The major challenge in developing commercial H2 generation by “dark” biotechnology, that is, by fermentation processes, has been the low productivity of natural H2- evolving microbes, and this can be resolved into two distinct limitations: the molar yield from a fermentable substrate and the expected low growth rates and cell densi­ties of microbial producers under energy-poor environmental conditions.

On the basis of data presented in table 7.2, a maximum of 4 mol of H2 per mole of fermentable glucose substrate equates (on a mass balance) to only 8 g/180 g of sugar consumed. In cell-free biotransformations, using mixtures of enzymes, a nearly threefold higher productivity has been demonstrated.41 If the pentose phosphate pathway (figure 3.2) can be run in a cyclic manner to completely oxidize glucose 6-phosphate to CO2 and H2O, each mole of glucose consumed (in six “turns” of the cycle) can generate 12 mol of reduced cofactor:

6C6H12O6 + 12 NADP+ ^ 5C6H12O6 + 6CO2 + 12NADPH + 12H+

Coupling the reoxidation of NADPH to the hydrogenase from Pyrococcus furiosus (table 7.2), one of only a few hydrogenases known to accept NADPH as a reducing agent, generated 116 mol H2/mol of phosphorylated glucose oxidized.41

Japanese researchers genetically modified a strain of E. coli to overexpress the gene for formate hydrogen lyase, an enzyme catalyzing the reaction:

HCOOH ^ CO2 + H2

By growing the cells to high cell densities under glucose-supported aerobic conditions before transfer to an anaerobic fermentor, a high catalytic potential for formate transformation to H2 was established, reaching 300 L H2/hr/l culture.42 This rate of H2 production could support a 1-kW fuel cell operating at 50% efficiency using only 2 l of culture medium maintained under continuous conditions by a feed of formic acid. Further strain construction (deleting lactate dehydrogenase and fumarate reductase genes) has improved the induction of the formate hydrogen lyase activity.43 The same genetic manipulations have eliminated side reactions of (phosphoenol)pyruvic acid in glucose-grown E. coli, maximizing the transformation of pyruvate to formate via the pyruvate formate lyase-catalyzed step:

CH3COCOOH + CoASH ^ CH3CO-SCoA + HCOOH

Such a genetic background (with formate hydrogen lyase as the next step) increases the production of H2 from glucose as the fermentation substrate, although only rates of approximately 20 l/hr/l culture have been achieved.44

Thermophilic and hyperthermophilic microbes are obvious choices for production strains to be cultured at high temperatures to accelerate H2 formation.

A strain of the bacterium Klebsiella oxytoca isolated from a hot spring in China could produce H2 even in the presence of 10% O2 in the gas phase but had a low molar yield (1 mol/mol glucose consumed).45 The extreme thermophile Caldicel — lulosiruptor saccharolyticus shows up to 92% of the theoretical H2 yield from glu­cose (4 mol/mol) at low growth rates at 72-73°C, indicating possible applications in long-term free or immobilized cultures.46 This organism also can produce H2 from hydrolyzed paper sludge industrial waste as the sole carbon source and is unusual in that it can utilize xylose faster than glucose.4748

An advanced bioprocess option for H2 production utilizes a membrane bioreactor to maintain the bacteria inside the reactor while allowing fluids to exit.49 This design could be the optimal methodology to restrict the growth of methanogenic bacteria that consume H2 and generate CH4, a gas with only 42 percent of the energy content of H2.50 Restricting the residence time of materials in a continuous flow reactor system allows H2 producers to outcompete the slower-growing methanogens. With a 12-hr residence time, glucose could be utilized as a substrate for H2 production with an over­all consumption of 98% and an efficiency (assuming 4 mol H2/mol glucose) of 25%, accumulating H2 at a concentration of 57-60% (volume basis) in the headspace.49

