Magnesium limitation and its role in apparent toxicity of ethanol during yeast fermentation

The rate of ethanol production per milligram of cell protein begins to decline in the early stage of batch fermentation before high concentrations of ethanol have accumulated. In yeast extract-peptone medium (20% glucose), this initial decline appears to be related to growth and to result in part from a nutrient deficiency. The addition of yeast extract, peptone, and ashed preparations of these restored the ability of glucose-reconstituted medium (in which cells had been previously grown) to support vigorous growth. Magnesium was identified as the active component. Supplementing fermentations with 0.5 mM magnesium prolonged exponential growth, resulting in increased yeast cell mass. The addition of magnesium also reduced the decline in fermentative activity (micromoles of CO2 evolved per hour per milligram of protein) during the completion of batch fermentations. These two effects reduced the time required for the conversion of 20% glucose into ethanol by 1/3 with no measurable loss in ethanol yield (98% of theoretical maximum yield). It is possible that some of the reported beneficial effects of complex nutrients (soy flour and yeast extract) for ethanol production also result from the correction of a simple inorganic ion deficiency, such as magnesium.     

Study of fermentation behavior and formulation of a semidefined nutrient medium based on acid-production measurements in Z. mobilis cultures

Batch fermentations of glucose to ethanol by Z. Mobilis.(ATCC 10988) were examined in several semidefined nutrient media. The measurement of acid produced by the microorganism was used to study its transient fermentation characteristics. Limitation of nitrogen source in the semidefined medium of Rogers and coworkers(2) was found to limit the growth of this microorganism in the late stages of batch fermentations, when the initial glucose concentration was 75 g/L and higher. The microorganism exhibits a preference for inorganic nitrogen over preformed organic nitrogen provided by yeast extract. The microbial growth occurs exponentially in the presence of ammonium sulfate and yeast extract. However, in the absence of ammonium sulfate, the growth occurs in a linear fashion. The "linear" growth phase is characterized by poor cell-mass yields, and during this phase, growth and ethanol production are decoupled. An improved semi-defined growth medium is established which supports better growth rate and cellular yield, without affecting the ethanol yield.     

Effects of yeast extract and glucose on xanthan production and cell growth in batch culture of Xanthomonas campestris

Although available kinetic data provide a useful insight into the effects of medium composition on xanthan production by Xanthomonas campestris, they cannot account for the synergetic effects of carbon (glucose) and nitrogen (yeast extract) substrates on cell growth and xanthan production. In this work, we studied the effects of the glucose/yeast-extract ratio (G/YE) in the medium on cell growth and xanthan production in various operating modes, including batch, two-stage batch, and fed-batch fermentations. In general, both the xanthan yield and specific production rate increased with increasing G/YE in the medium, but the cell yield and specific growth rate decreased as G/YE increased. A two-stage batch fermentation with a G/YE shift from an initial low level (2.5% glucose/0.3% yeast extract) to a high level (5.0% glucose/0.3% yeast extract) at the end of the exponential growth phase was found to be preferable for xanthan production. This two-stage fermentation design both provided fast cell growth and gave a high xanthan yield and xanthan production rate. In contrast, fed-batch fermentation with intermittent additions of glucose to the fermentor during the stationary phase was not favorable for xanthan production because of the relatively low G/YE resulting in low xanthan production rate and yield. It is also important to use a moderately high yeast extract concentration in the medium in order to reach a high cell density before the culture enters the stationary phase. A high cell density is also important to the overall xanthan production rate. Received: 30 September 1996 / Received revision: 21 January 1997 / Accepted: 10 February 1997     

Production of recombinant human growth hormone in Escherichia coli: expression of different precursors and physiological effects of glucose, acetate, and salts

