Many of the problems encountered in regulating both growth and metabolism in actively growing, high-cell-density systems could, in principle, be avoided by immobilizing the producer cells in or on inert supports and reusing them (if their viability can be maintained) for repetitive cycles of production. This is akin to trans­forming whole-cell biocatalysis to a more chemically defined form known for more than a century in the fine chemicals manufacturing industry. Cost savings in the power inputs required to agitate large-volume fermentors (100,000-500,000 l), in their cooling and their power-consuming aeration, spurred the fermentation industry at large to consider such technologies, and serious attempts to immobilize productive ethanologens began in the 1970s.

Alginate beads, prepared from the hydrophilic carbohydrate polymer alginic acid (extracted from kelp seaweed), are porous, compatible with an aqueous environ­ment, and could be loosely packed; in such a packed-bed bioreactor, perfused with a nutrient and substrate solution, a high volumetric productivity of ethanol produc­tion could be maintained for up to 12 days with a less than 10% loss of productive capacity.175 With cells of the yeast Kluyveromyces marxianus supplied with a Jeru­salem artichoke tuber extract, the maximum volumetric productivity was 15 times than for a conventional stirred tank fermentor. With S. cerevisiae in a fluidized bed contained in a closed circuit, ethanol production was possible with glucose solutions of up to 40% w/v at a relatively low temperature (18°C).176

As with continuous fermentation technologies, potable-alcohol manufacturers expressed interest in and actively developed demonstration facilities for immobi­lized yeast cells but failed to fully exploit the potential of the innovations — again because the product failed to meet organoleptic and other exacting specifications for the industry.177 Many different matrices were investigated beyond alginate and other seaweed polymers, including ceramic materials, synthetic polymers, and stainless steel fibers, to adsorb or entrap cells in up to five different reactor geometries:

1. The packed bed, the simplest arrangement and capable of upward or down­ward flow of the liquid phase

2. The fluidized bed, where mixing of gas, liquid, and solid phases occurs continuously

3. Airlift and bubble column bioreactors

4. Conventional stirred tank reactors

5. Membrane bioreactors where the cells are free but retained by a semiper­meable membrane

Beer producers have adopted immobilized cells to selectively remove unwanted aroma compounds from the primary fermentation product, “green” beer, and to both produce and remove aldehydes from alcohol-free and low-alcohol beers using strains and mutants of S. cerevisiae.178 For the main fermentation, however, immobilized cells remain a “promising” option for further development, but the added cost of immobilization techniques are impossible to justify for commercial processes with­out much stronger increases in volumetric productivity.

Cost-effective options have been explored for fuel ethanol production. In Brazil, improved fermentation practices and efficiencies have taken up the slack offered by low-intensity production system, and further progress requires technological innovations; immobilizing yeast ethanologens on sugarcane stalks is an eminently practical solution to providing an easily sourced and import-substituting support matrix:179 [39]

A summary of data from other studies of immobilized cells relevant to fuel ethanol production is given in table 4.2.180-184 The liquid phase can be recirculated in an immobilized system to maximize the utilization of the fermentable sugars; this is particularly useful to harmonize attaining maximal productivity (grams of ethanol produced per liter per hour) and maximum ethanol yield (as a percentage of the maximum theoretically convertible), with these optima occurring at different rates of flow with bacterial and yeast ethanologens.180

Among other recent developments, recombinant xylose-utilizing Z. mobilis cells were immobilized in photo-cross-linked resins prepared from polyethylene or poly­propylene glycols and shown to efficiently utilize both glucose and xylose from acid hydrolysates of cedar tree wood, rice straw, newspaper, and bagasse — unfortunately, the pretreatment was unfashionable, that is, a concentrated sulfuric process, and dif­ficult to precisely relate to modern trends in lignocellulosic processes.185 Alginate — immobilized S. cerevisiae cells have also been used in conjunction with a five-vessel cascade reactor in a continuous alcohol fermentation design.186 Sweet sorghum is a major potential source of fuel ethanol production in China where laboratory studies have progressed immobilized yeast cells to the stage of a 5-l bioreactor with stalk juice (i. e., equivalent to pressed cane sugar juice) as a carbon substrate.187

Insofar as immobilized cells represent a stationary phase or a population of very slowly growing cells, they may exhibit enhanced resistance to growth inhibitors and other impurities in substrate solutions that are more injurious to actively dividing cells. Immobilized cells of various microbial species, for example, are considered to be more resistant to aromatic compounds, antibiotics, and low pH, whereas immo­bilized S. cerevisiae is more ethanol-tolerant.178 This opens the novel possibility that immobilized yeast could be used to ferment batches of microbial toxin-containing feedstocks: a trichothecene mycotoxin inhibits protein synthesis and mitochon­drial function in S. cerevisiae and at 200 mg/l can cause cell death; conversely, fermentation is not affected and glucose metabolism may be positively redirected toward ethanol production, suggesting that trichothecene-contaminated grain could be “salvaged” for fuel ethanol manufacture.188 Alginate-encapsulated yeast cells are certainly able to withstand toxic sugar degradation products in acid hydrolysates prepared from softwoods (mainly spruce wood); such immobilized cells could fully ferment the glucose and mannose sugars within 10 hr, whereas free cells could not accomplish this within 24 hours, although the encapsulated cells lost their activity in subsequent batch fermentations.189