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The thermotolerant yeast Kluyveromyces marxianus is a promising bioethanol producer, but its fermentation arrested earlier at high temperatures. Untargeted metabolomic analysis was used to explore K. marxianus’s stress responses during high-temperature fermentation. Fermentation experiments were conducted at 45 °C, with a group conducted at 30 °C as control. Samples were collected from both groups at 14 and 22 h and were subjected to metabolomic analysis. The results show that pathways related to biosynthesis of amino acids, metabolism of purine, pyridoxine and riboflavin, etc. were induced at high temperature. More oleic acid, less palmitic acid and less palmitoleic acid were synthesized at 45 °C than at 30 °C, indicating that K. marxianus can adapt to high temperature by increasing the mean fatty acid chain length. In addition, most of the metabolic pathways stopped after the fermentation arrest, but pathways related to amino acids metabolism, riboflavin metabolism, etc. were still active. This is the first study about comprehensive metabolomic profiling of K. marxianus during high-temperature fermentation. The findings in this study offer deeper insight in mechanisms of K. marxianus’s stress responses during high-temperature fermentation at the metabolome level, and provide potential target pathways for further metabolic engineering towards improved stress tolerance and efficient bioethanol production.

During ethanol fermentation, ethanol accumulates in the medium and becomes a significant stress factor for yeast. Trehalose and glycogen are major carbohydrate storages and presumably protect cells from such stress conditions. In this study, batch cultivations of wild and adapted Saccharomyces cerevisiae were performed to evaluate the fermentation kinetics and carbohydrate storages, interrelatedly. Trehalose and glycogen were determined by both High Performance Liquid Chromatography (HPLC) and Fourier Transform-Infrared (FT-IR) spectroscopy for comparison. The obtained maximum trehalose and glycogen (7.5 and 10.5 %) contents of the adapted strain was remarkably higher compared to the wild strain (5 and 6.5 %). The adapted S. cerevisiae displayed an enhanced fermentation stresses coping ability resulting in a higher ethanol productivity (2.03 vs 1.65 g ethanol/h) as well as higher ethanol titer (7.7 vs 6.4 %). This study contributes to the understanding of the cellular response of S. cerevisiae cells to fermentation stresses in relation to culture history together with the evaluation of fermentation performance, as well as the process kinetics. This study also confirms that FT-IR spectroscopy of whole S. cerevisiae cells for determination of storage compounds without pretreatment has valuable advantages compared to HPLC with intensive sample handling.

The growth and ethanol fermentation of Saccharomyces cerevisiae are vulnerable to weak acid that coexists with sugar in lignocellulosic hydrolysate. In this study, the molecular basis of S. cerevisiae in response to acetic and formic acids was investigated when fermented with glucose and xylose at pH 5.0. Transcriptional profiling revealed that 294 (657 in total) differentially expressed genes (DEGs) were exclusive to mixed acetic and formic acids (Aa_Fa) group, the DEGs in acetic acid (Aa) and formic acid (Fa) group were 308 and 326, respectively. The general responses to Aa, Fa, and Aa_Fa mainly involved carbohydrate metabolism, amino acid metabolism, transcriptional regulation, and stress response. In Aa_Fa group, the enrichment ratio of each pathway was much higher than being exposed to Aa or Fa stress individually, and some enrichments not existed in Aa or Fa group were observed, suggesting the synergistic anti-inhibition responses of S. cerevisiae was triggered. In yeast cells, bioconversion, stress response, and transmembrane transport could be activated to resist acetic and formic acids accompanying with energy consumption. Except the putative transcriptional factor (TF) - Haa1p, the other three TFs, Hap4p, Yox1p, and Mag1p, might as well be considered to participate in acetic and formic acids resistance responses.

Weak acids are known to have a negative impact on yeast performance, restraining production efficiency during the production of bioethanol and other fermentative yeast-derived products. These acids, which might be hydrophilic or lipophilic exert negative effects on yeasts when they diffuse into the cell in their unionized state as a result of their pH being lower than the pka of yeast growth medium. Consequently, the unionized acids dissociate into their respective cations and anions, as intracellular pH is typically neutral. Further, proton accumulation tends to reduce intracellular pH. As a result, the anions destabilize the internal cell machinery, thus affecting cellular metabolism on various levels. Overcoming this acid-mediated stress in budding yeast would in part, harness the potential of using lignocellulosic biomass hydrolysate – which is typically acetic acid-rich – as a cheaper feedstock for large-scale bioethanol production. Since organic acids are key intermediates in ethanol fermentation, this review focuses on the prospects of bioethanol production from lignocellulosic biomass using weak acid-tolerant strains of yeasts derived by metabolic engineering.

