The use of plant cell vesicles for immobilization of yeast cells producing ethanol

The preparation of immobilized living yeast cells adsorbed into or onto delipided specimens of the dwarf duckweed Wolffia arrhiza (Fam. Lemnaceae) is reported. These yeast cell-plant cell conjugates were used for the repeated batch production of ethanol from glucose (143 to 246 g/l) or saccharose (150 g/l). Up to 25 fermentation cycles at 30°C were performed. The cycle time for complete substrate conversion to ethanol was reduced 10-fold by a 5-fold increase of the yeast cell Wolffia conjugate concentration (ε = 0.08 to ε =0.4) ε = volume of cell conjugate/totnl reaction volume. The corresponding ethanol production was 11.5 to 13.5 vol% and 9 vol% respectively. The reported results on the discontinuous ethanol fermentation with Wolffia-immobilized yeast cells open the field for their application in continuous ethanol production processes.     

Bacterial cellulose membrane – A new support carrier for yeast immobilization for ethanol fermentation

The aim of the work was to study the properties of the bacterial cellulose membrane (BCM) and the feasibility of using it as a new, environmentally friendly support carrier for yeast cell immobilization. It was observed that the morphology of BCM varied with different cultivation methods and the scanning electron microscopy (SEM) images confirmed that the yeast cells were entrapped in the porous network of BCM obtained from the static culture and stabilized by the cross-linked fibrils. Particularly, the research confirmed the effectiveness of yeast immobilization in BCM reflected by the high yield of alcohol (9.7% v/v, a 21.25% increase of those using free cells) and the high stability. The specific rate of ethanol production by the immobilized cells in BCM was 2.1 g g⁻¹ h⁻¹, 31.3% greater than that of the suspended cells. Results implied that applying BCM as the support carrier had little adverse effects on cell viability and proliferation. Instead, it facilitated the product leakage and nutrients transportation through the porous network.     

The role of cell immobilization in fermentation technology

The distinction between immobilized cell fermentation and immobilized cell biocatalysis is seldom made, though they are conceptually quite different. Unlike immobilized enzyme systems, immobilized viable cells can be used to carry out conventional fermentations. Microbial cells which would otherwise be freely dispersed (in almost colloidal suspension) within the fermentation environment can be encouraged to become attached in some way to a support (carrier), thus producing a discrete particulate solid phase. Such immobilization offers several potential advantages of a process engineering nature to the fermentation system. These include ease of handling and of cell separation, and lowering of bulk viscosity, as well as the obvious potential benefits of increased cell concentration.     

Effects of by-products of fermentation of banana pseudostem on ethanol separation by pervaporation

In this work, we performed recovery of ethanol from a fermentation broth of banana pseudostem by pervaporation (PV) as a lower-energy-cost alternative to traditional separation processes such as distillation. As real fermentation systems generally contain by-products, it was investigated the effects of different components from the fermentation broth of banana pseudostem on PV performance for ethanol recovery through commercial flat sheet polydimethylsiloxane (PDMS) membrane. The experiments were compared to a binary solution (ethanol/water) to determine differences in the results due to the presence of fermentation by-products. A real fermented broth of banana pseudostem was also used as feed for the PV experiments. Seven by-products from fermented broth were identified: propanol, isobutanol, methanol, isoamyl alcohol, 1-pentanol, acetic acid, and succinic acid. Moreover, the residual sugar content of 3.02 g/L¹ was obtained. The presence of methanol showed the best results for total permeate flux (0.1626 kg·m⁻²·h⁻¹) and ethanol permeate flux (0.0391 kg·m⁻²·h⁻¹) during PV at 25°C and 3 wt% ethanol, also demonstrated by the selectivity and enrichment factor. The lowest total fluxes of permeate were observed in the experiments containing the acids. Better permeance of 0.1171 from 0.0796 kg·m⁻²·h⁻¹ and membrane selectivity of 9.77 from 9.30 were obtained with real fermentation broth than with synthetic solutions, possibly due to the presence of by-products in the multicomponent mixtures, which contributed to ethanol permeation. The results of this work indicate that by-products influence pervaporation of ethanol with hydrophobic flat sheet membrane produced from the fermented broth of banana pseudostem.     

