Until the 1980s, the general brewing industry view of yeasts for alcohol production was that most could tolerate only low concentrations (7-8% by volume) of ethanol and, consequently, fermentation media (worts) could be formulated to a maximum of 15-16° (Plato, Brix, or Balling, depending on the industry subsector), equivalent to 15-16% by weight of a sugar solution; the events that radically changed this assessment of yeasts and their physiology were precisely and cogently described by one of the key players:116

• When brewers’ yeasts were grown and measured in the same way as the more ethanol-tolerant distillers’ and sake yeasts, differences in ethanol tol­erance were smaller than previously thought.

• “Stuck” fermentations, that is, ones with little or no active growth in supraopti — mal sugar concentrations, could easily be rescued by avoiding complete anaero — biosis and supplying additional readily utilizable nitrogen for yeast growth.

• By removing insoluble grain residues (to reduce viscosity), recycling clear mashes to prepare more concentrated media from fresh grain, optimizing yeast nutrition in the wort, and increasing cooling capacity, yeast strains with no previous conditioning and genetic manipulation could produce ethanol up to 23.8% by volume.

Very-high-gravity (VHG) technologies have great technical and economic advantages:

1. Water use is greatly reduced.

2. Plant capacity is increased, and fermentor tank volume is more efficiently utilized.

3. Labor productivity is improved.

4. Fewer contamination outbreaks occur.

5. The energy requirements of distillation are reduced because fermented broth is more concentrated (16-23% v/v ethanol).

6. The spent yeast can be more readily recycled.

7. The grain solids removed prefermentation can be a valuable coproduct.

With an increased volume of the yeast starter culture added to the wort (higher “pitching rate”) and a prolonged growth phase fueled by adequate O2 and free amino nitrogen (amino acids and peptides), high-gravity worts can be fermented to ethanol concentrations more than 16% v/v even at low temperatures (14°C) within a week and with no evidence of any ethanol “toxicity.”117-120

This is not to say, however, that high ethanol concentrations do not constitute a stress factor. High-alcohol-content worts do still have a tendency to cease fer­mentation, and high ethanol levels are regarded as one of the four major stresses in commercial brewing, the others being high temperature, infection (contamination, sometimes associated with abnormal pH values), and mycotoxins from grain car­rying fungal infections of Aspergillus, Penicillium, Fusarium, Claviceps, or Acre — monium species.121 To some extent, the individual stress factors can be managed and controlled — for example, in extremis, antimicrobial agents that are destroyed during distillation (so that no carryover occurs to the finished products) can be added even in potable alcohol production. It is when the major stresses combine that unique conditions inside a fermentor can be generated. For a potable alcohol producer, these can be disastrous because there is an essential difference between the products of fuel/industrial ethanol and traditional alcoholic beverages: the latter are operated for consistency in flavor and quality of the product; for the beverage producer, flavor and quality outweigh any other consideration — even distinct economic advantages associated with process change and improvement — because of the market risks, especially if a product is to be matured (“aged”) for several years before resale.122 Industrial ethanol is entirely amenable to changes in production practice, strain, trace volatile composition, and even process “excursions” when the stress factors result in out-of-tolerance conditions. Yeast (S. cerevisiae) cells may have the ability to reduce short-term ethanol toxicity by entering a “quiescent” state in their average popula­tion cell cycle, extending a phase of growth-unassociated ethanol production in a laboratory process developed to produce 20% ethanol by volume after 45 hours.123