Benzalkonium chloride (BAC) is highly abundant in wastewaters due to its wide application in household and industrial products. The aim of this research was to compare the resistance of two genetically related strains, Ogataea polymorpha (wild type) and O. polymorpha cat8Δ (recombinant strain), to enhanced concentrations of BAC (≥ 100 mg/L) in terms of its appearance in wastewaters. Inhibition of the respiration activity of wt and cat8Δ by 100 mg/L and 300 mg/L BAC in a xylose-containing broth (4 g/L xylose) was dependent on the carbon source used for obtaining the yeast biomass through ethanol fermentation. The respiration activity of wt in a liquid broth was inhibited by BAC, while cat8Δ strain cells were characterized by similar amounts of CO2 in the control and BAC-amended sets after 20 h of incubation and varied in range from 73.4 to 82.0 μgCO2/h for 108 CFU. In an agar diffusion assay, the resistance of both yeast strains to 12 μg BAC was dependent on the medium composition. BAC-removal activity was demonstrated for yeast cells immobilized on plastic units.

The aim of the research was to optimize the composition of the VHG (Very High Gravity) media by selecting the proportions of various plant materials, complementary in terms of the composition of substrates affecting the fermentation process and efficiency. The addition of 25, 33 and 50% of molasses in VHG mash resulted in higher ethanol concentration above 112 g L−1. The highest ethanol concentration of 129.96 ± 0.58 g L−1 and high actual fermentation yield as compared to theoretical one of 90.47 ± 0.40% was found for VHG mash made from all four plant materials used in the same proportions (25%). Raw spirit obtained by distillation of this medium was also characterized by the lowest content of acetaldehyde (332.8 ± 8.3 mg L−1 EtOH) and esters (94.3 ± 1.6 mg L−1 EtOH) in comparison with the spirit samples obtained from mash with a similar degree of attenuation. Addition of molasses to VHG mashes resulted also in lowering the concentration of higher alcohols in distillates to 3815 mg L−1 EtOH as compared to spirits obtained from substrates using starchy materials only. Use of only a complementary raw material composition ensuring that VHG mashes are rich in nutrients resulted in intensification of the alcoholic fermentation process.

The qualitative and quantitative profile of volatile compounds in agricultural distillate depends on numerous factors. Many such compounds, including higher alcohols, occur naturally, due to the metabolism of yeast during ethanol fermentation. Some are difficult to remove in the rectification process. For these reasons, it is desirable to reduce the synthesis of volatile compounds, among others higher alcohols, during the fermentation process. The results of the presented study showed that the raw materials, the method of starch liberation, and the initial pH of the sweet mashes affected the concentrations of higher alcohols in the distillates. Higher concentrations of 1-butanol, 1-propanol, and 2-propanol were found in distillates obtained from mashes prepared by the thermal-pressure method, while the remaining compounds occurred at higher concentrations in distillates obtained using the pressureless starch liberation method. The type of raw materials used had an influence on the concentration of the majority of higher alcohols. The content of 2-propanol, 2-methyl-1-propanol, 1-propanol, 1-butanol, 3-methyl-1-butanol, and 2-phenylethanol was lower in distillates obtained from mashes with an initial pH of 4, in comparison with those obtained from mashes with the pH set at 5.5.

In development of yeast cell factories, the adequate stress tolerance of the yeast strains is the universal requirement for industrial fermentation. Primary stress factor is the end product toxicity which has to be accumulated to obtain economically feasible performance in industrial processes. Second stress factor is the concentration and composition of substrate used for ethanol production and the third stress factor is temperature gradients in the fermentor which influence fermentation rate and productivity. This review encloses the development in understanding the response of yeast towards various environmental stresses: heat shock, ethanol stress and osmotic stress. These stresses are fundamental challenges that are experienced by various microbes. Physiological changes such as the synthesis of compatible solutes and heat shock proteins also contribute to cellular survival. Different methods are available to improve the strain’s survival under different stresses; thus, it will not only grow but also produce beneficial products in harsh environments. In combination with rational metabolic engineering to study and analyze processes of adaptive evolution, evolutionary engineering is a powerful strategy for improving industrially important, complex properties of yeast. Evolutionary engineering is an emerging field that draws on the most recent advances from a wide range of scientific and technical disciplines.