The production of H2 need not be based on pure bacterial cultures, mixed cul­tures, even ones with only indirect evidence of the microbial flora present, being suitable for wastewater treatment or for local production sites in isolated rural locali­ties. A clostridial population (on the basis of the spectrum of metabolites produced in parallel to H2) provided a system capable of stable and prolonged production, with H2 reaching 51 percent in the gas phase and with no methanogenesis observed.51 Some process control is, however, unavoidable to maintain H2-evolving capacity, particu­larly pH: maintaining a pH of 6.0 may inhibit the growth of lactobacilli in a mixed culture of Clostridium and Coprothermobacter species that could utilize untreated sludge and lake sediment material as substrates.52 The choice of pH regulant may, however, be crucial, and the accumulation of sodium to toxic levels was a severe limitation in a continuous biohydrogen system from sucrose-supplemented anaerobic sewage sludge.53 At a constant pH, the combination of substrate-material retention time and temperature (in the 30-37°C range) can have a marked effect on the bal­ance between different clostridial species, the appearance of nonclostridial bacterial species and the overall molar yield of H2 from carbohydrates.54 In a molasses waste­water treatment plant in China, the H2 production rate was highest in an ethanol­forming stage of the process, and at least six types of H2-producing microbe were present, predominantly a novel species, Ethanoligenes harbinese.55 Such a complex microbial ecology may be highly adaptable to differing types and compositions of carbon sources during production cycles or when seasonally available.

The use of advanced reactor types has been explored; for example, the fluidized bed reactor design has been explored with a mixed community that rapidly estab­lished H2 production from C. butyricum; instability developed during the course of time as propionate producers gradually took over, and biofilm-type reactors may not be the optimal design because of the efficient adhesion of H2-consuming microbial species to the carrier.56 A trickle-bed reactor packed with glass beads inoculated with a pure culture of C. acetobutylicum certainly gave high H2 gas concentrations but soon (60-72 hours) clogged because of bacterial growth.57

Irrespective of the long-term prospects for the industrial production of H2, dark fermentations are very likely to be permanent features of wastewater treatment technologies and as an alternative to methane for local “biogas” production — indeed an obvious application of the trickle-bed reactor may be for the treatment of high-carbohydrate wastewaters, requiring no energy input for stirring a conventional mixed tank and producing H2 as a recoverable fuel gas as well as a more dilute stream for conventional biogas production.57 The use of water streams with lower organic loading may, however, be advantageous for H2 production because supersaturation of the gas space inside bioreactors may feed back to inhibit H2 synthesis.58 Removing CO2 (e. g., by the use of KOH to absorb the gas) is also beneficial to H2 production, probably by minimizing the flow of utilizable substrates to acetogenic bacteria capable of synthesizing acetic acid from H2 and CO2.59 Animal waste — contaminated water can also be made acceptable to biological H2 producers if the ammonia concentration can be reduced and maintained below toxic concentrations in continuous flow systems, especially if the microbial community can be gradually adapted to increased ammonia levels.60 Food processing aqueous streams with high chemical oxygen demands can support biohydrogen production at 100 times the rates possible with domestic wastewaters — often reaching commercially viable amounts of H2 if used on-site as a heating fuel.61

As an excellent example of the opportunistic use of H2-producing microbes in biofuels production, a strain of Enterobacter aero genes was shown to be highly adept at producing both H2 and ethanol from glycerol-containing wastewaters from biodiesel production; continuous production in a packed bed reactor using porous ceramic support material maximized the H2 production rate.62 But, as a final twist, H2 producers may have an unexpected role in assisting a microbial community of methanogens to achieve full productivity, that is, a syntropic relationship may be established to provide the methanogens with a readily utilizable substrate; adding mesophilic or thermophilic H2-producing cultures increases biogas production from animal manure slurry, and the added species persist for several months of semicontinuous operation.63

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.”

In 2000 (but using data from 1996), a review, two of whose authors had affiliations to ExxonMobil, concluded that sugarcane grown under Brazilian conditions could generate above-ground and harvestable biomass of 932 GJ/hectare/year; a total of 9.3 x 108 hectares of land could substitute the full global primary energy of fossil fuels (3.2 x 1011 GJ/year), even if only sugarcane stems were used as a fuel with a quan­titative extraction of the energy inside the plant material, that is, 343 GJ/hectare.81 Based on a total area of land used to grow crops worldwide of 1.4 x 109 hectares, the fossil fuel demand could, in principle, be met with 67% of cultivatable land dedi­cated to sugarcane as an energy crop, or only 24% if all of the harvestable material were used. If, however, the sugarcane stems were to be used for ethanol production, only a third of the useful energy would be converted to the biofuel, and more than twice the global area of land used to grow crops would be needed, that is, ethanol production could not substitute for the world’s appetite for fossil fuels.

