Producing low-volume, high-cost products or, at least, middle-volume and middle — price coproducts from a biorefinery will be essential in establishing crop — and bio­mass-dependent biorefineries as components of the industrial landscape. The presence of up to 500 biorefineries in North America and 500-1000 across Europe brings the option of large numbers of small — to medium-capacity sugar streams in widely scat­tered locations, often in agricultural or afforested regions. Each site could house a fermentation facility for fine chemicals as well as being a site for biofuels — the costs of transporting biomass and of high-volume substrate solutions long distances by road or rail are unlikely to appeal either to industry or to environmentalists. The implication is that large numbers of production sites will arise where many commer­cial products for the chemical, food, and other industries will be synthesized. In turn, this suggests a reversal of the process by which large-scale fermentation production for antibiotics, enzymes, vitamins, food flavors, and acids has been exported from the United States and Europe to areas with lower labor and construction costs (India, China, etc.). This has been described as “restructuring the traditional fermentation industry into viable biorefineries.”82 Such a vision will almost certainly challenge the imaginations of industrial fermentation companies and demand that much closer attention is paid to the practical economics of the biorefinery concept(s).

Nevertheless, long-term strategic research around the world has begun to explore what might be achieved with such an abundant supply of pentose sugars from lig- nocellulosic biomass and of the metabolic and biosynthetic uses that might result.

Corynebacterium glutamicum has, as its name suggests, been much used for the industrial production of amino acids such as glutamic acid and lysine for the food and agricultural feed sectors. To broaden its substrate utilization range to include xylose, a two-gene xylose catabolic pathway was constructed using the E. coli xylA gene (encoding xylose isomerase) with either the E. coli xylB gene (for xylulokinase) or a corynebacterial gene for this enzyme; recombinants could grow in minimal media with xylose as the sole carbon source under aerobic conditions or, when O2- limited, utilize xylose alone or in combination with glucose.83

An unusual Lactobacillus sp. strain MONT4, isolated from a high-temperature fermenting grape must, is uniquely capable of fermenting L-arabinose to a mixture of d — and l-lactic acids; the organism contains two separate genes encoding lactate dehydrogenase with differing stereochemistries.84 D-lactic acid, along with “unnatu­ral” D-acids and amino acids, is contained in a series of bioactives developed as antiworming agents.85 Chemical transformations of lactic acids can yield a variety of chemical intermediates and feedstocks, notably acrylic acid and propylene glycol, compounds with major existing petrochemical-derived markets.86

Few (if any) major industrial-scale processes could fail to utilize individual sugars or mixtures of the carbohydrates emanating from lignocellulosic biomass processing or biodiesel production — even as atypical a microbe as Streptomyces clavuligerus (unable to metabolize glucose) can use glycerol for fermentations for the medically important в-lactam inhibitor clavulanic acid.87 The extensive experience of adapt­ing microbes to growing and functioning in lignocellulosic hydrolysates (chapters 3 and 4) should be readily transferable to industrial strains that already are expected to produce large amounts of high-value products in extremely concentrated media with sugars, oligosaccharides, or plant oils as substrates and with vegetable pro­teins or high concentrations of ammonium salts as nitrogen sources. Conversely, the genomes of already adapted ethanologens could (with removal of genes for ethanol formation) provide platform hosts for the expression of other biosynthetic pathways.

Only biopharmaceuticals, that is, recombinant proteins expressed in and produced by animal cell cultures, yeast, or E. coli, are impossible to merge with biorefineries because of regulatory restrictions and the absolute requirement for sterile cleanliness at the production facility; with the rise of biogeneric products, the production of biopharmaceuticals, already becoming global, will have completely left its early exclusive bases in southern California and western Europe.88 Less highly regulated bioprocesses, including all fermentation-derived chemicals not intended for biomed­ical use, are likely to migrate to sites of cheap and abundant carbon, nitrogen, and mineral nutrients, probably assisted by grants and incentives to provide employment in rural or isolated areas.