To decrease alkaline dosage and increase the enzymatic saccharification of sugarcane bagasse (SB), dilute mixed alkali salts soaking (0.5% K2CO3, 0.068% K2SO3) in an autoclave at 100–125 °C for 10–60 min was attempted to pretreat SB. The increased pretreatment severity at elevated temperatures resulted in the increase of delignification and enzymatic saccharification. It was found the optimum pretreatment temperature and pretreatment time were 120 °C for 40 min. Furthermore, FTIR, XRD and SEM characterized the porosity, morphology, and crystallinity changes of the pretreated SB samples. After the enzymatic in situ saccharification of 50 g/L SB in dilute K2CO3/K2SO3 (0.35% K2CO3, 0.048% K2SO3) media for 72 h, the reducing sugars and glucose could be obtained at 32.94 and 20.18 g/L, respectively. Finally, the SB-hydrolysates containing 20 g/L glucose were used for ethanol fermentation in the presence of dilute alkali salts. After 48 h, the ethanol yield was 0.394 g ethanol/g glucose, which represents 77.25% of the theoretical yield. In conclusion, an effective process about the biomass pretreatment, enzymatic saccharification and ethanol fermentation was successfully demonstrated in “one-pot” media containing low loading of alkali salts K2CO3/K2SO3.

Control of rheological characteristics of substrates is important in ethanol fermentation, particularly in a very high gravity (VHG) process. In this study, the synergism of cell wall degrading enzymes on rheology of cassava root mash was studied under VHG condition. The root mash exhibited a shear-thinning behavior. Crude enzyme mixture from Aspergillus aculeatus showed a dose-dependent effect on reduction of complex viscosity and storage modulus of mash. The enzyme showed strong synergism with Trichoderma reesei cellulase and Aspergillus pectinase leading to marked reduction in mash viscosity compared with individual enzymes. The optimal mixture comprising the three enzymes at a 1:1:1 ratio at 0.05 mg protein/g dried solid led to a remarkable decrease of complex viscosity to 1.01 Pas at 15.79 rad/s compared with 832.40 Pas of the control after incubation at 45 °C for 2 h. Synergism could be related to complementary activities of glycosyl hydrolases from different microbial sources, according to proteomic analysis. Simultaneous saccharification and fermentation of the pretreated mash by Saccharomyces cerevisiae led to a final ethanol concentration of 15.47% (w/w) corresponding to 88.14% theoretical yield. The work showed for the first time the enzymatic modification of cassava root mash rheology related to degradation of cell wall polysaccharides.

Makgeolli is a traditional fermented rice wine in Korea containing complex mixture of metabolites. GC–MS datasets coupled with multivariate statistical analysis were used for the first time to investigate metabolic changes in Makgeolli during fermentation and aging, and to assess the fermentative behaviors of yeast strains. Makgeolli fermentation progressed very quickly during the early fermentation period, whereas Makgeolli metabolites barely changed during the aging period for up to 70 days. During alcohol fermentation, decreased levels of glucose and phosphoric acid, along with increased levels of the other identified metabolites were observed. The levels of tryptophan, myo-inositol, malic acid, tyrosine, ornithine, leucine, glutamic acid, glutamine, citric acid, and 2-hydroxyglutaric acid in Makgeolli with the Saccharomyces cerevisiae, as well as the levels of glycerol, lactic acid, glycine, 4-hydroxyphenylethanol, and erytritol in Makgeolli with the Saccharomyces coreanus had the highest significance. This study highlights the applicability of GC–MS based metabolite profiling for monitoring Makgeolli fermentation and evaluating the fermentative characteristics of yeast strains.

Bioethanol is a renewable biofuel that has a strong inhibitory effect on cells in bioethanol fermentation by yeast. In this study, carbon nanotube (CNT)-mixed polydimethylsiloxane (PDMS) membranes were used for ethanol recovery from model solutions as well as for fermentation by self-flocculating yeast. Imbedding CNTs into the PDMS membrane led to enhanced ethanol recovery, with a maximum total flux of 128.7 g/m2 h and an ethanol titer of 615.1 g/L in permeate. The CNTs can provide a flexible route for ethanol transport through the inner tubes or along the smooth surface. In fed-batch fermentation incorporating pervaporation, 112.3 g/L of ethanol was produced with an overall ethanol productivity and yield of 2.23 g/L h and 0.45 g/g, respectively. The membrane produced a highly concentrated condensate containing 400.3–487.5 g/L of ethanol. Furthermore, as yeast flocs can be throttled down in the bioreactor, self-flocculating yeast can be used to prevent membrane fouling induced by cell adsorption on the membrane. Therefore, the CNT-mixed membrane coupled with ethanol fermentation by self-flocculating yeast not only reduces ethanol-mediated inhibition of cells but also saves the production cost because of the reduced fouling risk. Thus, this combination approach has potential in industrial bioethanol production for long-time operation.