This is an interesting but disingenuous set of calculations because ethanol had never been advocated as anything other than a convenient energy carrier as a replace­ment (partial or otherwise) for gasoline in automobiles. From the most recent data published by the International Energy Agency, oil used for transport is expected to continue being approximately 20% of the yearly fossil fuel demand or to slowly increase to more than 50% of the crude oil extraction rate (figure 5.8). To substitute the global demand for oil as a transport fuel, therefore, sugarcane grown for ethanol would need to occupy 40% of the world’s arable land (stems only as a feedstock) or 20-25% if the entire harvestable biomass were to be used for ethanol production. The long-perceived potential conflict between land use for food crop production and for bioenergy is highly likely to be a reality, and genetic engineering or other innovative

technologies cannot avoid this without massive increases over current bioprocess — limited abilities to transform biomass into ethanol (or any other liquid biofuel).82 Any conventional or molecular “improvement” of a major crop species must avoid greater dependence on fertilizer and other agrochemical application to achieve greater yields. A particular drawback is the reliance on energy crops with large inputs of nonrenew­able energy for the production of biofuels: corn-derived ethanol and, for biodiesel, rape (canola) and soybean seeds (see chapter 6, section 6.1).83

Equally, the use of agricultural residues for biofuel production has severe agro­nomic limitations. Although a worldwide potential residue harvest of 3.8 x 109 tonnes/year has been estimated, equivalent to 7.5 billion barrels of diesel, removing more than 30% of this could greatly increase soil erosion as well as depleting the soil organic carbon content. One solution to this problem is to establish new bioenergy plantations on uncultivated land, perhaps 250 million hectares worldwide.84 This could be supplemented by the exploitation of weed infestations, for example, water hyacinth in lakes and waterways, naturally occurring sugar-rich forest flowers, or other adventitious resources.85 86 Of the major candidate energy crops, switchgrass and other prairie grasses appear to offer the best balance among high net energy yields, low nutrient demands, and high soil and water conservation; with its high total root mass, switchgrass can replace soil carbon lost during decades of previous tilling within (perhaps) 20 years.87 Under U. S. farm conditions, there is mounting evidence that dedicated energy crops would significantly reduce erosion and chemi­cal runoff in comparison with conventional monoculture crops.88-90 Replacing con­tinuous corn cropping with a corn-wheat rotation and no-till field operations might maximize agricultural residue availability.91

Doubts persist, however, that any of these options represent true sustainability, even when consideration is extended to prolific plantations of acacia and eucalyptus
trees, both of which rapidly exhaust tropical soils of nutrients and fall far short of even 100 years of high productivity.79 The nearest approach to true sustainability could be the immediate-locale use of sun-dried wood from well-managed energy planta­tions, using highly efficient wood-burning stoves; the necessary high rates of biomass “mining” (probably requiring some method of energy-dependent biomass drying) to support predicted rates of growth of gasoline and biofuel consumption appears to be mathematically unsustainable.79 In any case, intensive energy crop plantations would certainly require a high degree of environmental monitoring and management to avoid biological collapse unless lavish amounts of fertilizers are applied and maxi­mum yields are ensured by equally large supplies of insecticides and pesticides.

A final twist is the impact of global climate change: could global warming (at least for a limited time, perhaps as long as 50-100 years) increase plant growth to such an extent that biomass extraction targets would be more closely met? Even accurately predicting biomass yields from monocultures of fast-growing tree species such as willow in the northeastern United States suffers from methodological weaknesses.92 The effects of changes in daily minimum and maximum temperatures are complex because they differentially influence crop yield parameters; this is a major area of uncertainty for projecting yield responses to climate change.93 For three major cereal species (wheat, corn, and barley), and an increase in annual global temperatures since 1980 of approximately 0.4°C, there is evidence for decrease in yield; the magnitude of the effect is small in comparison with the technological yield gains during the same period but suggests that rising temperature might cancel out the expected increase in yield because of increased CO2 concentrations.9495 Using the global environment as an uncontrolled experimental system has its methodological drawbacks, but the results of the one long-term field trial to attempt to isolate the effects of temperature on rice growth indicate that a 15% reduction in yield could result from each 1°C rise in temperature, a much greater effect than predicted by simulation models.96 The International Panel on Climate Change has, in stark contrast, predicted that CO2 benefits will exceed temperature-induced yield reductions with a modest rise in tem­perature. Because this conclusion has received wide media coverage, it is important to examine the detailed conclusions for food, fiber, and forest products: [53]

• Globally, commercial timber productivity rises modestly with climate change in the short to medium term, with large regional variability around the global trend.