The constitutive cytoplasmic expression in E. coli of human growth hormone (hGH) with different N-terminal extensions (3 or 4 amino acids) has been studied. These hGH precursors were used for in vitro cleavage to obtain the mature, authentic hormone. Small changes in the amino acid extensions of the hGH precursors led to three-fold differences in specific expression rates. The specific expression rate of the hGH precursors was inversely proportional to the ratios of the specific growth rates of plasmid containing and plasmid free cells (micro(+)/micro(-)) and also to the genetic stability. To ensure a satisfactory genetic stability in production fermentors, an hGH precursor with a moderate expression efficiency was chosen.The medium composition and growth conditions were studied, resulting in the choice of a glucose fed batch fermentation process using a complex medium. In this process a yield of 2000 mg/L of met-ala-glu-hGH (MAE-hGH) was obtained. The fermentation process comprised a glucose-limited growth phase followed by a second phase with increased glucose feed and exhaustion of phosphate from the medium. The second phase is characterized by an MAE-hGH production, whereas further biomass formation is blocked. High concentrations of glucose led to reduced specific expression of MAE-hGH--the specific and total yield in batch glucose fermentations is only about 30% of the yield in optimized fed batch fermentations. The physiological background for this was investigated. Chemostat experiments showed that the glucose concentration and the metabolic condition of the cells--i.e. with or without formation of acetate--was not critical per se in order to obtain a high specific yield of MAE-hGH. Therefore it is unlikely that formation of MAE-hGH is catabolite repressed by glucose. Furthermore it was shown that the specific production rate of MAE-hGH was independent of the specific growth rate and it was further demonstrated that the decrease in expression efficiency in glucose batch fermentation was a result of an inhibitory effect of acetic acid. In batch fermentations this inhibitory effect was enhanced by a salt effect caused by increased consumption of acid and base used to control pH. The identity of the acid and the base used are not important in this context. From studies of the expression of other proteins in E. coli. with constitutive as well as inducible promoters we conclude that glucose fed batch processes are often superior to batch processes in the production of heterologous proteins E. coli.     

Kinetics of sugars consumption and ethanol inhibition in carob pulp fermentation by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in batch and fed-batch cultures

The waste materials from the carob processing industry are a potential resource for second-generation bioethanol production. These by-products are small carob kibbles with a high content of soluble sugars (45–50%). Batch and fed-batch Saccharomyces cerevisiae fermentations of high density sugar from carob pods were analyzed in terms of the kinetics of sugars consumption and ethanol inhibition. In all the batch runs, 90–95% of the total sugar was consumed and transformed into ethanol with a yield close to the theoretical maximum (0.47–0.50 g/g), and a final ethanol concentration of 100–110 g/l. In fed-batch runs, fresh carob extract was added when glucose had been consumed. This addition and the subsequent decrease of ethanol concentrations by dilution increased the final ethanol production up to 130 g/l. It seems that invertase activity and yeast tolerance to ethanol are the main factors to be controlled in carob fermentations. The efficiency of highly concentrated carob fermentation makes it a very promising process for use in a second-generation ethanol biorefinery.     

Osmotic pressure effects and intracellular accumulation of ethanol in yeast during fermentation

Summary The intracellular accumulation of ethanol in yeast and its potential effects on growth and fermentation have been topics of controversy for the past several years. The determination of intracellular ethanol based on the exclusion of [¹⁴C]sorbitol to estimate aqueous cell volume was used to examine the question of intracellular ethanol accumulation. An intracellular accumulation of ethanol inSaccharomyces cerevisiae was observed during the early stages of fermentation. However, as fermentation continued, the intracellular and extracellular concentrations of ethanol became similar. Increasing the osmotic pressure of the medium with glucose or sorbitol was observed to cause an increase in the intracellular ethanol concentration. Associated with this was a decrease in yeast growth and fermentation rates. In addition, increasing the osmotic pressure of the medium was observed to cause an increase in glycerol production. Supplementation of the media with excess peptone, yeast extract, magnesium sulfate and potassium phosphate was found to relieve the detrimental effects of high osmotic pressure. Under these conditions, though, no effect on the intracellular and extracellular ethanol distribution was observed. These results indicate that nutrient limitation, and not necessarily intracellular ethanol accumulation, plays a key role during yeast fermentations in media of high osmolarity.     

