The proteomic response of Saccharomyces cerevisiae in very high glucose conditions with amino acid supplementation

Ethanol yield by Saccharomyces cerevisiae in very high glucose (VHG) media with an amino acid supplement was investigated. Amino acid supplementation led to positive cell responses, including reduced lag time and increased cell viability in VHG media. A quantitative shotgun proteomic analysis was used to understand how amino acid supplemented S. cerevisiae responds to high osmotic conditions. iTRAQ data revealed that most proteins involved in glycolysis and pentose phosphate pathways were up-regulated under high glucose shock. Reactivation of amino acid metabolism was also observed at the end of the lag phase. The relative abundance of most identified proteins, including aminoacyl-tRNA biosynthesis proteins, and heat-shock proteins, remained unchanged in the hours immediately following application of glucose shock. However, the expression of these proteins increased significantly at the end of the lag phase. Furthermore, the up-regulation of trehalose and glycogen biosynthesis proteins, first maintaining then latterly increasing glycolysis pathway activity was also observed. This was verified by enhanced ethanol yields at 10 and 12 h (0.43 and 0.45 g ethanol/g glucose) compared to 2 h (0.32 g ethanol/g glucose). These data combined with relevant metabolite measurements demonstrates that enhanced ethanol fermentation under VHG conditions can be achieved with the aid of amino acid supplementation.     

Very high gravity ethanol fermentation by flocculating yeast under redox potential-controlled conditions

ABSTRACT: BACKGROUND: Very high gravity (VHG) fermentation using medium in excess of 250 g/L sugars for more than 15 % (v) ethanol can save energy consumption, not only for ethanol distillation, but also for distillage treatment; however, stuck fermentation with prolonged fermentation time and more sugars unfermented is the biggest challenge. Controlling redox potential (ORP) during VHG fermentation benefits biomass accumulation and improvement of yeast cell viability that is affected by osmotic pressure and ethanol inhibition, enhancing ethanol productivity and yield, the most important techno-economic aspect of fuel ethanol production. RESULTS: Batch fermentation was performed under different ORP conditions using the flocculating yeast and media containing glucose of 201 [PLUS-MINUS SIGN] 3.1, 252 [PLUS-MINUS SIGN] 2.9 and 298 [PLUS-MINUS SIGN] 3.8 g/L. Compared with ethanol fermentation by non-flocculating yeast, different ORP profiles were observed with the flocculating yeast due to the morphological change associated with the flocculation of yeast cells. When ORP was controlled at [MINUS SIGN]100 mV, ethanol fermentation with the high gravity (HG) media containing glucose of 201 [PLUS-MINUS SIGN] 3.1 and 252 [PLUS-MINUS SIGN] 2.9 g/L was completed at 32 and 56 h, respectively, producing 93.0 [PLUS-MINUS SIGN] 1.3 and 120.0 [PLUS-MINUS SIGN] 1.8 g/L ethanol, correspondingly. In contrast, there were 24.0 [PLUS-MINUS SIGN] 0.4 and 17.0 [PLUS-MINUS SIGN] 0.3 g/L glucose remained unfermented without ORP control. As high as 131.0 [PLUS-MINUS SIGN] 1.8 g/L ethanol was produced at 72 h when ORP was controlled at [MINUS SIGN]150 mV for the VHG fermentation with medium containing 298 [PLUS-MINUS SIGN] 3.8 g/L glucose, since yeast cell viability was improved more significantly. CONCLUSIONS: No lag phase was observed during ethanol fermentation with the flocculating yeast, and the implementation of ORP control improved ethanol productivity and yield. When ORP was controlled at [MINUS SIGN]150 mV, more reducing power was available for yeast cells to survive, which in turn improved their viability and VHG ethanol fermentation performance. On the other hand, controlling ORP at [MINUS SIGN]100 mV stimulated yeast growth and enhanced ethanol production under the HG conditions. Moreover, the ORP profile detected during ethanol fermentation with the flocculating yeast was less fluctuated, indicating that yeast flocculation could attenuate the ORP fluctuation observed during ethanol fermentation with non-flocculating yeast.     