Taken together, these International Panel on Climate Change prognoses suggest some short-term improvement in the productivity of a range of plant species, including timber grown as feedstocks for lignocellulosic ethanol, but the predictions become more unreliable as the geographical area narrows and related effects of climate are considered. This highlights the obvious conclusion that geostatistical surveys and controlled experiments should both be pursued vigorously as priority issues for agronomy and plant physiology in the next ten years.

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

Xylose reductase, the first step in the pathway of xylose catabolism in most yeast species, functions as an enzyme equally well with L-arabinose as with D-xylose, with a slightly higher affinity (lower Km) and a higher maximal rate (Vmax) for l-arabinose.59 Polyol dehydrogenases, active on xylitol, on the other hand, find either L — or D-arabinose to be a poor substrate.103 Although the XDH activity from P. stipitis was kinetically investigated in 1989, little is known about its func­tional physiology; the catalyzed reaction is reversible but activity is unlikely to be regulated by the NAD/NADH balance inside the cell.104 This yeast also con­tains a second XDH, quite distinct from the well characterized xyl2 gene product, but its role is presently undefined in either xylose catabolism or ethanol produc — tion.105 The NAD-specific XR from S. cerevisiae itself is even less well charac­terized, although the enzyme activity is induced by xylose with the wild-type organism.106

An outline of known enzyme-catalyzed metabolic relationship for pentitols and pentoses is given in figure 3.6; some of these pathways are of increasing contempo­rary interest because either they or their engineered variants could lead to the syn­thesis by whole cells (or in biotransformations with isolated enzymes) of “unnatural” or rare sugars useful for the elaboration of antibiotic or antiviral drugs — this is discussed later in chapter 8 when the Green Chemistry of the biorefinery concept for processing agricultural residues is discussed in depth.

Progress in defining the actual pathways operating in known ethanologenic yeasts was rapid after the year 2000

• gene encoding an L-xylulose reductase (forming xylitol; NADP-depen — dent) was then demonstrated in H. jecorina and overexpressed in S. cere — visiae; the l-arabinose pathway uses as its intermediates l-arabinitol, l-xylulose, xylitol, and (by the action of XDH) D-xylulose; the xylulose reductase exhibited the highest affinity to l-xylulose, but some activity was shown toward d-xylulose, d-fructose, and l-sorbose.109

• In H. jecorina, deletion of the gene for XDH did not abolish growth because ladl-encoded l-arabinitol 4-dehydrogenase compensated for this loss — however, doubly deleting the two dehydrogenase genes abolished the ability to grow on either d-xylose or xylitol.110

With this knowledge, expressing the five genes for L-arabinose catabolism in S. cere — visiae enabled growth on the pentose and, although at a low rate, ethanol production from l-arabinose under anaerobiosis.111 In the same year (2003), the genes of the shorter bacterial pathway for L-arabinose catabolism were inserted into S. cerevi — siae.112 The bacterial pathway (active in, e. g., B. subtilis and E. coli) proceeds via l-ribulose, l-ribulose 5-phosphate, and d-xylulose 5-phosphate (figure 3.6), using the enzymes L-arabinose isomerase, L-ribulokinase, and L-ribulose 5-phosphate epimerase. The coexpression of an arabinose-transporting yeast galactose permease allowed the selection on L-arabinose-containing media of an L-arabinose-utilizing yeast transformant capable of accumulating ethanol at 60% of the theoretical maxi­mum yield from L-arabinose under O2-limiting conditions.112

How to define yield with energy crops? This is both more flexible and less precise than with, for example, crop yield as measured by grain size or weight per plant or per unit area. For a feedstock such as wheat straw, increasing plant biomass accumulation per plant may suffice, and this implies a change in nutrient utilization or absorption from the soil, but this may paradoxically reverse the trend toward “dwarf” crops (i. e., more grain, less stem/stalk) that has been emphasized in the green revolution type of agronomy.290 In principle, simply achieving higher leaf, stalk, and stem mass per plant is a straightforward target that is not limited by considerations of morphology for crops dedicated to energy supply and/or biofu­els. Mass clonal propagation of commercial trees is now well advanced, and gene transfer technologies have been devised for conifers and hardwoods, that is, forest biotechnology has emerged (probably irreversibly) out of the laboratory and into the global ecosystem.291