Lactic acid fermentation in a recycle batch reactor using immobilized Lactobacillus casei

Lactic acid production by recycle batch fermentation using immobilized cells of Lactobacillus casei subsp. rhamnosus was studied. The culture medium was composed of whey treated with an endoprotease, and supplemented with 2.5 g/L of yeast extract and 0.18 mM Mn(2+) ions. The fermentation set-up comprised of a column packed with polyethyleneimine-coated foam glass particles, Pora-bact A, and connected with recirculation to a stirred tank reactor vessel for pH control. The immobilization of L. casei was performed simply by circulating the culture medium inoculated with the organism over the beads. At this stage, a long lag period preceded the cell growth and lactic acid production. Subsequently, for recycle batch fermentations using the immobilized cells, the reducing sugar concentration of the medium was increased to 100 g/L by addition of glucose. The lactic acid production started immediately after onset of fermentation and the average reactor productivity during repeated cycles was about 4.3 to 4.6 g/L . h, with complete substrate utilization and more than 90% product yield. Sugar consumption and lactate yield were maintained at the same level with increase in medium volume up to at least 10 times that of the immobilized biocatalyst. The liberation of significant amounts of cells into the medium limited the number of fermentation cycles possible in a recycle batch mode. Use of lower yeast extract concentration reduced the amount of suspended biomass without significant change in productivity, thereby also increasing the number of fermentation cycles, and even maintained the D-lactate amount at low levels. The product was recovered from the clarified and decolorized broth by ion-exchange adsorption. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:841-853, 1997.     

Effect of yeast extract on Escherichia coli growth and acetic acid production

Fed batch cultures were performed to investigate the effect of yeast extract concentration on the kinetics of growth and acetic acid production of recombinant Escherichia coli BL21 in a synthetic medium. Three runs were performed with 40g/l total glucose concentration. The yeast extract/glucose ratio (YE/G; w/w), was 0.1, 0.05 and 0.025 in the feed. These decreasing YE/G values did not affect growth kinetics, but reduced the final cell concentration by about 10%, and also reduced the cell yield. Experiments with 60g/l total glucose concentration, one with a YE/G of 0.025 in the feed and the other without yeast extract, showed final acetic acid concentrations of 5.1 and 0.5g/l respectively, without any difference in cellular concentration. Although there was no significant influence on growth kinetics and final cellular concentration, the cell fermentative capacity was enhanced by yeast extract. The feed medium without yeast extract was the best condition for control purposes in high cell density cultures and for recombinant gene expression.     

The cause of "acid-crash" and "acidogenic fermentations" during the batch acetone-butanol-ethanol (ABE-) fermentation process

Experiments were performed to determine the cause of "acid crash", a phenomenon which occasionally occurs in pH-uncontrolled batch fermentations resulting in premature cessation of ABE (acetone butanol) production. The results indicate that "acid crash" occurs when the concentration of undissociated acids in the broth exceeds 57 - 60 mmol/l. Prevention can be achieved by introducing some limited pH control to minimize the concentration of undissociated acids or by slowing the metabolic rate, and thus the rate of acid production, by, for example, lowering the fermentation temperature. "Acidogenic fermentations", which occur when batch fermentations are performed at pH values close to neutrality, are due to rapid production of acids followed by inhibition of solventogenesis when the total acid concentration reaches 240 - 250 mmol/l. Solventogenesis can be achieved at these pH values by lowering the glucose uptake rate / acid production rate by use of e.g. elevated glucose or lowered yeast extract concentrations in the growth medium.     

Use of carbohydrate-supplemented distillery spent wash as a medium for ethanol production by a thermotolerant strain of yeast at 45°C

Summary Spent wash from the Old Bushmill's Distillery Co. Ltd. was supplemented with either glucose (10% [w/v]) or cellulose (5% [w/v]) and used as a medium for the thermotolerant yeast strain Kluyveromyces marxianus IMB3. There was no significant difference in ethanol production during growth on these media at 45° C, compared with that produced during growth on conventional, pre-defined laboratory media. On glucose supplemented spent wash ethanol yields were in the region of 45 g/L, representing 87% of the maximum theoretical yield. Analysis of spent media from the glucose-containing fermentations demonstrated that the total organic carbon (TOC) content was reduced by 36%. The results suggest a novel means of utilizing whiskey distiller spent wash.     

