Biomass — as described in the preceding section — has a calorific value that can be at least partly “captured” in other, more immediately useful forms of energy. Two of the examples included in figure 2.2 were fermentations, biological processes involving microbial bioenergetics in the chemical transformations. “Fermentation” has come a long way semantically since its coining by Louis Pasteur as life without air (la vie sans l’air).8 Pasteur was referring to yeast cells and how they altered their respiratory functions when air (or oxygen) became limiting. The modern use of the term is best understood by considering how microbes oxidize (or metabolize or combust) a substrate such as glucose released by the hydrolysis of, for example, starch or cellulose (figures 1.13 and 1.23). The complete biological oxidation of glucose can be written as

+ 6O2 ^ 6CO2 + 6H2O

As written, this chemical transformation is of little biological use to a population of microbial cells (yeast, fungi, or bacteria) because it implies only the generation of heat (metabolic heat) by the cells and that all the carbon is evolved as CO2 and cannot be utilized in cell replication. In a real microbiological scenario, glucose represents a valuable organic carbon supply as well as an energy source; part of that carbon is transformed by biochemical reactions inside the cells into the materials for new cells (before cell division), and much of the free energy that is released by the energetically favorable (exothermic) oxidation of the remainder of the glucose is used to drive the energetically unfavorable (endothermic) reactions of biosynthetic pathways; only a small portion of the total energy available is “wasted” as heat by biochemical reactions having mechanisms with thermodynamic efficiencies less than 100%:

nC6H12O6 + mO2 ^ cells + pCO2 + qH2O + evolved heat (where p, q << 6n).

Such a system is, to the microbial physiologist, simply “growth.” If, however, a metabolic output takes the form of a chemical product — especially where that product is commercially desirable or useful — then the process generates cells and product, whereas the CO2 evolved is (from an industrial or bioprocess standpoint) “waste.” In large industrial fermentors (with volumes in excess of 500,000 L), the generation of “metabolic” heat by dense cultures of microbial cells necessitates the expenditure of large amounts of energy to cool the liquid mass and, in turn, stimulates considerable efforts to recycle the “waste” heat on the industrial plant.9 Microbial processes producing antibiotics, enzymes, amino acids, and other products, recombinant proteins, flavors, and pharmaceutical active ingredients are generally termed “fermentations” although large volumes of oxygen-bearing air are supplied (via heavy-duty compressors) at high rates to the vessels so that microaerobic or anaerobic conditions are deliberately prevented, large quantities of glucose (or other carbon and energy sources) are utilized, and commercial products are accumulated as rapidly as the nutritional and physical conditions allow.

The primary fermentation in ethanol production is, on the other hand, a classic fermentation, that is, one where the ability of the cells to absorb glucose and other sugars from the medium (and to complete the early steps in their metabolic oxidation) exceeds the supply of oxygen required to fully complete the oxidative reactions. Under such conditions, yeasts such as Saccharomyces cerevisiae will accumulate (mostly) ethanol, whereas other yeasts, fungi, and bacteria produce combinations of the following:10

• Alcohols (ethanol, glycerol, я-propanol, я-butanol)

• Acids (formic, acetic, lactic, propionic, butyric)

• Decarboxylated acids (acetoin, acetone, diacetyl, 2,3-butanediol)