Traditional breeding and marker-assisted selection can identify genes involved in nutrient use efficiency that can then be used in gene transfer programs to improve features of plant nutrition — for crop plants in intensive agriculture, nitrogen assimilation and recycling within the plant over the stages of plant development are crucial.292 Although plant biochemistry is increasingly well understood at the molecular level, what is much less clear is how to accurately modulate gene expres­sion (single genes or whole pathways) to achieve harvestable yield increases.290 As with other “higher” organisms, a greater understanding of how regulatory circuits and networks control metabolism at organ and whole-plant levels as an exercise in systems biology will be necessary before metabolic engineering for yield in crop plants becomes routine.294

Nevertheless, successes are now being reported for “gene therapy” with the goal of improving the assimilation of CO2 into biomass, however this is defined:

• Transgenic rice plants with genes for phosphoeno/pyruvate carboxylase and pyruvate, orthophosphate dikinase from maize, where the two enzymes are key to high photosynthetic carbon fixation under tropical conditions (“C4 metabolism”), increases photosynthetic efficiency and grain yield by up to 35% and has the potential to enhance stress tolerance.295

• Once CO2 is “fixed” by green plants, some of the organic carbon is lost by respiratory pathways shared with microorganisms, and there are several reports that partially disabling the oxidative pathways of glucose metabo­lism enhances photosynthetic performance and overall growth: for exam­ple, in transgenic tomato plants with targeted decreases in the activity of mitochondrial malate dehydrogenase, CO2 assimilation rates increased by up to 11% and total plant dry matter by 19%.296

• Starch synthesis in developing seeds requires ADP-glucose phosphorylase; expressing a mutant maize gene for this enzyme in wheat increases both seed number and total plant biomass, these effects being dependent on increased photosynthetic rates early in seed development.297

Much attention has been given to improving the catalytic properties of the primary enzyme of CO2 fixation, ribulose 1,5-bisphosphate carboxylase (Rubisco), the most abundant single protein on Earth and one with a chronically poor kinetic efficiency for catalysis; although much knowledge has been garnered pertaining to the natural variation in rubisco’s catalytic properties from different plant species and in developing the molecular genetics for gene transfer among plants, positive effects on carbon metabolism as a direct result of varying the amounts of the enzyme in leaves have proved very slow to materialize.298 A more radical approach offers far greater benefits: accepting the inevitable side reaction catalyzed by Rubisco, that is, the formation of phosphoglyceric acid (figure 4.15), transgenic plants were constructed to contain a bacterial pathway to recycle the “lost” carbon entirely inside the chloro — plast rather than the route present in plant biochemistry that involved the concerted actions of enzyme in three plant cell organelles (chloroplast, mitochondrion, and peroxisome); transgenic plants grew faster, produced more biomass (in shoot and roots), and had elevated sugar contents.299

Is there an upper limit to plant productivity? A temperate zone crop such as wheat is physiologically and genetically capable of much higher productivity and efficiency of converting light and CO2 into biomass than can be achieved in a “real”

environment, that is, in hydroponics and with optimal mineral nutrition; again, most studies focus on yield parameters such as grain yield (in mass per unit area) but total leaf mass (a component of straw or stover) will also increase under such ideal conditions.300 The higher the light intensity, the greater the plant response, but natural environments have measurable total hours of sunshine per year, and the climate imposes average, minimum, and maximum rainfall and temperatures; supplementary lighting is expensive — but, given unlimited renewable energy resources, substantial increases in plant productivity are theoretically possible. From a shorter-term perspective, however, the choice of biomass refuses to be erased from the agenda, and agricultural wastes simply cannot compete with the “best” energy crops. For example, using biofuel yield as the metric, the following ranking can be computed from relative annual yields (liters per hectare):301

Corn stover (1.0) < poplar (2.9) < switchgrass (3.2) < elephant grass (Miscanthus, 4.4)

Rather than mobilizing molecular resources against the vagaries of climate, concen­trating effort on maximizing biomass supply from a portfolio of crops other than those most presently abundant would pay dividends. Overreliance on a few species (vulnerable to pests and climatic variability) would be minimized by expanding the range of such crops.

