Fuel alcohol production: effects of free amino nitrogen on fermentation of very-high-gravity wheat mashes

Although wheat mashes contain only growth-limiting amounts of free amino nitrogen, fermentations by active dry yeast (Saccharomyces cerevisiae) were completed (all fermentable sugars consumed) in 8 days at 20 degrees C even when the mash contained 35 g of dissolved solids per 100 ml. Supplementing wheat mashes with yeast extract, Casamino Acids, or a single amino acid such as glutamic acid stimulated growth of the yeast and reduced the fermentation time. With 0.9% yeast extract as the supplement, the fermentation time was reduced from 8 to 3 days, and a final ethanol yield of 17.1% (vol/vol) was achieved. Free amino nitrogen derived in situ through the hydrolysis of wheat proteins by a protease could substitute for the exogenous nitrogen source. Studies indicated, however, that exogenously added glycine (although readily taken up by the yeast) reduced the cell yield and prolonged the fermentation time. The results suggested that there are qualitative differences among amino acids with regard to their suitability to serve as nitrogen sources for the growth of yeast. The complete utilization of carbohydrates in wheat mashes containing very little free amino nitrogen presumably resulted because they had the "right" kind of amino acids.     

Fuel alcohol production: effects of free amino nitrogen on fermentation of very-high-gravity wheat mashes.

Although wheat mashes contain only growth-limiting amounts of free amino nitrogen, fermentations by active dry yeast (Saccharomyces cerevisiae) were completed (all fermentable sugars consumed) in 8 days at 20 degrees C even when the mash contained 35 g of dissolved solids per 100 ml. Supplementing wheat mashes with yeast extract, Casamino Acids, or a single amino acid such as glutamic acid stimulated growth of the yeast and reduced the fermentation time. With 0.9% yeast extract as the supplement, the fermentation time was reduced from 8 to 3 days, and a final ethanol yield of 17.1% (vol/vol) was achieved. Free amino nitrogen derived in situ through the hydrolysis of wheat proteins by a protease could substitute for the exogenous nitrogen source. Studies indicated, however, that exogenously added glycine (although readily taken up by the yeast) reduced the cell yield and prolonged the fermentation time. The results suggested that there are qualitative differences among amino acids with regard to their suitability to serve as nitrogen sources for the growth of yeast. The complete utilization of carbohydrates in wheat mashes containing very little free amino nitrogen presumably resulted because they had the "right" kind of amino acids.     

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.     

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.     

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

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

Effects of particulate materials and osmoprotectants on very-high-gravity ethanolic fermentation by Saccharomyces cerevisiae.

The effects of osmoprotectants (such as glycine betaine and proline) and particulate materials on the fermentation of very high concentrations of glucose by the brewing strain Saccharomyces cerevisiae (uvarum) NCYC 1324 were studied. The yeast growing at 20 degrees C consumed only 15 g of the sugar per 100 ml from a minimal medium which initially contained 35% (wt/vol) glucose. Supplementing the medium with a mixture of glycine betaine, glycine, and proline increased the amount of sugar fermented to 30.5 g/100 ml. With such supplementation, the viability of the yeast cells was maintained above 80% throughout the fermentation, while it dropped to less than 12% in the unsupplemented controls. Among single additives, glycine was more effective than proline or glycine betaine. On incubating the cultures for 10 days, the viability decreased to only 55% with glycine, while it dropped to 36 and 27%, respectively, with glycine betaine and proline. It is suggested that glycine and proline, known to be poor nitrogen sources for growth, may serve directly or indirectly as osmoprotectants. Nutrients such as tryptone, yeast extract, and a mixture of purine and pyrimidine bases increased the sugar uptake and ethanol production but did not allow the population to maintain the high level of cell viability. While only 43% of the sugar was fermented in unsupplemented medium, the presence of particulate materials such as wheat bran, wheat mash insolubles, alumina, and soy flour increased sugar utilization to 68, 75, 81, and 82%, respectively.     

Influence of cultivation procedure for <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> used as pitching agent in industrial spent sulphite liquor fermentations

The cell viability and fermentation performance often deteriorate in fermentations of spent sulphite liquor (SSL). This investigation therefore addresses the question of how different cultivation conditions for yeast cells influence their ability to survive and boost the ethanol production capacity in an SSL-based fermentation process. The strains used as pitching agents were an industrially harvested Saccharomyces cerevisiae and commercial dry baker’s yeast. This study therefore suggests that exposure to SSL in combination with nutrients, prior to the fermentation step, is crucial for the performance of the yeast. Supplying 0.5 g/l fresh yeast cultivated under appropriate cultivation conditions may increase ethanol concentration more than 200%.     