Continuous ethanol fermentation using immobilized yeast cells

Growing cells of Saccharomyces cerevisiae immobilized in calcium alginate gel beads were employed in fluidizedbed reactors for continuous ethanol fermentation from cane molasses and other sugar sources. Some improvements were made in order to avoid microbial contamination and keep cell viability for stable long run operations. Notably, entrapment of sterol and unsaturated fatty acid into immobilized gel beads enhanced ethanol productivity more than 50 g ethanol/L gel h and prolonged life stability for more than one-half year. Cell concentration in the carrier was estimated over 250 g dry cell/L gel. A pilot plant with a total column volume of 4 kL was constructed and has been operated since 1982. As a result, it was confirmed that 8-10%(v/v)ethanol-containing broth was continuously produced from nonsterilized diluted cane molasses for over one-half year. The productivity of ethanol was calculated as 0.6 kL ethanol/kL reactor volume day with a 95% conversion yield versus the maximum theoretical yield for the case of 8.5% (v/v) ethanol broth.     

Genetic immobilization of proteins on the yeast cell surface

A genetic system has been exploited to immobilize proteins in their active and functional forms on the cell surface of yeast, Saccharomyces cerevisiae. DNAs encoding proteins with a secretion signal peptide were fused with the genes encoding yeast agglutinins, a- and alpha-type proteins involved in mating. The fusion gene was introduced into S. cerevisiae and expressed under the control of several promoters. Appearance of the fused proteins expressed on the cell surface was demonstrated biochemically and by immunofluorescence and immunoelectron microscopy techniques. Alpha-galactosidase from Cyamopsis tetragonoloba seeds, peptide libraries including scFv and variable regions of the T cell receptor from mammalian cells have been successfully immobilized on the yeast cell wall in the active form. Recently, surface-engineered yeasts have been constructed by immobilizing the enzymes and a functional protein, for example, green fluorescent protein (GFP) from Aequorea victoria. The yeasts were termed 'arming yeasts' with biocatalysts or functional proteins. Such arming cells displaying glucoamylase from Rhizopus oryzae and alpha-amylase from Bacillus stearothermophilus, or carboxymethylcellulase and beta-glucosidase from Aspergillus acleatus, could assimilate starch or cellooligosaccharides as the sole carbon source, although S. cerevisiae cannot intrinsically assimilate these substrates. GFP-arming cells can emit green fluorescence from the cell surface in response to the environmental conditions. The approach described in this review will enable us to endow living cells, including yeast cells, with novel additional abilities and to open new dimensions in the field of biotechnology.     

Novel supplements enhance the ethanol production in cane molasses fermentation by recycling yeast cell

Summary Eight alcohol producing yeast strains were screened for their sedimentation rates and it was found thatS.cerevisiae NCIM 3526 was a better flocculant strain. This strain was employed in cane molasses fermentation with yeast recycle, supplemented with skim milk, chitin and fungal mycelium individually or in combination, at 30°C, using fermentable sugars 15%. On the completion of ten 16 h cycles, 20–30% more ethanol was produced in presence of these supplements and the efficiency of the process was improved from 66 to 87%.     

Recovery of yeast invertase from ethanol fermentation broth

Summary Ethanol fermentation broth produced by an aggregated form ofSaccharomyces uvarum strain contained invertase when sucrose-based raw materials were used. The amount of invertase in the borth was in the range of 1.4 to 4.8 units/ml, which was affected by the dilution rate, the concentration of corn steep liquor, and the type of sugar used. The activity of invertase in the broth could be maintained at 0.8 units/ml over two months. When the broth was passed through DEAE-cellulose beads and eluted with a NaCl-Tris-HCl buffer solution, a 75% recovery yield of invertase with 9-fold purification and 30-fold concentration could be achieved.     

Online monitoring and characterization of flocculating yeast cell flocs during continuous ethanol fermentation

Both intrinsic and observed kinetic investigations for those ethanol fermentations using self-flocculated yeast strains have been hindered by the lack of real online monitoring techniques and proper characterization methods for the flocs. An optical detecting technique, the focused beam reflectance measurement probe developed by Lasentec (Redmond, WA) was inserted into a fermentor to monitor the floc chord length distributions. Using a simulating system composed of the floc-buffer suspensions, the total floc chord length counts per second were directly correlated with the floc biomass concentrations so that the floc biomass concentrations can be in situ detected. Furthermore, a characterization method of the flocs was established by properly weighted treatments of the detected floc chord length distributions. When a real yeast floc ethanol fermentation system was detected during its intrinsic kinetic investigations in which the floc size needed to be controlled at a level of micrometer scale to eliminate inner mass transfer limitations, it was found and validated that CO(2) produced during fermentation exerted significant disturbances. By applying 1/length-weighted treatment, these disturbances were effectively overcome.     