Alcoholic fermentation using yeast strains with flocculent characteristics results in a higher biomass concentration in the reactor than does the use of a culture without flocculation ability. In addition, this approach is economically competitive due to its simplicity and low cost, it requires no centrifugal separation for the soluble yeast cells and does not need support, as is the case with immobilized cells. This paper evaluates the behavior of a Saccharomyces cerevisiae strain with flocculent characteristics and the effect of altering the inlet sucrose concentration, flow cell recycle and residence time using a central composite design. A novel experimental process for ethanol fermentation was designed and evaluated by employing a cell-recycling two-tower-reactor system in which each tower was individually equipped with a settler for recycling flocculent yeast. The fermentation experiments were conducted in a system with two tower reactors, operated continuously in series. Fermentation yields of approximately 86% and productivity up to 18 g/L h were obtained in this process. The system was effective for continuous ethanol fermentation from sucrose, cane juice and molasses obtained from sugar manufacturing.

For the purpose of designing an automatic repeated batch ethanol fermentation process, a self-settlement feature of flocculating yeast was used along with the development of a relevant kinetic model to describe such an operation. The frequency of repeated batch operation was controlled by the measured fermentation redox potential. As the redox potential profile changed its slope from zero or from negative to positive, repeated operation was triggered. The ethanol productivity varied between 3.26 ± 0.22 and 6.72 ± 0.24 g/L h for ∼200 g glucose/L case, and between 3.88 ± 0.37 and 4.68 ± 0.15 g/L h for ∼240 g glucose/L cases during the course of repeated batch fermentation. Although the biomass built up as fermentation proceeded, the yeast viability decreased, consequently limiting the number of batch operation that could be repeated. The number of repeated fermentation depended on the initial glucose concentration used. The proposed model predicted that the maximum repeatable batch operation when flocculating yeast was used was 14 and 8 for ∼200 and ∼240 g glucose/L case, respectively. Although the biomass increased significantly comparing with single batch, the cell viability decreased along with the batch number. To overcome this problem, a partial removal of yeast during the repeated batch fermentation would extend the operation.

Cassava is an important feedstock for bioethanol production; however, its use in very high gravity (VHG) fermentation is limited by the high viscosity of mash due to the presence of plant cell wall derived polysaccharides. In this study, viscosity reduction of cassava root mash, chips, and pulp was achieved using a mixture of cell wall degrading enzymes prepared from solid state fermentation of Aspergillus aculeatus BCC17849. Proteomic analysis showed the mixture contained endo- and exo-acting cellulases, hemicellulases, and pectinases from various glycosyl hydrolase families. Enzymatic pretreatment of cassava substrates with the enzyme mixture containing endoglucanase, FPase, xylanase, polygalacturonase, β-glucanase, and mannanase activities at 45 °C, pH 5.0 for 2 h reduced viscosity to the operating level of

The fermentability of four different side streams produced in sulfite pulping has been compared in ethanol production with Saccharomyces cerevisiae. The results show that the fermentability of the different side streams varies, depending on where in the process they are produced, and the additional treatment applied to them. Side streams spent sulfite liquor, spent sulfite liquor derivative and spent sulfite liquor after ethanol fermentation that were fermentable benefited from the main cooking process, during which 90% of the sulfite was removed, whereas the side stream produced in the first cooking step, containing 11.0 g/L sulfite, was unfermentable. The fermentation of the side streams resulted in lower yields and productivity than fermentation in a defined medium. Furthermore, the fermentability of the side streams was improved after over-liming, evaporation, and laccase treatment. Over-liming was the most efficient means of detoxifying the side-streams, resulting in better fermentability. Sulfite treatment, however, had a counterproductive effect on fermentation due to the toxicity of this chemical to yeast metabolism. When the side-streams were detoxified by over-liming, loss of sugars was observed. Laccase treatment was less efficient, but it should be further explored as it offers a sustainable method of detoxifying side streams in situ.