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.     

Grain pearling and very high gravity (VHG) fermentation technologies for fuel alcohol production from rye and triticale

A SATAKE laboratory abrasive mill was used for rye and triticale grain processing. About 12% of dry grain mass was removed after three and five successive abrasions for triticale and rye, respectively. Starch contents in the pearled grain were increased by 8·0% for triticale, and by 7·1% for rye. The pearled rye and triticale were ground and fermented by active dry yeast for fuel alcohol production by very high gravity (VHG) fermentation at 20°C. VHG technology was applied to increase final ethanol concentrations in the fermentors from 9·5–10·0% (v/v) (normal gravity) to 12·9–15·1% (v/v). The grain pearling process coupled with VHG technology further raised the ethanol concentration to 15·7–16·1% (v/v). Partial removal of outer grain solids in an alcohol plant would improve plant efficiency and decrease energy requirements for mash heating, mash cooling, and ethanol distillation.     

Enhanced ethanol and chitosan production from wheat straw by Mucor indicus with minimal nutrient consumption

Recently, Mucor indicus was introduced as a promising ethanol producing microorganism for fermentation of lignocellulosic hydrolysates, showing a number of advantages over Saccharomyces cerevisiae. However, high nutrient requirement is the main drawback of the fungus in efficient ethanol production from lignocelluloses. In this study, application of fungal extract as a potential nutrient source replacing all required nutrients in fermentation of wheat straw by M. indicus was investigated. Wheat straw was pretreated with N-methylmorpholine-N-oxide (NMMO) at 120 °C for 1–5 h prior to enzymatic hydrolysis. Hydrolysis yield was improved at least by 6-fold for 3 h pretreated straw compared with that of untreated one. A fungal extract was produced by autolysis of M. indicus biomass, an unavoidable byproduct of fermentation. Maximum free amino nitrogen (2.04 g/L), phosphorus (1.50 g/L), and total nitrogen (4.47 g/L) as well as potassium, magnesium, and calcium in the fungal extract were obtained by autolysis of the biomass at 50 °C and pH 5.0. The fungal extract as a nutrient-rich supplement substituted yeast extract and all other required minerals in fermentation and enhanced the ethanol yield up to 92.1% of the theoretical yield. Besides, appreciate amounts of chitosan were produced as another valuable product of the autolysis.     

Acetone-butanol-ethanol (ABE) fermentation in an immobilized cell trickle bed reactor

Acetone-butanol-ethanol (ABE) fermentation was successfully carried out in an immobilized cell trickle bed reactor. The reactor was composed of two serial columns packed with Clostridium acetobutylicum ATCC 824 entrapped on the surface of natural sponge segments at a cell loading in the range of 2.03-5.56 g dry cells/g sponge. The average cell loading was 3.58 g dry cells/g sponge. Batch experiments indicated that a critical pH above 4.2 is necessary for the initiation of cell growth. One of the media used during continuous experiments consisted of a salt mixture alone and the other a nutrient medium containing a salt mixture with yeast extract and peptone. Effluent pH was controlled by supplying various fractions of the two different types of media. A nutrient medium fraction above 0.6 was crucial for successful fermentation in a trickle bed reactor. The nutrient medium fraction is the ratio of the volume of the nutrient medium to the total volume of nutrient plus salt medium. Supplying nutrient medium to both columns continuously was an effective way to meet both pH and nutrient requirement. A 257-mL reactor could ferment 45 g/L glucose from an initial concentration of 60 g/L glucose at a rate of 70 mL/h. Butanol, acetone, and ethanol concentrations were 8.82, 5.22, and 1.45 g/L, respectively, with a butanol and total solvent yield of 19.4 and 34.1 wt %. Solvent productivity in an immobilized cell trickle bed reactor was 4.2 g/L h, which was 10 times higher than that obtained in a batch fermentation using free cells and 2.76 times higher than that of an immobilized CSTR. If the nutrient medium fraction was below 0.6 and the pH was below 4.2, the system degenerated. Oxygen also contributed to the system degeneration. Upon degeneration, glucose consumption and solvent yield decreased to 30.9 g/L and 23.0 wt %, respectively. The yield of total liquid product (40.0 wt %) and butanol selectivity (60.0 wt %) remained almost constant. Once the cells were degenerated, they could not be recovered.     