Inulin enrichment by fermentation in a flocculating yeast reactor

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

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.     

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

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

Improving the ethanol yield by reducing glycerol formation using cofactor regulation in Saccharomyces cerevisiae

To increase ethanol yield and decrease glycerol production in Saccharomyces cerevisiae, the strategies of direct cofactor-regulation were explored. During anaerobic batch fermentations, the yeast expressing Bacillus cereus gapN gene, encoding non-phosphorylating NADP(+)-dependent glyceraldehyde-3-phosphate dehydrognease, produced 73.8?g ethanol?l(-1), corresponding to 96% of theoretical maximum yield compared to 92% for the wild type. The yeast expressing Escherichia coli frdA gene encoding the NAD(+)-dependent fumarate reductase, exhibited a 22% (relative to the amount of substrate consumed) increase in glycerol yield in medium containing 2?g fumarate?l(-1). The yeast expressing mhpF gene, encoding acetylating NAD(+)-dependent acetaldehyde dehydrogenase, produced 74.5?g ethanol?l(-1), corresponding to 97.4% of theoretical maximum yield while glycerol decreased by 40% when acetic acid was added before inoculation. This strain represents a promising alternative for ethanol production with lignocellulosic hydrolysates where acetate is available at significant amounts.     

Combined use of three methods for high concentration ethanol production by Saccharomyces cerevisiae

Summary Ethanol concentration and fermentation productivity using Saccharomyces cerevisiae were substantially increased in shake flask cultures with a normal inoculum by combining 3 methods: (a) by making nutrient additions to the standard medium for ethanol production, (b) by immobilizing the cells in alginate beads and (c) by using a glucose step-feeding batch process. Ethanol concentration by free yeast was improved from 5.9% (w/w) to 9.6% (w/w) when a further 0.8% yeast extract and 1% animal peptone were added to the standard 30% (w/v) glucose nutrient medium. This was further increased to 12.8% (w/w) by using alginate immobilized yeast. The ethanol concentration was increased again, to 15.0% (w/w) by using the glucose step-feeding batch process.     

Repeated use of yeast biomass in ethanol fermentation of juniper berry sugars

Summary The object of this study was to establish the possibility of using the yeast biomass separated from the fermentation broth at the end of ethanol fermentation of juniper berry sugars as an inoculum in successive batch fermentation processes. A part of the fermentation broth (10% v/v) and a suspension of yeast biomass (separated from the same broth) into the water extract of juniper berries (2 g of wet yeast biomass per liter of water extract) were used as inocula. It was shown that the suspension of yeast biomass could be used as inoculum in successive batch processes without negative effects on the kinetics and ethanol yield, but with positive effects on the capacity and economy of the bioprocess. The addition of ammonium salts at optimum levels did not affect the final ethanol concentrations (4.3–4.4% v/v), but enhanced the specific rate of ethanol production, thus reducing the process duration by about five times.     

Proof-of-concept of a novel micro-bioreactor for fast development of industrial bioprocesses

The experimental performance of a novel micro-bioreactor envisaged for parallel screening and development of industrial bioprocesses has been tested in this work. The micro-bioreactor with an internal volume of 4.5 mL is operated under oscillatory flow mixing (OFM), where a controllable mixing and mass transfer rates are achieved under batch or continuous laminar flow conditions. Several batch fermentations with a flocculent Saccharomyces cerevisiae strain were carried out at initial glucose concentrations (S(0)) range of approximately 5-20 g/L and compared to yeast growth kinetics in a stirred tank (ST) bioreactor. Aerobic fermentations were monitored ex situ in terms of pH, DO, glucose consumption, and biomass and ethanol production (wherever applicable). An average biomass production increase of 83% was obtained in the micro-bioreactor when compared with the ST, with less 93.6% air requirements. It also corresponded to a 214% increase on biomass production when compared with growth in a shaken flask (SF) at S(0) = 20 g/L. Further anaerobic fermentations at the same initial glucose concentration ranges gave the opportunity to use state-of-the-art fiber optics technology for on-line and real-time monitoring of this bioprocess. Time profiles of biomass concentration (measured as optical density (OD)) were very similar in the ST bioreactor and in the micro-bioreactor, with a highly reproducible yeast growth in these two scale-down platforms.     