Very high gravity ethanol and fatty acid production of <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> without amino acid and vitamin

Very high gravity (VHG) fermentation is the mainstream technology in ethanol industry, which requires the strains be resistant to multiple stresses such as high glucose concentration, high ethanol concentration, high temperature and harsh acidic conditions. To our knowledge, it was not reported previously that any ethanol-producing microbe showed a high performance in VHG fermentations without amino acid and vitamin. Here we demonstrate the engineering of a xylose utilizing recombinant Zymomonas mobilis for VHG ethanol fermentations. The recombinant strain can produce ethanol up to 136 g/L without amino acid and vitamin with a theoretical yield of 90 %, which is significantly superior to that produced by all the reported ethanol-producing strains. The intracellular fatty acids of the bacterial were about 16 % of the bacterial dry biomass, with the ratio of ethanol:fatty acids was about 273:1 (g/g). The recombinant strain was achieved by a multivariate-modular strategy tackles with the multiple stresses which are closely linked to the ethanol productivity of Z. mobilis. The over-expression of metB/yfdZ operon enabled the growth of the recombinant Z. mobilis in a chemically defined medium without amino acid and vitamin; and the fatty acids overproduction significantly increased ethanol tolerance and ethanol production. The coupled production of ethanol with fatty acids of the Z. mobilis without amino acid and vitamin under VHG fermentation conditions may permit a significant reduction of the production cost of ethanol and microbial fatty acids.     

An innovative consecutive batch fermentation process for very high gravity ethanol fermentation with self-flocculating yeast

An innovative consecutive batch fermentation process was developed for very high gravity (VHG) ethanol fermentation with the self-flocculating yeast under high biomass concentration conditions. On the one hand, the high biomass concentration significantly shortened the time required to complete the VHG fermentation and the duration of yeast cells suffering from strong ethanol inhibition, preventing them from losing viability and making them suitable for being repeatedly used in the process. On the other hand, the separation of yeast cells from the fermentation broth by sedimentation instead of centrifugation, making the process economically more competitive. The VHG medium composed of 255 g L⁻¹ glucose and 6.75 g L⁻¹ each of yeast extract and peptone was fed into the fermentation system for nine consecutive batch fermentations, which were completed within 8–14 h with an average ethanol concentration of 15% (v/v) and ethanol yield of 0.464, 90.8% of its theoretical value of 0.511. The average ethanol productivity that was calculated with the inclusion of the downstream time for the yeast flocs to settle from the fermentation broth and the supernatant to be removed from the fermentation system was 8.2 g L⁻¹ h⁻¹, much higher than those previously reported for VHG ethanol fermentation and regular ethanol fermentation with ethanol concentration around 12% (v/v) as well.     

Application of multistage continuous fermentation for production of fuel alcohol by very-high-gravity fermentation technology

A fermentation system to test the merging of very-high-gravity (VHG) and multistage continuous culture fermentation (MCCF) technologies was constructed and evaluated for fuel ethanol production. Simulated mashes ranging from 15% to 32% w/v glucose were fermented by Saccharomyces cerevisiae and the dilution rates were adjusted for each glucose concentration to provide an effluent containing less than 0.3% w/v glucose (greater than 99% consumption of glucose). The MCCF can be operated with glucose concentrations up to 32% w/v, which indicates that the system can successfully operate under VHG conditions. With 32% w/v glucose in the medium reservoir, a maximum of 16.73% v/v ethanol was produced in the MCCF. The introduction of VHG fermentation into continuous culture technology allows an improvement in ethanol productivity while producing ethanol continuously. In comparing the viability of yeast by methylene blue and plate count procedures, the results in this work indicate that the methylene blue procedure may overestimate the proportion of dead cells in the population. Ethanol productivity (Yps) increased from the first to the last fermentor in the sequence at all glucose concentrations used. This indicated that ethanol is more effectively produced in later fermentors in the MCCF, and that the notion of a constant Yps is not a valid assumption for use in mathematical modeling of MCCFs. Journal of Industrial Microbiology & Biotechnology (2001) 27, 87–93. Received 20 January 2001/ Accepted in revised form 28 April 2001     