As an example of a tightly closed circular biorefinery process, consider biodiesel production from a vegetable oil — or, as work in Brazil indicates, intact oil-bearing seeds89 — using enzyme-catalyzed transesterification rather than alkaline hydroly- sis/methanolysis (chapter 6, section 6.1.2). The glycerol-containing effluent could be a carbon source for the methylotrophic yeast Pichia pastoris (able also to catabo — lize any contaminating methanol) that is widely used for expressing heterologous proteins, including enzymes; if the esterase used in the biodiesel process were to be produced on-site by a Pichia fermentation, the facility could be self-sufficient biotechnologically (figure 8.7). More ambitiously, any solid materials from the fer­mentation together with any “waste” plant material could provide the substrates for solid-state fermentations producing bioinsecticides, biopesticides, and biofertilizers for application to the farmland used to grow the oil crop itself.90

Microbial enzymes and single-cell proteins (as well as xylitol, lactic acid, and other fermentation products) have become targets for future innovations in utiliz­ing sugarcane bagasse in Brazil.91 The bioproduction of bacterial polyhydroxyal — kanoate polymers to replace petrochemical-based plastics is another goal of projects aiming to define mass-market uses of agricultural biomass resources in Brazil and elsewhere.92 Polyhydroxybutyrate and related polymers have had a long but unsuc­cessful record of searching for commercialization on a large scale on account of their uncompetitive economics and not always entirely industry-friendly chemical and physical properties, but high oil prices will increasingly sideline the former, whereas continued research into the vast number of possible biosynthetic structures may alight on unsuspected properties and uses — this also illustrates an impor­tant distinction made by the founders of “biocommodity engineering,” that is, that replacement of a fossil resource-derived product by a biomass-derived compound of identical composition is a conservative strategy, whereas a more radical one is that of substituting the existing chemical product with a biochemical with equivalent functionalities but with a distinct composition, which involves a more protracted transition but could be more promising in the long term.93

Using blue-green algae, not only to generate photobiohydrogen, but as microor­ganisms capable of much wider (and mostly unexplored) metabolic and biosynthetic capabilities, also offers innovative polymeric products, that is, exopolysaccharides whose massive cellular production has elicited interest in exploring their properties

and uses as industrial gums, bioflocculents, soil conditioners, emulsifiers and stabi­lizers, and vehicles for the removal and recovery of dissolved heavy metals.94

The biggest and most challenging leap for feedstocks for biorefineries, however, is that of using known microbial biochemistry to adsorb excess CO2 in the atmo­sphere. This is one of the two means — the other being the genetic manipulation of higher plants to increase their photosynthetic efficiencies (chapter 4, section 4.7) — that biotechnology could make decisive contributions to the global campaign to reduce atmospheric CO2 levels. Although plants use the Calvin-Benson cycle to fix CO2 into organic carbon compounds (initially sugar phosphates), CO2 fixation by “dark” metabolic processes are considerably less well publicized. The most recently discovered pathway was defined as recently as 1989 in an archeon, a type of single — celled microbe that is bacteria-like but evolved as an ancient line quite separately from the eubacteria and blue-green algae and whose members usually inhabit extreme environments (figure 8.8).95 The extensive cultivation of photobioreactors anywhere but in climates and locations with long guaranteed daily hours of intense sunlight is inefficient except in thin films, energy-requiring (e. g., to maintain an optimum temperature of 24°C or more[67]), and always limited by variations in the light/dark cycle.96 Light-independent bacterial bioprocesses avoid such limitations; if combined with biosynthetic pathways for high-value chemicals, bioreactors supplied with CO2 pumped into underground storage or with chemically adsorbed and released CO2 could be a primary technology for the later twenty-first century, whereas known and planned biofuels (other than H2) may prove disappointing in the extent to which their production and use over their full life cycles actually reduces transportation green­house gas emissions (chapter 1, section 1.6.2, and chapter 6, section 6.1.4), applying biotechnology to use accumulated CO2 as a process input would be a more widely applauded achievement.

Chemical routes to carbon capture and sequestration have already begun to establish themselves, especially for the most pertinent application of removing (as far as possible) CO2 emissions from power stations; trapping CO2 with amine-based systems has, however, been questioned as to its economics and its use of energy.97,98 Pumping trapped CO2 into sandstone deposits underneath the North Sea is practiced by the Norwegian oil industry but is of doubtful legality as well as requiring constant monitoring — it is, in many ways, a Faustian bargain with uncertain implications for the future despite its technical feasibility.99