The production of a two-layer composite biocatalyst for immobilization of two different microorganisms for simultaneous alcoholic and malolactic fermentation (MLF) of wine in the same bioreactor is reported. The biocatalyst consisted of a tubular delignified cellulosic material (DCM) with entrapped Oenococcus oeni cells, covered with starch gel containing the alcohol resistant and cryotolerant strain Saccharomyces cerevisiae AXAZ-1. The biocatalyst was found effective for simultaneous low temperature alcoholic fermentation resulting to conversion of malic acid to lactic acid in 5 days at 10 °C. Improvement of wine quality compared with wine fermented with S. cerevisiae AXAZ-1 immobilized on DCM was attributed to MLF as well as to increased ester formation and lower higher alcohols produced at low fermentation temperatures (10 °C) as shown by GC and headspace SPME GC/MS analysis. Scanning electron microscopy showed that the preparation of a three-layer composite biocatalyst is also possible. The significance of such composite biocatalysts is the feasibility of two or three bioprocesses in the same bioreactor, thus reducing production cost in the food industry

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.

In this study, the saccharification and fermentation of the by-product of starch manufacture, potato pulp, were investigated. Analytic results of the components show that the potato pulp contains large amounts of starch, cellulose, and pectin. A commercial enzyme from Acremonium cellulolyticus was found to be highly efficient in the saccharification of potato pulp, since it exhibited high pectinase, α-amylase, and cellulase activities. Hydrothermal treatment of the potato pulp increased the saccharification rate, with a corresponding glucose concentration of 114 g/L and yield of 68% compared to the glucose concentration of 47 g/L and yield of 28% in the untreated case. The hydrolyzate could be used as both nitrogen and carbon sources for ethanol fermentation, showing that bioconversion of potato pulp to ethanol is feasible.

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.

An immobilized cell microchannel bioreactor was designed to test continuous fermentation. The fermentation set-up included a bottom hydrophilic quartz channel to immobilize cells using 0.4 wt% polyethyleneimine and a top channel designed to continuously remove metabolically generated carbon dioxide using hydrophobic polypropylene. To evaluate fermentation characteristics of immobilized cells, ethanol fermentation was carried out using Saccharomyces cerevisiae and Pichia stipitis . The immobilized cell microchannel bioreactor was used to identify long-term activity of immobilized S. cerevisiae cells. The continuous flow microchannel bioreactor was operated stably over a period of 1 month. The immobilized cell microchannel bioreactor was used to examine the characteristics cells that consumed mixed substrates. The concentration ratio of glucose to xylose for simultaneous utilization of hemicellulosic sugars was evaluated using the microchannel bioreactor and the results were compared with those obtained by using conventional batch fermentation with P. stipitis .

When Saccharomyces cerevisiae was cultivated under ∼200 g glucose/l condition, the time point at which glucose was completely utilized coincided with the moment at which the slope of a redox potential profile changed from negative or zero to positive. Based on this feature, a redox potential-driven glucose-feeding fermentation operation was developed, and resulted in a self-cycling period of 14.25 ± 0.4 h. The corresponding ethanol concentration was maintained at 88.4 ± 1.0 g/l with complete glucose conversion, and the cell viabilities increased from 80% in the transition period to 97.2 ± 1.1%, implying the occurrence of yeast acclimatization. In contrast, a pre-determined 36-h manually adjusted period was chosen to oscillate yeast cells under ∼250 g glucose/l conditions, which resulted in 106.76 ± 0.7 g ethanol/l and 15.19 ± 1.3 g glucose/l remaining at the end of each cycle. Compared to the equivalent batch and continuous ethanol fermentation processes, the annual ethanol productivity of the reported fermentation operation is 2.4% and 13.2% greater, respectively in ∼200 g feeding glucose/l conditions.

Eastern gamagrass (Trypsacum dactyloides) is a C4 perennial grass, native to the USA with desirable characteristics that warrants further investigation as a new lignocellulosic crop for bioethanol production. Chemical composition assays showed that eastern gamagrass had comparable cellulose, hemicellulose and lignin compositions to those of switchgrass (Panicum virgatum). With the cellulose solvent-based lignocellulose fractionation (CSLF) pretreatment and subsequent enzymatic saccharification, 80.5–99.8% of cellulosic glucose was released from the gamagrass biomass, which was 10–17% greater than the glucose release efficiency from switchgrass (73.5–87.1%). Furthermore, the hydrolysate of gamagrass supported greater ethanol fermentation yield (up to 0.496 g/g glucose) than the hydrolysates of switchgrass. As such, in the whole process of biomass-to-ethanol conversion, gamagrass could yield 13–35% more ethanol per gram of biomass than switchgrass, indicating that gamagrass has high potential as an alternative energy feedstock for lignocellulosic ethanol production.