Glycerol represents 10% by weight of typical triglycerides, and biodiesel production generates large amounts of this coproduct (figure 6.3). This obviously marginalizes any chemical routes to glycerol synthesis that are either cost-inefficient or otherwise suboptimal in a competitive manufacturing environment.82 By any manufacturing route, glycerol production provides a good input to FT conversion to liquid alkanes because glycerol can be converted over platinum-based catalysts into syngas at rela­tively low temperatures, 225-350°C.104 The gas mixture from glycerol conversion at 300-450°C has a molar excess of H2 over CO (up to 1.83:1), a high ratio (up to 90:1) between CO and CO2, and only traces of methane. With a subsequent FT step, the overall conversion of glycerol to hydrocarbons can be written as

25C3O3H8 ^ 7C8H8 + 19CO2 + 37H2O

and is a mildly exothermic process (enthalpy change -63 kJ/mol glycerol). High rates of conversion of glycerol into syngas were observed using glycerol concentra­tions of 20-30% (w/w).

Unlike ethanol, a major fermentation product of only a selected few microbial species, glycerol is ubiquitous because of its incorporation into the triglycerides that are essential components of cellular membranes, as well as being accumulated in vegetable oils (figure 6.1). Unlike methanol, which is toxic to microbial species as well as higher animals and has no biosynthetic pathway, glycerol is benign and has a well-characterized route from glucose and other sugars (figure 2.3). In fermentations for potable ethanol, the priority is to regulate glycerol accumulation as its formation is a waste of metabolic potential in fuel alcohol production — and much research effort has, therefore, been devoted to minimizing glycerol formation by yeasts by, for example, regulating the glucose feeding rate to maintain an optimal balance of CO2 production and O2 consumption.105

Conversely, maximizing glycerol production by yeasts is also straightforward, successful strategies including:106

• Adding bisulfite to trap acetaldehyde (an intermediate in the formation of ethanol), thus inhibiting ethanol production and forcing glycerol accumula­tion to restore the balance of intracellular redox cofactors

• Growing yeast cultures at much higher pH values (7 or above) than tradi­tionally used for ethanol fermentations

• Using osmotolerant yeasts — glycerol is often accumulated inside yeast cells to counteract the adverse effects of high osmotic pressures

Suitable osmotolerant strains can accumulate 13% (w/v) glycerol within four to five days. Even more productive is the osmophilic yeast Pichia farinosa that was reported to produce glycerol at up to 30% (w/v) within 192 hours in a fed-batch fermentation with glucose as the carbon source as a molar yield of glycerol from glucose of 0.90.107

Fixing a maximum theoretical yield of glycerol from glucose is difficult because it is highly dependent on the totality of biochemical routes available to the producer cells. With Saccharomyces cerevisiae genetically manipulated to overproduce glycerol, the glycerol yield was 0.50 g/g of glucose, that is, a molar yield of 0.98.108 With this genetic background, a yield of 1 mol of glycerol/mol of glucose consumed may be the maximum obtainable (figure 6.10). Although an osmotolerant Saccharomyces strain isolated from sugarcane molasses could accumulate higher levels of glycerol (up to 260 g/l), the molar yield was still 0.92.109 An osmotolerant Candida glycerinogenes was, however, reported to produce glycerol with a molar yield of 1.25.110 Breaking through the limitation imposed by redox cofactors can, therefore, be accomplished by natural biochemistry. Some yeasts harbor a well-characterized “short circuit” for NADH oxidation, bypassing an energy conservation mechanism via an alternative oxidase, a mitochondrial enzyme constitutively expressed in industrial strains of Aspergillus niger used in citric acid prorduction.111113 Similar enzymes are known in plants, fungi, and many types of yeast; overexpressing the alternative oxidase gene in another yeast of industrial relevance (Pichia pastoris) resulted in a small increase in growth rate.114 Because the functioning of the alternative oxidase system is thought to circumvent any restrictions imposed on productivity by redox cofac­tor regeneration in citric acid producers, a bioconversion of each mole of glucose to produce nearly 2 mol of glycerol might be achievable.