Very high gravity (VHG) ethanolic brewing and fermentation: a research update

There have been numerous developments in ethanol fermentation technology since the beginning of the new millennium as ethanol has become an immediate viable alternative to fast-depleting crude reserves as well as increasing concerns over environmental pollution. Nowadays, although most research efforts are focused on the conversion of cheap cellulosic substrates to ethanol, methods that are cost-competitive with gasoline production are still lacking. At the same time, the ethanol industry has engaged in implementing potential energy-saving, productivity and efficiency-maximizing technologies in existing production methods to become more viable. Very high gravity (VHG) fermentation is an emerging, versatile one among such technologies offering great savings in process water and energy requirements through fermentation of higher concentrations of sugar substrate and, therefore, increased final ethanol concentration in the medium. The technology also allows increased fermentation efficiency, without major alterations to existing facilities, by efficient utilization of fermentor space and elimination of known losses. This comprehensive research update on VHG technology is presented in two main sections, namely VHG brewing, wherein the effects of nutrients supplementation, yeast pitching rate, flavour compound synthesis and foam stability under increased wort gravities are discussed; and VHG bioethanol fermentation studies. In the latter section, aspects related to the role of osmoprotectants and nutrients in yeast stress reduction, substrates utilized/tested so far, including saccharide (glucose, sucrose, molasses, etc.) and starchy materials (wheat, corn, barley, oats, etc.), and mash viscosity issues in VHG bioethanol production are detailed. Thereafter, topics common to both areas such as process optimization studies, mutants and gene level studies, immobilized yeast applications, temperature effect, reserve carbohydrates profile in yeast, and economic aspects are discussed and future prospects are summarized.     

Consolidated bioprocessing strategy for ethanol production from Jerusalem artichoke tubers by Kluyveromyces marxianus under high gravity conditions

Aims: Developing an innovative process for ethanol fermentation from Jerusalem artichoke tubers under very high gravity (VHG) conditions. Methods and Results: A consolidated bioprocessing (CBP) strategy that integrated inulinase production, saccharification of inulin contained in Jerusalem artichoke tubers and ethanol production from sugars released from inulin by the enzyme was developed with the inulinase‐producing yeast Kluyveromyces marxianus Y179 and fed‐batch operation. The impact of inoculum age, aeration, the supplementation of pectinase and nutrients on the ethanol fermentation performance of the CBP system was studied. Although inulinase activities increased with the extension of the seed incubation time, its contribution to ethanol production was negligible because vigorously growing yeast cells harvested earlier carried out ethanol fermentation more efficiently. Thus, the overnight incubation that has been practised in ethanol production from starch‐based feedstocks is recommended. Aeration facilitated the fermentation process, but compromised ethanol yield because of the negative Crabtree effect of the species, and increases the risk of contamination under industrial conditions. Therefore, nonaeration conditions are preferred for the CBP system. Pectinase supplementation reduced viscosity of the fermentation broth and improved ethanol production performance, particularly under high gravity conditions, but the enzyme cost should be carefully balanced. Medium optimization was performed, and ethanol concentration as high as 94·2 g l^?1 was achieved when 0·15 g l^?1 K₂HPO₄ was supplemented, which presents a significant progress in ethanol production from Jerusalem artichoke tubers. Conclusions: A CBP system using K. marxianus is suitable for efficient ethanol production from Jerusalem artichoke tubers under VHG conditions. Significance and Impact of the Study: Jerusalem artichoke tubers are an alternative to grain‐based feedstocks for ethanol production. The high ethanol concentration achieved using K. marxianus with the CBP system not only saves energy consumption for ethanol distillation, but also significantly reduces the amount of waste distillage discharged from the distillation system.     

Improvement of ethanol production in very high gravity fermentation by horse gram (Dolichos biflorus) flour supplementation

AIMS: To determine the effect of osmotic stress on yeast and to investigate the protective role of horse gram flour during very high gravity (VHG) ethanol fermentation. METHODS AND RESULTS: Saccharomyces cerevisiae was inoculated into high sugar (30-40%, w/v) containing medium with and without supplementation of horse gram flour. The fermentation experiments were carried out in batch mode. The effect of 4 or 6% of horse gram flour to the medium on the metabolic behaviour and viability of yeast was studied. Significant increase in ethanol yield up to 50% and dramatic decrease in glycerol production up to 100% was observed in the presence of horse gram flour. The fermentation rate was increased from 3 to 5 days with increased viable cell count. The physical and chemical factors of horse gram flour may aid in reducing the osmotic stress of high gravity fermentation of ethanol as well as enhancing ethanol yield. CONCLUSIONS: It was found that horse gram flour not only reduced fermentation time but also enhanced ethanol production by better utilization of sugar. SIGNIFICANCE AND IMPACT OF THE STUDY: Production of high ethanol concentration by using VHG sugar fermentation eliminates the expensive steps in the conventional process and saves time.     