A continuous very-high-gravity (VHG) ethanol fermentation process design, consisting of a chemostat vessel connected to several equal-sized ageing vessels configured in parallel, was developed. The objective of the developed process is to have complete glucose utilization during fermentation stage. The process design integrates the conservation of mass principle and the experimental data of collected residual glucose profiles measured under VHG conditions. An ageing vessel involves three consecutive time periods: filling, ageing and operating. The ageing time is biological relevance, and is affected by the initial glucose concentration, the ethanol concentration, and the yeast viability in an ageing vessel. The operating time period is adjustable; a short operating time means a high discharge rate in order to empty an ageing vessel. The filling time links to the selection of the number of equal-sized ageing vessels that are installed downstream to a chemostat device. The developed process features the use of equal sized fermenters for all chemostat and ageing vessels so that the vessel exchangeability and the flexibility of fermentation operation are increased.

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.

Based on the shapes of the redox potential profile and the growth curve of Saccharomyces cerevisiae, five distinct time intervals were observed: lag phase (ca. 0–8 h), logarithmic (or log) phase (ca. 8–18 h), late log to mid stationary phase (ca. 18–28 h), mid stationary to the commencement of death phase (ca. 28–38 h), and death phase (ca. 38–48 h). Respectively, the first four intervals along with two other extreme aeration schemes, no aeration and full-course aeration (0–48 h), were chosen to aerate very-high-gravity (VHG) fermentation. The objectives were to locate the optimal aeration timing and aeration interval that could effectively promote yeast growth and enhance ethanol productivity. Results show that the aeration rate of 0.82 l/min, and aerating yeast in its log phase were the most effective aeration schemes among others. It could generate 2.14-fold more viable yeast cells as compared to the no aeration scheme, and was able to completely utilize glucose in 36 h as compared to 48 h when using a no aeration scheme. Four aeration rates (0, 0.4, 0.82, and 1.3 l/min) were provided under the same aeration interval (i.e., 8–18 h) in order to find out the proper aeration rate. Results show that high yeast viability and high ethanol productivity were attained when VHG fermentation was aerated at 0.82 l/min. Microscopic observations illustrate that larger and lustrous yeast cells were obtained when cultivating under the optimal aeration time interval as compared to no aeration and 28–38 h aeration interval, where the yeast morphology appeared smaller and dull.

Poly(vinyl alcohol) (PVA) hydrogel beads crosslinked with boric acid have been widely utilized for microorganism immobilization. However, a drawback of the method for preparing conventional PVA–boric acid beads is that microorganisms enclosed in the PVA matrix are drastically damaged by boric acid during the bead preparation process. In the present study, we utilized sodium sulfate as an inducer for crosslinkage of PVA for fabricating microorganism-enclosing PVA hydrogel beads to avoid the drastic decrease in cell viability caused by saturated boric acid solution. First, PVA was crosslinked by boric acid for a short time. Subsequently, the molecules were further crosslinked using sodium sulfate. X-ray diffraction analysis indicated that sodium sulfate promoted crystallization of PVA, which contributed to gelation of PVA solution. Moreover, the toxicity of sodium sulfate to Saccharomyces cerevisiae was lower than that of boric acid. The stability in water of the PVA–sodium sulfate beads was superior to the stability of PVA–boric acid beads. The ethanol fermentation rate of the PVA–sodium sulfate hydrogel beads enclosing the yeast cells was 2–3 times as fast as that of PVA–boric acid beads. The PVA–sodium sulfate beads could be reused 10 times in repeated ethanol fermentation tests.

Response surface methodology (RSM) based on the 23 factorial central composite design (CCD) was applied to optimize the conditions of enzymatic saccharification and ethanol fermentation using food waste. Optimum conditions were found to be saccharification pH of 5.20, enzyme reaction temperature of 46.3 °C, enzyme concentration of 0.16% (v/v), fermentation pH of 6.85, fermentation temperature of 35.3 °C, and fermentation time of 14 h. The model predicted that maximum concentration of reducing sugar and ethanol under the above optimum conditions were 117.0 g reducing sugar/L and 57.6 g EtOH/L, respectively. Experimental results were in close agreement with model prediction with 120.1 g reducing sugar/L and 57.5 g EtOH/L, respectively.