All these products (including ethanol) are the products (or intermediates) of a cluster of closely linked metabolic pathways and most of them are formed biosyn­thetically by reduction, thus regenerating the finite pool of redox carriers — in par­ticular, nicotinamide adenine dinucleotide (NAD) — in their oxidized forms inside the cell (figure 2.3). With the exception of ethanol (a much earlier development), all of these products were also industrially produced by microbial processes that were commercialized in the twentieth century; they represent “overflow” products of metabolism, given the supply of a high-value carbon and energy source such as glucose and an imbalanced nutritional environment where only part of the available carbon and energy can be fully used in cell replication and cell division. In particular, any cell population has, as determined by its precise genetic profile, a finite maximum specific oxygen uptake rate (i. e., grams of O2 per grams of cells per hour); feeding glucose as the growth-limiting nutrient to induce specific growth rates requiring the cells to exceed this maximum specific oxygen uptake rate causes the accumulation of “overflow” metabolites such as acetic and formic acids (figure 2.3).11 Ethanol produc­tion by S. cerevisiae, however, exhibits a highly important relationship: even under aerobic conditions (where the capacity to fully metabolize, or combust, glucose is not compromised by the lack of O2), feeding glucose at an increasingly rapid rate eventually swamps the ability of the cells to both grow and respire the glucose, that is, to provide the energy required to support growth and all the required ana­bolic, biosynthetic reactions. Below this crucial rate of glucose supply (most readily demonstrated in continuous cultures where the rate of glucose entry, and therefore, growth, is determined by the experimenter without any limitation of O2 or other nutrient supply), no ethanol is formed;[11] above this threshold, ethanol is accumulated, the “Crabtree effect” (see section 3.1.1), which is so vital to the long historical use of “wine” and other yeasts for ethanol production:10

яC6H12O6 + mO2 ^ cells + pCO2 + qH2O + rC2H5OH + evolved heat

Much has been written quasi-philosophically (and from a distinctly anthropo­centric viewpoint) on how the central metabolic pathways in microbes and plants have evolved and on their various thermodynamic inefficiencies.13 The single most pertinent point is, however, very straightforward: in a natural environment, there is no selective advantage in microbes having the capability of extracting the full thermo­dynamic energy in foodstuffs; the problem is that of kinetically utilizing (or using up) any available food source as rapidly as possible, thereby extracting as much energy

glucose

Enzyme pathways for glucose metabolism in microbial fermentations (+[2H], -[2H] represent reductive and oxidative steps,

On

On

and nutritional benefit in the shortest possible time in periods of alternating “feast and famine.”14 Many microbes are adventitious feeders, being capable of adapting even to extremes of oxygen availability in aerobic and anaerobic lifestyles. It is merely a human description that overspill products such as ethanol, acetic and lactic acids, and solvents such as acetone are “useful”; in a natural ecosystem, each might be the food­stuff for successive members of microbial consortia and lower and higher plants — in that sense of the term, nature is never wasteful. Axenic cultures of microbes in shake flasks and industrial fermentors are (in the same way as fields of monoculture crops such as soybean and sugarcane) “unnatural” because they are the products of modern agricultural practice and industrial microbiology and offer the economic advantages and potential environmental drawbacks of both of those twentieth-century activities.

Once the growth rate in a fermentation of glucose by an ethanol-forming organism becomes very low, and if glucose continues to be supplied (or is present in excess), the biochemistry of the process can be written minimally:

C6H12O6 ^ 2CO2 + 2C2H5OH

This represents a fully fermentative state where no glucose is respired and no fermentation side products are formed: for every molecule of glucose consumed, two molecules of ethanol are formed; for every 180 g of glucose consumed, 92 g of ethanol are formed, or 51.1 g of ethanol are produced per 100 g of glucose utilized. Any measurable growth or side-product formation will reduce the conversion efficiency of glucose to ethanol from this theoretical maximum.

Microbial energetics and biochemistry can, therefore, predict maximum yields of ethanol and other fermentation products from the sugars available in cane juices, starch, and cellulose (figure 2.3). How closely this theoretical maximum is reached is a function of fermentation design, control, engineering, and the biochemistry of the microorganism used for ethanol production. If a lignocellulosic substrate is to be used, however, the range of biochemical pathways required increases greatly both in scope and complexity as more sugars (pentoses as well as hexoses, plus sugar acids such as glucuronic acid) are potentially made available; this introduces substantially more variables into the putative industrial process, and preceding even this, there is the major hurdle of physically and chemically/enzymically processing biomass materials to yield — on an acceptable mass and time basis — a mixture of fermentable sugar substrates.