas nitrogenlimitation was found to be essential for the higher production of curdlan byAgrobacterium sp. ATCC 31749. As the amount of the inoculum increased, the cell growth as well as the production of curdlan also increased in the MSM without a nitrogen source. The cell growth and production of curdlan increased as the initial pH of the medium decreased as low as 5.0. The conversion rate and concentration of curdlan from 2% (w/v) glucose in the MSM with concentrated cells under nitrogen deletion was 67% and 13.4 g/L, respectively. The highest conversion rate of curdlan under the conditions optimized in this study was 71% when the glucose concentration was 1% (w/v).