Supplementation effect of compatible solute ectoine on the ethanol fermentation by Zymomonas mobilis CICC10232 was examined in the presence of high glucose concentration. Addition of ectoine promoted the cell growth as well as volumetric ethanol productivity in the presence of 250 g/l of glucose. At the end of ethanol fermentation, cell dry weight resulted in 1.8 and 1.5 g CDW/l in the presence and absence of 1 mM ectoine, respectively. Volumetric ethanol productivity in the presence of 1 mM ectoine became 1.1 g/l/h, which resulted in 57.1% higher than that in the absence of ectoine, 0.7 g/l/h. In addition, fermentation time was shortened by 24 h when 1 mM ectoine existed in the medium. In the presence of 250 g/l of glucose together with various concentrations of ectoine, relative enzyme activities of glucokinase (GK), glucose-6-phosphate dehydrogenase (G-6-PDH), and alcohol dehydrogenase (ADH) increased by 29.9, 11.6, and 7.7% in the presence of 0.5, 0.5, and 0.25 mM ectoine compared to those without ectoine supplemented, respectively. The promoting function of ectoine on ethanol fermentation may be related to the protection of these enzyme activities under osmotic stress.

Corn fiber (CF) is a potential raw material for the production of various products because it is widely available in corn-producing countries. Corn fiber is a byproduct of the corn wet-milling industry and a very large amount of it (approximately 130 t/day) is produced in Hungary. The major component of corn fiber is the pericarp that consists of 35% hemicellulose, 18% cellulose and 20% remaining starch (protein, fiber oil and lignin are also present in this material). Corn fiber is presently used as animal feed. However, with continuous growth in corn processing to ethanol, there might be problems with the utilization of the surplus fibrous byproducts. In this paper the conversion of corn fiber to ethanol or other products was examined. Destarched corn fiber was pretreated by using different alkaline solutions and dissolved hemicellulose was precipitated with ethanol for the recovery of a valuable coproduct. The residual material consisting mostly of cellulose was hydrolyzed with cellulolytic enzymes and fermented into ethanol by using Saccharomyces cerevisiae.

“Propagation” or “conditioning” of active dry yeast (ADY) for the production of fuel ethanol is thought to reduce lag times in fermentation and reduce overall fermentation times. The objectives of this study were to determine the optimal time that ADY should be conditioned prior to fermentation and to determine how well yeast and bacterial contaminants present in ADY are propagated. A new term, metabolic acclimatization (MAcc), was introduced to describe events occurring during conditioning. It was observed that MAcc is not necessary prior to alcoholic fermentation. Lag times were reduced but overall fermentation times were unaffected. In fact, fermentation rates were slower than control conditions where ADY was directly inoculated into fermentors. In addition, if MAcc systems were to be used, it is recommended that only batch MAcc systems be practiced. Continuous MAcc (continuous yeast propagation) leads to increased bacterial numbers and organic acid levels, which become stressful to yeast.

Phenolic compounds affect the rate of fermentation, quality and stability of opaque beer produced from sorghum. The levels of proanthocyanidins (PAs) were determined photometrically at various stages during the preparation of opaque beer from high polyphenol sorghum. Decreases in the levels of proanthocyanidins were 52 and 34% during the lactic and second alcoholic fermentation stages, respectively. Quantitative and qualitative changes in the low molecular weight phenolic compounds of sorghum during the preparation process of opaque beer were determined by gas chromatography coupled with mass spectrometric detection. The main low molecular weight phenolic compounds detected in sorghum malt were p -hydroxybenzoic acid, p -hydroxybenzaldehyde and p -hydroxybenzoyl alcohol. Levels of p -hydroxybenzaldehyde decreased during both lactic acid and alcoholic fermentation while the levels of p -hydroxybenzoic acid and p -hydroxybenzoyl alcohol increased during alcoholic fermentation. Protocatechuic acid, caffeic acid and p -coumaric acid were detected at relatively low levels at various stages of opaque beer preparation.