Production of ethanol by immobilized Saccharomyces bayanus in an extractive fermentation system

An extractive fermentation system using immobilized yeast cells was developed to study the ethanol production at high sugar concentrations. Organic acids were used as extracting solvents of ethanol and their toxicity was tested in free and k-carrageenan entrapped cell preparations. Immobilization seems to protect cells against solvent toxicity, when long-chain organic acids, e.g., oleic acid, were used, probably due to steric and diffusional limitations, the free cells not being viable at high oleic acid concentrations. The entrapped cells also present a higher metabolic activity than their free counterparts at high glucose concentrations. A solution of 300 g/L of glucose was totally fermented by the immobilized yeast cells, which when free cannot normally convert more than 200 g/L. In situ recovery of ethanol by oleic acid in a batch immobilized cell system led to higher ethanol productivities and to the fermentation of 400 g/L, when an oleic acid/medium ratio of 5 was used.     

Metabolite profiling for analysis of yeast stress response during very high gravity ethanol fermentations

A laboratory strain and an industrial strain of Saccharomyces cerevisiae were grown at high substrate concentration, so-called very high gravity (VHG) fermentation. Simultaneous saccharification and fermentation (SSF) was applied in a batch process using 280 g/L maltodextrin as carbon source. It was shown that known ethanol and osmotic stress responses such as decreased growth rate, lower viability, higher energy consumption, and intracellular trehalose accumulation occur in VHG SSF for both strains when compared with standard laboratory medium (20 g/L glucose). The laboratory strain was the most affected. GC-MS metabolite profiling was applied for assessing the yeast stress response influence on cellular metabolism. It was found that metabolite profiles originating from different strains and/or fermentation conditions were unique and could be distinguished with the help of multivariate data analysis. Several differences in the metabolic responses to stressing conditions were revealed, particularly the increased energy consumption of stressed cells was also reflected in increased intracellular concentrations of pyruvate and related metabolites.     

Ethanol fermentation in a magnetically fluidized bed reactor with immobilized Saccharomyces cerevisiae in magnetic particles

Ethanol fermentation by immobilized Saccharomyces cerevisiae cells in magnetic particles was successfully carried out in a magnetically stabilized fluidized bed reactor (MSFBR). These immobilized magnetic particles solidified in a 2 % CaCl(2) solution were stable and had high ethanol fermentation activity. The performance of ethanol fermentation of glucose in the MSFBR was affected by initial particle loading rate, feed sugar concentration and dilution rate. The ethanol theoretical yield, productivity and concentration reached 95.3%, 26.7 g/L h and 66 g/L, respectively, at a particle loading rate of 41% and a feed dilution rate of 0.4 h(-1) with a glucose concentration of 150 g/L when the magnetic field intensity was kept in the range of 85-120 Oe. In order to use this developed MSFBR system for ethanol production from cheap raw materials, cane molasses was used as the main fermentation substrate for continuous ethanol fermentation with the immobilized S. cerevisiae cells in the reactor system. Molasses gave comparative ethanol productivity in comparison with glucose in the MSFBR, and the higher ethanol production was observed in the MSFBR than in a fluidized bed reactor (FBR) without a magnetic field.     

Ethanol fermentation in an immobilized cell reactor using Saccharomyces cerevisiae

Fermentation of sugar by Saccharomyces cerevisiae, for production of ethanol in an immobilized cell reactor (ICR) was successfully carried out to improve the performance of the fermentation process. The fermentation set-up was comprised of a column packed with beads of immobilized cells. The immobilization of S. cerevisiae was simply performed by the enriched cells cultured media harvested at exponential growth phase. The fixed cell loaded ICR was carried out at initial stage of operation and the cell was entrapped by calcium alginate. The production of ethanol was steady after 24 h of operation. The concentration of ethanol was affected by the media flow rates and residence time distribution from 2 to 7 h. In addition, batch fermentation was carried out with 50 g/l glucose concentration. Subsequently, the ethanol productions and the reactor productivities of batch fermentation and immobilized cells were compared. In batch fermentation, sugar consumption and ethanol production obtained were 99.6% and 12.5% v/v after 27 h while in the ICR, 88.2% and 16.7% v/v were obtained with 6 h retention time. Nearly 5% ethanol production was achieved with high glucose concentration (150 g/l) at 6 h retention time. A yield of 38% was obtained with 150 g/l glucose. The yield was improved approximately 27% on ICR and a 24 h fermentation time was reduced to 7 h. The cell growth rate was based on the Monod rate equation. The kinetic constants (K(s) and mu(m)) of batch fermentation were 2.3 g/l and 0.35 g/lh, respectively. The maximum yield of biomass on substrate (Y(X-S)) and the maximum yield of product on substrate (Y(P-S)) in batch fermentations were 50.8% and 31.2% respectively. Productivity of the ICR were 1.3, 2.3, and 2.8 g/lh for 25, 35, 50 g/l of glucose concentration, respectively. The productivity of ethanol in batch fermentation with 50 g/l glucose was calculated as 0.29 g/lh. Maximum production of ethanol in ICR when compared to batch reactor has shown to increase approximately 10-fold. The performance of the two reactors was compared and a respective rate model was proposed. The present research has shown that high sugar concentration (150 g/l) in the ICR column was successfully converted to ethanol. The achieved results in ICR with high substrate concentration are promising for scale up operation. The proposed model can be used to design a lager scale ICR column for production of high ethanol concentration.     