Starch hydrolysis and its effect on product yield and microbial contamination in yeast ethanol fermentation

The influence of temperature and incubation time on starch gelatinization in wheat, rye and corn grain were studied. Rye starch was the most susceptlble to enzyme hydrolysls. Heat treatment of ground grain during starch gelatinlzation significantly reduced microblal contamination. In the batch fermentation of wheat, a high sugar utillization ranged from 92 to 94%. The highest alcohol yield was 65% from rye starch. The results obtained show that the high pressure cooking used for mash preparation can be replaced successfully by low temperature treatment.The authors are with the Institute of Food Technology of Plant Origin, The University of Agriculture, 60-624 Poznan, ul. Wojska Polskiego 31, Poland.     

Inhibitory effect of lignin by-products of pulping on yeast growth

The antimicrobial properties of lignin by-products obtained by organic solvent, neutral sulfite semichemical and kraft pulping were shown in a series of yeasts. Lignin can act as an inhibitor of growth ofSporobolomyces roseus, Candida tropicalis, Trichosporon cutaneum andCandida albicans. Oxidation of all lignin samples tested depresses their inhibitory effects.     

自絮凝酵母高浓度重复批次乙醇发酵

利用发酵性能优良的自絮凝酵母Saccharomyces cerevisiaeflo,研究开发了重复批次高浓度乙醇发酵系统,以节省下游加工过程的能耗。在终点乙醇浓度达到120g/L左右的条件下,发酵系统的乙醇生产强度达到8.2g/(L·h)。然而实验中发现,随着发酵批次的增多,自絮凝酵母沉降性能逐渐下降,从发酵液中沉降分离所需时间相应延长,导致发酵液中高浓度乙醇对酵母的毒害作用加剧,影响其发酵活性和发酵系统运行的稳定性,发酵装置运行11个批次后无法继续运行。实验结果表明,絮凝能力下降导致的酵母絮凝颗粒尺度减小是其沉降性能下降的主要原因。进一步研究发现,酵母的絮凝能力通过再培养可以恢复。在此基础上对发酵系统操作进行改进,每批发酵结束后可控采出一定比例菌体,调节系统的酵母细胞密度和乙醇生产强度以刺激酵母增殖,保持其絮凝能力。在达到相同发酵终点乙醇浓度条件下,虽然发酵系统的乙醇生产强度降低到4.0g/(L·h),但运行10d后絮凝颗粒酵母尺度趋于稳定,继续运行14d,未发现絮凝颗粒酵母尺度继续下降的现象,系统可以稳定运行。     

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.     

Stillage backset and its impact on ethanol fermentation by the flocculating yeast

The second largest cost in fuel ethanol production is from energy consumption with ethanol distillation and stillage treatment, particularly when stillage is treated by the multi-evaporation process. Therefore, stillage backset is the most economically competitive strategy for reducing discharge and saving energy consumption. In this article, continuous ethanol fermentation was performed by the flocculating yeast under stillage backset conditions. Compared to regular yeast, immobilized yeast within the fermentor through flocculation reduced byproducts formation in the stillage, since heat lysis of yeast during ethanol distillation was prevented, and many side reactions were thus eliminated, making more stillage backset within the fermentation system possible. Although pyruvic acid, succinic acid, citric acid, α-ketoglutaric acid, fumaric acid and glycerol from yeast metabolism, furfural and 5-hydroxymethyl furfural from process operations, and acetic acid and lactic acid from slight contamination were accumulated with the stillage backset, they had no significant impact on yeast growth and ethanol fermentation due to low concentrations accumulated within the fermentation system. However, propionic acid that was generated mainly during hydrolysate sterilization and distillation of the fermentation broth was detected as the major inhibitor, but this byproduct would be significantly reduced under industrial conditions without hydrolysate sterilization, making the stillage backset more reliable for industrial application.     

pH inhibition of yeast ethanol fermentation in continuous culture

Summary In a low dilution rate study an unexpected pH-related inhibition of yeast fermentation was found. A higher volumetric rate of ethanol production occurred at lower pH values (2.8 to 3.2), suggesting a low optimum pH.Notation Ki product inhibition constant, L/g - Ks substrate saturation constant, g/L - P product (ethanol) concentration, g/L - S substrate (glucose) concentration, g/L - specific growth rate, h^–1 - ₀ maximum specific growth rate, h^–1