From the work on VHG fermentations, the realization was gained that typical media were seriously suboptimally supplied with free amino acids and peptides for the crucial early growth phase in the fermentation; increasing the free amino nitrogen content by more than fourfold still resulted in the exhaustion of the extra nitrogen within 48 hours (figure 4.6). With the correct supplements, brewer’s yeast could consume all the fermentable sugars in a concentrated medium (350 g/l) within eight days at 20°C or accumulate 17% (v/v) ethanol within three days.124 Fresh yeast autolysate was another convenient (and cost-effective) means of nitrogen supplemen­tation with an industrial distillery yeast from central Europe — although, with such a strain, while nitrogen additions improved final ethanol concentration and glucose utilization, none of them increased cell viability in the late stages of the fermentation, ethanol yield from sugar, or the maximum rate of ethanol formation.125 Commercial

proteases can liberate free amino acids and peptides from wheat mash, the low — molecular-weight nitrogen sources increasing the maximal growth (cell density) of the yeast cells and reducing the fermentation time in VHG worts from nine to three days — and without (this being an absolute priority) proteolytic degradation of the glucoamylase added previously to the mash to saccharify the wheat starch; rather than adding an extra nitrogen ingredient, a one-time protease digestion could replace medium supplementation.126 Not all amino acids are beneficial: lysine is severely inhibitory to yeast growth if the mash is deficient in freely assimilated nitrogen, but adding extra nitrogen sources such as yeast extract, urea, or ammonium sulfate abol­ishes this effect, promotes uptake of lysine, increases cell viability, and accelerates the fermentation.127

Partial removal of bran from cereal grains (wheat and wheat-rye hybrids) is an effective means of improving the mash in combination with VHG tech­nology with or without nitrogen supplementation (figure 4.7); in a fuel alcohol plant, this would increase plant efficiency and reduce the energy required for heating the fermentation medium and distilling the ethanol produced from the VHG process.128 Conversely, adding particulate materials (wheat bran, wheat mash insolubles, soy or horse gram flour, even alumina) improves sugar utiliza­tion in VHG media: the mechanism may be to offer some (undefined) degree of osmoprotection.129,130

A highly practical goal was in defining optimum conditions for temperature and mash substrate concentration with available yeast strains and fermentation hardware: with a wheat grain-based fermentation, a temperature of 30°C and an initial mash specific gravity of 26% (w/v) gave the best balance of high ethanol productivity, final ethanol concentration, and shortest operating time.131 The conclusions from such investigations are, however, highly dependent on the yeast strain employed and on the type of beer fermentation being optimized: Brazilian investigators working with

FIGURE 4.7 Grain pearling and very high-gravity grain mash fermentations for fuel ethanol production. (Data from Wang et al.128)

a lager yeast strain found that a lower sugar concentration (20% w/v) and temperature (15°C) were optimal together with a triple supplementation of the wort with yeast extract (as a peptidic nitrogen source), ergosterol (to aid growth), and the surfactant Tween-80 (possibly, to aid O2 transfer in the highly concentrated medium).132

For VHG fermentations, not only is nitrogen nutrition crucial (i. e., the sup­ply of readily utilizable nitrogen-containing nutrients to support growth) but other medium components require optimization: adding 50 mM of a magnesium salt in tandem with a peptone (to supply preformed nitrogen sources) increased ethanol concentrations from 14.2% to 17% within a 48-hr fermentation.133 These results were achieved with a medium based on corn flour (commonly used for ethanol produc­tion in China), the process resembling that in corn ethanol (chapter 1, section 1.4) with starch digestion to glucose with amylase and glucoamylase enzyme treatments. With a range of nutrients tested (glycine, magnesium, yeast extract, peptone, bio­tin, and acetaldehyde), cell densities could dramatically differ: the measured ranges were 74-246 x 106 cells/g mash after 24 hours and 62-392 x 106 cells/g mash after 48 hours. A cocktail of vitamins added at intervals in the first 28-37 hours of the fermentation was another facile strategy for improving final ethanol concentration, average ethanol production rate, specific growth rate, cell yield, and ethanol yield — and a reduced glycerol accumulation.134 Small amounts of acetaldehyde have been claimed to reduce the time required to consume high concentrations of glucose (25% w/v) in VHG fermentations; the mechanism is speculative but could involve increas­ing the intracellular NAD:NADH ratio and accelerating general sugar catabolism by glycolysis (figure 3.1).135 Side effects of acetaldehyde addition included increased accumulation of the higher alcohols 2,3-butanediol and 2-methylpropanol, exem­plifying again how immune fuel ethanol processes are to unwanted “contaminants”
and flavor agents so strictly controlled in potable beverage production. Mutants of brewer’s yeast capable of faster fermentations, more complete utilization of wort carbohydrates (“attenuation”), and higher viability under VHG conditions are eas­ily selected after UV treatment; some of these variants could also exhibit improved fermentation characteristics at low operating temperature (11°C).136