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.     

Relationship of low lysine and high arginine concentrations to efficient ethanolic fermentation of wheat mash.

Very high gravity wheat mashes containing 20 or more grams of carbohydrates per 100 mL were fermented completely by Saccharomyces cerevisiae, even though these mashes contained low amounts of assimilable nitrogen. Supplementation of wheat mashes with various amino acids or with yeast extract, urea, or ammonium sulfate reduced the fermentation time. However, lysine or glycine added as single supplements, inhibited yeast growth and fermentation. With lysine, yeast growth was severely inhibited, and a loss of cell viability as high as 80% was seen. Partial or complete reversal of lysine-induced inhibition was achieved by the addition of a number of nitrogen sources. All nitrogen sources that relieved lysine-induced inhibition of yeast growth also promoted uptake of lysine and restored cell viability to the level observed in the control. They also increased the rate of fermentation. Experiments with minimal media showed that for lysine to be inhibitory to yeast growth, assimilable nitrogen in the medium must be in growth-limiting concentrations or totally absent. In the presence of excess nitrogen, lysine stimulated yeast growth and fermentation. Results indicate that supplementing wheat mash with other nitrogen sources increases the rate of fermentation not only by providing extra nitrogen but also by reducing or eliminating the inhibitory effect of lysine on yeast growth.     

Role of phosphatidylinositol (PI) in ethanol production and ethanol tolerance by a high ethanol producing yeast

The effect of inositol addition on phospholipids, cell growth, ethanol production and ethanol tolerance in a high ethanol producing Saccharomyces sp were studied. Addition of inositol greatly influenced major phospholipid synthesis. With inositol in the fermentation medium, phosphatidylinositol (PI) content was increased, while phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were decreased. However, without inositol in the fermentation medium, PI content dropped down within 24 h, then increased, but was lower than in the presence of inositol. When yeast cells had a higher content of PI, they produced ethanol much more rapidly and tolerated higher concentrations of ethanol. During ethanol shock treatment at 18% (v/v) ethanol, yeast cells with a higher concentration of PI lost their viability much more slowly than those with a lower concentration of PI, indicating that the PI content in these yeast cells can play an important role in ethanol production and ethanol tolerance. Fatty acids and ergosterol were not responsible for high ethanol tolerance and high ethanol production in this yeast strain. Received 22 September 1998/ Accepted in revised form 20 December 1998     

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

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

Effects of heat shock and ethanol stress on the viability of aSaccharomyces uvarum (carlsbergensis) brewing yeast strain during fermentation of high gravity wort

Summary The effects of heat shock and ethanol stress on the viability of a lager brewing yeast strain during fermentation of high gravity wort were studied. These stress effects resulted in reduced cell viability and inhibition of cell growth during fermentation. Cells were observed to be less tolerant to heat shock during the fermentation of 25°P (degree Plato) wort than cells fermenting 16°P wort. Degree Plato (^oP) is the weight of extract (sugar) equivalent to the weight of sucrose in a 100 g solution at 20°C. Relieving the stress effects of ethanol by washing the cells free of culture medium, improved their tolerance to heat shock. Cellular changes in yeast protein composition were observed after 24 h of fermentation at which time more than 2% (v/v) ethanol was present in the growth medium. The synthesis of these proteins was either induced by ethanol or was the result of the transition of cells from exponential phase to stationary phase of growth. No differences were observed in the protein composition of cells fermenting 16°P wort compared to those fermenting 25°P wort. Thus, the differences in the tolerance of these cells to heat shock may be due to the higher ethanol concentration produced in 25°P wort which enhanced their sensitivity to heat shock.     