Development and application of co-culture for ethanol production by co-fermentation of glucose and xylose: a systematic review

This article reviews current co-culture systems for fermenting mixtures of glucose and xylose to ethanol. Thirty-five co-culture systems that ferment either synthetic glucose and xylose mixture or various biomass hydrolysates are examined. Strain combinations, fermentation modes and conditions, and fermentation performance for these co-culture systems are compared and discussed. It is noted that the combination of Pichia stipitis with Saccharomyces cerevisiae or its respiratory-deficient mutant is most commonly used. One of the best results for fermentation of glucose and xylose mixture is achieved by using co-culture of immobilized Zymomonas mobilis and free cells of P. stipitis, giving volumetric ethanol production of 1.277 g/l/h and ethanol yield of 0.49–0.50 g/g. The review discloses that, as a strategy for efficient conversion of glucose and xylose, co-culture fermentation for ethanol production from lignocellulosic biomass can increase ethanol yield and production rate, shorten fermentation time, and reduce process costs, and it is a promising technology although immature.     

Production of ethanol by alginate-entrapped Saccharomyces cerevisiae strain "14-12"

Cells of S. cerevisiae strain "14-12" of different ages were immobilized in sodium alginate and used for conversion of glucose to ethanol. Immobilized cells of 48 hr old were the most potential. Employment of high counts of alginate-entrapped cells shortened the period required for production of the maximal alcohol yield. However, the percentage surviving cells decreased with increasing initial cell counts. Maximal accumulation of ethanol (4.18 g/100 ml) was obtained after 4 days of static fermentation with 1.8 X 10(8) immobilized yeast cells. The residual viable cell count was found to represent 3-fold the surviving percentage in a control experiment using an inoculum of the free yeast cells. Immobilized yeast cells could convert about 85% of the available sugars to ethanol over 28 days of the repeated-batch fermentation. The immobilized cells retained 50% of their viability for 16 days. After 48 days of repeated fermentation only 6% of the yeast cells were viable, and on the 52nd day no viable cells could be detected.     

Immobilized <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> cells in carboxymethyl cellulose for production of ethanol from cheese whey: experimental and kinetic studies

Cheese whey fermentation to ethanol using immobilized Kluyveromyces marxianus cells was investigated in batch and continuous operation. In batch fermentation, the yeast cells were immobilized in carboxymethyl cellulose (CMC) polymer and also synthesized graft copolymer of CMC with N-vinyl-2-pyrrolidone, denoted as CMC-g-PVP, and the efficiency of the two developed cell entrapped beads for lactose fermentation to ethanol was examined. The yeast cells immobilized in CMC-g-PVP performed slightly better than CMC with ethanol production yields of 0.52 and 0.49 g ethanol/g lactose, respectively. The effect of supplementation of cheese whey with lactose (42, 70, 100 and 150 g/l) on fermentative performance of K. marxianus immobilized in CMC beads was considered and the results were used for kinetic studies. The first order reaction model was suitable to describe the kinetics of substrate utilization and modified Gompertz model was quite successful to predict the ethanol production. For continuous ethanol fermentation, a packed-bed immobilized cell reactor (ICR) was operated at several hydraulic retention times; HRTs of 11, 15 and 30 h. At the HRT of 30 h, the ethanol production yield using CMC beads was 0.49 g/g which implies that 91.07 % of the theoretical yield was achieved.     