Ethanol diffuses freely across cell membranes, and it seems to be impossible for yeast cells to accumulate ethanol against a concentration gradient.137 This implies that ethanol simply floods out of the cell during the productive phases of alcoholic fermen­tations; the pioneering direct measurement of unidirectional rate constants through the lipid membrane of Z. mobilis confirmed that ethanol transport does not limit etha­nol production and that cytoplasmic ethanol accumulation is highly unlikely to occur during glucose catabolism.138 Nevertheless, even without such an imbalance between internal and external cellular spaces, product inhibition by ethanol is still regarded as an inhibitor of yeast cell growth, if not of product yield, from carbohydrates.139 Yeasts used for the production of sake in Japan are well known as able to accumulate ethanol in primary fermentations to more than 15% (v/v), and both Japanese brewing compa­nies and academic centers have pursued the molecular mechanisms for this:

• With the advent of genomics and the complete sequencing of the S. cerevi — siae genome, whole-genome expression studies of a highly ethanol-tolerant strain showed that ethanol tolerance was heightened in combination with resistance to the stresses imposed by heat, high osmolarity, and oxida­tive conditions, resulting in the accumulation internally of stress protec­tant compounds such as glycerol and trehalose and the overexpression of enzymes, including catalase (catalyzing the degradation of highly reactive hydrogen peroxide).140

• Inositol synthesis as a precursor of inositol-containing glycerophospholip — ids in cellular membranes is a second factor in membrane properties alter­ing (or altered by) ethanol tolerance.141

• Disrupting the FAA1 gene encoding a long-chain fatty acid acyl-CoA syn­thetase and supplying exogenously the long-chain fatty acid palmitic acid were highly effective in stimulating growth of yeast cells in the presence of high ethanol concentrations.142

• Ethanol stress provokes the accumulation of the amino acid L-proline, otherwise recognized as a defense mechanism against osmotic stress; disrupting a gene for proline catabolism increased proline accumulation and ethanol tolerance.143

• Part of the proline protective effect involves proline accumulation in inter­nal vacuoles — heat shock responses are, however, not changed, and this clearly differentiates cellular and biochemical mechanisms in the various stress reactions.144

Multiple sites for how sake yeasts have adapted (and, presumably, can further adapt) to high ethanol concentrations strongly suggest that continued “blind” selection of mutants that are fitter (in an imposed, Darwinian sense) to function despite the stresses of VHG media might be fruitful in the short to medium term.145 Eventually, however, the need to rationally change multiple sites simultaneously to continue improving the biological properties of yeast ethanologens will require a more proactive use of genomic knowledge.146 The positive properties of sake yeasts can, however, be easily transmitted to other yeast strains to ferment high-gravity worts.147 A compromise between “scien­tific” and traditional methodologies for fuel ethanol production may be to generate fus — ants between recombinant ethanologens and osmo — and ethanol-tolerant sake strains.

A last footnote for sake brewing (but not for bioethanol production) is that the high ethanol concentrations generated during the fermentation extract the antioxi­dant protein thioredoxin from the producing cells so that readily detectable levels of the compound persist in the final sake product.148 In addition to its antioxidant func­tion, thioredoxin is anti-inflammatory for the gastric mucosa and, by cleaving disul­fide bonds in proteins, increases protein digestibility, and sake can be considered as a development stage for “functional foods.”