Interaction effects of lactic acid and acetic acid at different temperatures on ethanol production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in corn mash

The combined effects of lactic acid and acetic acid on ethanol production by S. cerevisiae in corn mash, as influenced by temperature, were examined. Duplicate full factorial experiments (three lactic acid concentrations × three acetic acid concentrations) were performed to evaluate the interaction between lactic and acetic acids on the ethanol production of yeast at each of the three temperatures, 30, 34, and 37°C. Corn mash at 30% dry solids adjusted to pH 4 after lactic and acetic acid addition was used as the substrate. Ethanol production rates and final ethanol concentrations decreased (P<0.001) progressively as the concentration of combined lactic and acetic acids in the corn mash increased and the temperature was raised from 30 to 37°C. At 30°C, essentially no ethanol was produced after 96 h when 0.5% w/v acetic acid was present in the mash (with 0.5, 2, and 4% w/v lactic acid). At 34 and 37°C, the final concentrations of ethanol produced by the yeast were noticeably reduced by the presence of 0.3% w/v acetic acid and ≥2% w/v lactic acid. It can be concluded that, as in previous studies with defined media, lactic acid and acetic acid act synergistically to reduce ethanol production by yeast in corn mash. In addition, the inhibitory effects of combined lactic and acetic acid in corn mash were more apparent at elevated temperatures.     

Lactic acid production from agricultural resources as cheap raw materials

Agricultural resources such as barley, wheat, and corn were hydrolyzed by commercial amylolytic enzymes and fermented into lactic acid by Enterococcus faecalis RKY1. Although no additional nutrients were supplemented to those resources, lactic acid productivities were obtained at >0.8 g/l h from barley and wheat. When 200 g/l of whole wheat flour was hydrolyzed by amylolytic enzymes after the pre-treatment with 0.3% (v/v) sulfuric acid and sterilized by filtration, E. faecalis RKY1 efficiently produced lactic acid with 2.6 g/l h of lactic acid productivity and 5.90 g/l of maximal dry cell weight without additional nutrients. Lactic acid productivity and cell growth could be enhanced to 31% and 12% higher values than those of non-adapted RKY1, by adaptation of E. faecalis RKY1 to CSL-based medium. When the medium contained 200 g/l of whole wheat flour hydrolyzate, 15 g/l of corn steep liquor, and 1.5 g/l of yeast extract, lactic acid productivity and maximal dry cell weight were obtained at 5.36 g/l h and 14.08 g/l, respectively. This result represented an improvement of up to 106% of lactic acid productivity and 138% of maximal dry cell weight in comparison to the fermentation from whole wheat flour hydrolyzate only.     

Effect of oxygen on ethanol fermentation in packed-bed tapered-column reactor

Summary In ethanol production with immobilized yeast a major problem is the provision of nutrients to these highly concentrated cells. O₂ being one of the nutrients of utmost importance to yeast cells, was fed into a column packed with beads with a cell loading of more than 40 g/l. Since addition of large volume of air or O₂ to a cylindrical column reactor would aggravate the problems of pressure build up and channelling caused by the evolving CO₂ gas, a tapered-column reactor and pulsed flow of oxygen gas was used. The supplement of O₂ gas to the tapered column increased the productivity from 21.1 g ethanol x (l gel x h)^-1 to 26.7 g x (l gel x h)^-1, when the ethanol concentration at the outlet was about 80 g/l. The yield coefficient of ethanol was also increased from 0.41 g ethanol/g glucose to 0.43 after O₂ supplement was started. The effects of frequency and duration of O₂ supplement were also determined.     

In situ detoxification and continuous cultivation of dilute-acid hydrolyzate to ethanol by encapsulated S. cerevisiae

Dilute-acid lignocellulosic hydrolyzate was successfully fermented to ethanol by encapsulated Saccharomyces cerevisiae at dilution rates up to 0.5h(-1). The hydrolyzate was so toxic that freely suspended yeast cells could ferment it continuously just up to dilution rate 0.1h(-1), where the cells lost 75% of their viability measured by colony forming unit (CFU). However, encapsulation increased their capacity for in situ detoxification of the hydrolyzate and protected the cells against the inhibitors present in the hydrolyzate. While the cells were encapsulated, they could successfully ferment the hydrolyzate at tested dilution rates 0.1-0.5h(-1), and keep more than 75% cell viability in the worst conditions. They produced ethanol with yield 0.44+/-0.01 g/g and specific productivity 0.14-0.17 g/(gh) at all dilution rates. Glycerol was the main by-product of the cultivations, which yielded 0.039-0.052 g/g. HMF present in the hydrolyzate was converted 48-71% by the encapsulated yeast, while furfural was totally converted at dilution rates 0.1 and 0.2h(-1) and partly at the higher rates. Continuous cultivation of encapsulated yeast was also investigated on glucose in synthetic medium up to dilution rate 1.0 h(-1). At this highest rate, ethanol and glycerol were also the major products with yields 0.43 and 0.076 g/g, respectively. The experiments lasted for 18-21 days, and no damage in the capsules was detected.