Inhibition of fermentation and growth in batch cultures of the yeast Brettanomyces intermedius upon a shift from aerobic to anaerobic conditions (Custers effect)

Aerobic growth of the yeast Brettanomyces intermedius CBS 1943 in batch culture on a medium containing glucose and yeast extract proceeded via a characteristic pattern. In the first phase of growth glucose was fermented to nearly equal amounts of ethanol and acetic acid. After glucose depletion, growth continued while the ethanol produced in the first phase was almost quantitatively converted to acetic acid. Finally, after a long lag phase, growth resumed with concomitant consumption of acetic acid.When the culture was made anaerobic during the first phase, growth, glucose consumption and metabolite production stopped immediately. This Custers effect (inhibition of alcoholic fermentation as a result of anaerobic conditions) was transient. After 7–8 h the culture was adapted to anaerobiosis, and growth and ethanol production resumed. The lag phase could be shortened at will by the introduction of hydrogen acceptors, such as oxygen or acetoin, into the culture. Glycerol production was not observed during any phase of growth. These results support the hypothesis that the Custers effect in this yeast is due to a disturbance of the redox balance, resulting from the tendency of the organism to produce acetic acid, and its inability to restore the balance by production of glycerol.     

Reasons for the apparent difference in the effects of produced and added ethanol on culture viability during rapid fermentation by Saccharomyces cerevisiae

By feeding ethanol at various high rates to low cell density cultures of Saccharomyces cerevisiae it was shown that the sharp fall in viability when ethanol is produced during rapid fermentations is in part a direct consequence of the high rate of change of extracellular ethanol concentration. Nevertheless, the fall in viability in high cell density rapid fermentations which produced 98 g L(-1) ethanol in 3 h considerably exceeded that of control low cell density cultures to which ethanol was added at the same rate. This difference was shown to be not due to intracellular ethanol accumulation or to differences in glucose concentration between the cultures. The concentrations of a range of potentially toxic fatty acids, higher alcohols, and esters were measured during rapid fermentations, but when added at these concentrations to control cultures in the presence of ethanol they had no significant toxic effect. However, when rapid fermentations were conducted in rich medium containing 80 g L(-1) yeast extract, the apparent difference in toxicity of produced and added ethanol virtually disappeared. Magnesium was shown to be the component of yeast extract primarily responsible for this effect. The high rate of fall of viability when ethanol is rapidly produced is suggested to be partly due to the inability of the cells to adapt quickly enough to the rising ethanol concentration and partly to an increased demand for magnesium at higher ethanol concentrations which cannot be met in Mg-unsupplemented high cell density fermentations.     

Optimizing alcohol production from whey using computer technology

This study was undertaken with the major goal of optimizing the ethanol production from whey using computer technology. To reach this goal, a mathematical model that would describe the fermentation and that could be used for the optimization was developed. Kluyveromyces fragilis was the microorganism used to ferment the lactose in the whey into ethanol. Preliminary studies showed that K. fragilis produced about 90% of the theoretical ethanol yield when grown in whey-complemented media. However, when this yeast is grown in nonsupplemented whey media, it does not produce more than 32% of that yield. Comparative batch fermentations of lactose and whey-complemented media showed that whey possibly contains enhancing components for yeast growth and ethanol production. To obtain the mathematical model, the one-to-one effect of the process variables (lactose and yeast extract concentrations, air flowrate, pH, and dilution rate) on the ethanol production were first investigated. Experiments on the pH effect showed that a decrease in pH from 7 to 4 produced an increase in ethanol concentration from 16.5 to 26.5 g/L (50 g/L initial lactose). The results obtained from modeling of the continuous fermentation using the previously listed variables showed that air flowrate, pH, and dilution rate were the process variables that most influence the production of ethanol.