Characterization of ethanol fermentation waste and its application to lactic acid production by Lactobacillus paracasei

In this study, an ethanol fermentation waste (EFW) was characterized for use as an alternative to yeast extract for bulk fermentation processes. EFW generated from a commercial plant in which ethanol is produced from cassava/rice/wheat/barley starch mixtures using Saccharomyces cerevisiae was used for lactic acid production by Lactobacillus paracasei. The effects of temperature, pH, and duration on the autolysis of an ethanol fermentation broth (EFB) were also investigated. The distilled EFW (DEFW) contained significant amounts of soluble proteins (2.91 g/l), nitrogen (0.47 g/l), and amino acids (24.1 mg/l). The autolysis of the EFB under optimum conditions released twice as much amino acids than in the DEFW. Batch fermentation in the DEFW increased the final lactic acid concentration, overall lactic acid productivity, and lactic acid yield on glucose by 17, 41, and 14 %, respectively, in comparison with those from comparable fermentation in a lactobacillus growth medium (LGM) that contained 2 g/l yeast extract. Furthermore, the overall lactic acid productivity in the autolyzed then distilled EFW (ADEFW) was 80 and 27 % higher than in the LGM and DEFW, respectively.     

Effect of the size of yeast flocs and zinc supplementation on continuous ethanol fermentation performance and metabolic flux distribution under very high concentration conditions

Taking continuous ethanol fermentation with the self‐flocculating yeast SPSC01 under very high concentration conditions as an example, the fermentation performance of the yeast flocs and their metabolic flux distribution were investigated by controlling their average sizes at 100, 200, and 300 µm using the focused beam reflectance online measurement system. In addition, the impact of zinc supplementation was evaluated for the yeast flocs at the size of 300 µm grown in presence or absence of 0.05 g L^?1 zinc sulfate. Among the yeast flocs with different sizes, the group with the average size of 300 µm exhibited highest ethanol production (110.0 g L^?1) and glucose uptake rate (286.69 C mmol L^?1 h^?1), which are in accordance with the increased flux from pyruvate to ethanol and decreased flux to glycerol. And in the meantime, zinc supplementation further increased ethanol production and cell viability comparing with the control. Zinc addition enhanced the carbon fluxes to the biosynthesis of ergosterol (28.6%) and trehalose (43.3%), whereas the fluxes towards glycerol, protein biosynthesis, and tricarboxylic acid cycle significantly decreased by 37.7%, 19.5%, and 27.8%, respectively. This work presents the first report on the regulation of metabolic flux by the size of yeast flocs and zinc supplementation, which provides the potential for developing engineering strategy to optimize the fermentation system. Biotechnol. Bioeng. 2010;105: 935–944. © 2009 Wiley Periodicals, Inc.     

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

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

Ageing vessel design and optimization for continuous very-high-gravity ethanol fermentation processes

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.     

Acetic acid production from lactose by an anaerobic thermophilic coculture immobilized in a fibrous-bed bioreactor

An anaerobic thermophilic coculture consisting of a heterofermentative bacterium (Clostridium thermolacticum) and a homoacetogen (Moorella thermoautotrophica) was developed for acetic acid production from lactose and milk permeate. The fermentation kinetics with free cells in conventional fermentors and immobilized cells in a recycle batch fibrous-bed bioreactor were studied. The optimal conditions for the cocultured fermentation were found to be 58 degrees C and pH 6.4. In the free-cell fermentation, C. thermolacticum converted lactose to acetate, ethanol, lactate, H(2) and CO(2), and the homoacetogen then converted lactate, H(2), and CO(2) to acetate. The overall acetate yield from lactose ranged from 0.46 to 0.65 g/g lactose fermented, depending on the fermentation conditions. In contrast, no ethanol was produced in the immobilized-cell fermentation, and the overall acetate yield from lactose increased to 0.8-0.96 g/g lactose fermented. The fibrous-bed bioreactor also gave a higher final acetate concentration (up to 25. 5 g/L) and reactor productivity (0.18-0.54 g/L/h) as compared to those from the free-cell fermentation (final acetate concentration, 15 g/L; productivity, 0.06-0.08 g/L/h). The superior performance of the fibrous-bed bioreactor was attributed to the high cell density (20 g/L) immobilized in the fibrous-bed and adaptation of C. thermolacticum cells to tolerate a higher acetate concentration. The effects of yeast extract and trypticase as nutrient supplements on the fermentation were also studied. For the free-cell fermentation, nutrient supplementation was necessary for the bacteria to grow in milk permeate. For the immobilized-cell fermentation, plain milk permeate gave a high acetate yield (0.96 g/g), although the reactor productivity was lower than those with nutrient supplementation. Balanced growth and fermentation activities between the two bacteria in the coculture are important to the quantitative conversion of lactose to acetic acid. Lactate and hydrogen produced by C. thermolacticum must be timely converted to acetic acid by the homoacetogen to avoid inhibition by these metabolites.