Bacillus sp. PS-7, isolated from the hot springs of Manikaran (H.P.), India, produced very high levels of thermostable α-amylase by solid state fermentation (SSF) in Erlenmeyer flasks and enamel coated metallic trays. Productivity was affected by the nature of the solid substrate, nature of the moistening agent, level of moisture content, incubation temperature, presence or absence of surfactant, carbon, nitrogen, mineral, amino acid and vitamin supplements. Maximum enzyme production of 4,64,000 U/g dry bacterial bran was obtained on wheat bran supplemented with glycerol (1.0%, w/w), soyabean meal (1.0%, w/w), l -proline (0.1%, w/w), vitamin B-complex (0.01%) and moistened with tap water containing 1% Tween-40 and 1 mM MgSO 4 ·7H 2 O at ratio of 1:1.5 after 48 h incubation at 37 °C. The enzyme was partially purified to 12.7-fold by ammonium sulphate precipitation, gel filtration on Sephadex G-75 column followed by Phenyl agarose hydrophobic interaction chromatography. The kinetic characterisation of the partially purified enzyme exhibited maximum activity at 60 °C and pH 6.5. The thermal stability profile revealed the half-life of more than 6 h at 60° and 5(1/2) h at 70 °C respectively in presence of Ca 2+ . The enzyme did not exhibit marked increase in activity in presence of any metal ion. It could effectively act in synergism with the commercial amyloglucosidase during direct malt starch hydrolysis for ethanol fermentation.

Ethanol fermentation under non-sterilized condition was developed in this paper. An acid-tolerant mutant of Zymomonas mobilis, which can begin fermentation at the low pH of 4.5, was selected. At this low pH, potential contamination of ethanol production was controlled at the normal level without autoclaving or filter sterilization. Compared with the filter sterilization process, the same ethanol yield of 73.1 g/l indicated that no contamination occurred in the non-sterilized process. Compared with the autoclaved process, ethanol yield increased from 70.3 to 73.2 g/l and residual sugar concentration decreased from 5.3 to 1.3 g/l with 40 h fermentation. The theoretical yield of ethanol from glucose was 0.488 g/g in this new ethanol process.

Glycerol formation is reported in alcoholic fermentation with an alcohol resistant and psychrophylic Saccharomyces cerevisiae strain immobilized on delignified cellulosic material (DCM) and gluten pellets (GP). The immobilized biocatalysts were used for successive fermentation batches of synthetic media (glucose, fructose and sucrose) and grape must. Generally, immobilization affects glycerol production positively. Glycerol concentration in the range 11.9–14.9 g/l in wines was produced by immobilized cells and 10.2–12.8 g/l was produced by free cells at fermentation temperatures 5–30 °C. Glycerol concentration was affected by fermentation temperature and decreased as the temperature decreased for every fermenting medium. Glycerol concentration was higher in fermentations of grape must with immobilized or free cells than in those of synthetic media. The increased glycerol concentrations produced by immobilized yeast cells on DCM and GP support their use in wine making given that glycerol affects wine flavour and gives an impression of fullness and softness.

A study of the aerobic degradation of beet molasses alcoholic fermentation wastewater diluted to 50% (chemical oxygen demand, COD: 82 g/l) was carried out using the following fungi: Penicillium sp., Penicillium decumbens , Penicillium lignorum and Aspergillus niger . These four microorganisms produce a decolorization of the wastewater from the first day of incubation, achieving the maximum decolorization level at the fourth day of treatment in all cases. P. decumbes showed the maximum decolorization with a percentage of 40%. Simultaneously, a significant reduction in the phenolic content of the wastewater was also observed in all cases, reaching average removals of 70% for the four microorganisms studied. Average COD removals were similar in the four cases, achieving maximum values of 52.1 and 50.7%, respectively, on the fifth day of fermentation with Penicillium sp. and P. decumbens . Finally, a comparative study of the mesophilic anaerobic digestion of untreated and previously fermented (with P. decumbens ) beet molasses was carried out in laboratory-scale suspended cell bioreactors. Average COD removals of 93% and methane yield coefficient of 305 ml methane at STP conditions per g of COD removed were found in the anaerobic digestion of pre-treated molasses. The combined aerobic/anaerobic process showed the following advantages in relation to the single anaerobic digestion process: higher average percentages of COD removal (96.5 compared with 90.0%) and a decrease of the hydraulic retention time (HRT) necessary to achieve these COD reductions, increasing the decolorization of the wastewater. # 2002 Elsevier Science Ltd. All rights reserved.

In this work a SISO non-linear predictive controller was developed for an extractive alcoholic fermentation process. The internal model of the controller was represented by a functional link network (FLN). This model was identified using simulated data generated from a deterministic mathematical model whose kinetic parameters were determined experimentally. The FLN structure presented good non-linear approximation ability, with the advantage that the estimation of its weights is a linear optimization problem. The results show that the proposed algorithm has a great potential to identification and control of non-linear processes.