Even with presently known levels of glycerol production, it is technically feasible to process a harvested filtered culture broth directly for syngas formation from glyc­erol.104 Because 50% of the annual costs of FT fuels from biomass were considered to be capital-related, combined syngas/FT conversion of glycerol would be expected to entail markedly lower costs, eliminating the need for a biomass gasifier and gas­cleaning steps.89,104

4.1 THE IOGEN CORPORATION PROCESS AS A TEMPLATE AND PARADIGM

The demonstration process operated since 2004 is outlined in figure 4.1. In many of its features, the Iogen process is relatively conservative:

• Wheat straw as a substrate — a high-availability feedstock with a low lig­nin content in comparison with tree wood materials (figure 4.2)12

• A dilute acid and heat pretreatment of the biomass — the levels of acid are sufficiently low that recovery of the acid is not needed and corrosion problems are avoided

• Separate cellulose hydrolysis and fermentation with a single sugar substrate product stream (hexoses plus pentoses) for fermentation

• Cellulase breakdown of cellulose — Iogen is an enzyme producer

• A Saccharomyces yeast ethanologen — relatively ethanol-tolerant and engineered for xylose consumption as well as offering a low incidence of contamination, the ability to recycle the cells, and the option for selling on the spent cells for agricultural use1

In the first description of the process (written in and before July 1999), agricultural residues such as wheat straw, grasses, and energy crops (aspen, etc.) were equally “pos­sible” or a “possibility.”3 By the next appearance of the article in 2006[31] — and as dis­cussed in chapter 2, section 2.6 — cereal straws had become the substrates of choice. Lignin does not form a seriously refractory barrier to cellulase access with wheat straw; this renders organic solvent pretreatment unnecessary. More than 95% of the cellulosic glucose is released by the end of the enzyme digestion step, the remainder being included in the lignin cake that is spray-dried before combustion (figure 4.1).

The Iogen process is viewed as a sequential evolution of the bioethanol paradigm, no more complex than wet mill and dry mill options for corn ethanol production (figures 1.20 and 2.21), substituting acid pretreatment for corn grinding steps, and

adding on-site cellulase generation, the latter mostly as a strategy to avoid the costs of preservatives and stabilizer but possibly also to use a small proportion of the hydro­lyzed cellulose as a feedstock for the enzyme fermentation itself. Salient features of the technology were present in Canadian initiatives from the 1970s and 1980s. The Bio-hol process, financially supported by the Ontario Ministry of Energy and Energy, Mines, and Resources, Canada, opted for Zymomonas mobilis as the ethanologen and had established acid hydrolysis pretreatments for wheat straw, soy stalks, corn stover, canola stalks, pine wood, and poplar wood.4 For both Z. mobilis and S. cere — visiae, pretreated wheat straw had the distinct advantage of presenting far less of a toxic mixture to the producer organism (figure 4.3); methods for removing growth inhibitors from the biomass acid hydrolysates could reduce the effect by >20-fold.

Minimum Inhibitory Concentration (% w/v)

FIGURE 4.3 Growth of Z. mobilis on biomass hydrolysates. (Data from Lawford et al.4)

The Stake Company Ltd. was founded in 1973 to develop and market a process for biomass conversion to sugar streams for both biofuels and animal feeds as well as chemicals derived from lignin and hemicellulose.5 A continuous feedstock processing system was constructed to handle 4-10 tons of wood chips/hr and licensed to end — users in the United States and France.

Before 2004 (or 1999), moreover, more radical processes were examined in detail — including being upscaled to pilot plant operations — for lignocellulosic ethanol. These proposals included those to avoid the need for cellulase fermentations
independent of the main ethanolic fermentation as well as the use of thermophilic bacteria in processes that more closely resembled industrial chemistry than they did the traditional potable alcohol manufacture. Indeed, it is clear that Iogen consid­ered sourcing thermophilic bacteria[32] and nonconventional yeasts during the 1990s.3 The achieved reality of the Iogen process will, therefore, be used as a guide to how innovations have successfully translated into practical use — or have failed to do so — reviewing progress over (mostly) the last three decades and offering predictions for new solutions to well-known problems as the bioethanol industry expands geo­graphically as well as in production scale.

How small can a viable rural ethanol production site be? The final section of the 1978-1980 collection of case studies projected fuel ethanol production from corn on a family-run farm in the Midwest. The cost calculations were very different, however, because there was already a working market for fuel alcohol — and a known price ($1.74/gallon in Iowa, November 1979). Using this figure,[47] a net operat­ing profit of total revenues could be projected for the first year of operation, ignoring factors such as finished goods and work-in-process inventories that would reduce the actual production cost of ethanol (table 5.4).

property taxes). Also designed for farm-scale use in South Dakota, a solid-state fer­mentation process (chapter 4, section 4.6.2) using sweet sorghum as the feedstock and upscaled to produce in principle 83 l of 95% ethanol/hr was predicted to have produc­tion costs of $1.80/gallon; the single largest contributor (61%) to production costs was that of the sorghum feedstock.15

In comparison with fermentor-based H2 production, the use of photosynthetic organisms has received wider publicity because envisaged bioprocesses have convincing environmental credentials, that is, the ability to produce a carbonless fuel using only water, light, and air (CO2) as inputs. Hydrogen photobiology is, how­ever, highly problematic because of the incompatibility of the two essential steps:

• In the first stage, water is split to produce O2.

• In the second stage, the photoproduced electrons are combined with proteins to form H2 by either a hydrogenase or a nitrogenase — and O2 is a potent inhibitor of such an “anaerobic” system.

Nature provides two related solutions to this dilemma.64 First, filamentous cyanobacteria (e. g., Anabaena cylindrica) that compartmentalize the two reactions into different types of cell: vegetative cells for generating O2 from water and using the reducing power to fix CO2 into organic carbon compounds that then pass to specialized nitrogenase-containing heterocyst cells that evolve H2 when N2 reduction is blocked by low ambient concentrations of N2. The second scenario is that of non­heterocystous cyanobacteria that separate O2 and H2 evolution temporally (in day and night cycles), although the same overall effect could be achieved using sepa­rate light and dark reactors. With either type of nitrogen-fixing organism, however, the high energy requirement of nitrogenase would lower solar energy conversion efficiencies to unacceptably low levels.

Hydrogenase is the logical choice of biocatalyst for H2 production, and nearly 35 years have now passed since the remarkable experimental demonstration that simply mixing chloroplasts isolated from spinach leaves with hydrogenase and ferredoxin isolated by cells of Clostridium kluyveri generated a laboratory system capable of direct photolysis of water and H2 production.65 The overall reaction sequence in that “hybrid” biochemical arrangement was

H2O + light ^ /02 + 2e — + 2H+ ^ ferredoxin ^ hydrogenase ^ H2

Light-induced photolysis of water produced electrons that traveled via the photo­systems of the chloroplast preparations to reduce ferredoxin before hydrogenase catalyzing the reunion of electrons and protons to form molecular hydrogen. For over a quarter of a century, therefore, the nagging knowledge that direct photolytic H2 production is technically feasible has both tantalized and spurred on research into solar energy conversion.

Thirty years ago, the abilities of some unicellular green (chlorophyll-containing) algae, that is, microalgae, to generate H2 under unusual (02-free) conditions where hydrogenase was synthesized had already been defined.66 Such microalgae can, when illuminated at low light intensities in thin films (5-20 cellular monolayers), show conversion efficiencies of up to 24% of the photosynthetically active radiation.67

What is the biological function of hydrogenase in such highly aerobic organisms? An induction period with darkness and anaerobiosis appears to be essential.68 Photosynthetic H2 production is also enhanced if the concentration of C02 is low, suggesting that the hydrogenase pathway is competitive with the normal CO2- fixing activity of chloroplasts.69 Because the electron transport via the hydrogenase pathway is still coupled to bioenergy conservation (photosynthetic phosphorylation), hydrogenase may represent an “emergency” strategy in response to adverse environmental conditions, for example, in normally above-ground plant parts subject to water logging and anaerobiosis where essential maintenance and cellular repair reactions can still operate with a continuing source of energy. It naturally follows that reintroduction of O2 and CO2 would render such a function of hydrogenase superfluous — and explains the inhibitory effect of 02 on hydrogenase and the ability of even background levels of O2 to act as an electron acceptor in direct competi­tion with hydrogenase-mediated H2 production.70 Genetic manipulation and directed evolution of algal hydrogenases with reduced or (in the extreme case) no sensitivity to O2 is, therefore, unavoidable if maximal and sustained rates of photohydrogen production can be achieved in microalgal systems.

Progress has begun to be made on the molecular biology of microalgal hydrog — enases, including the isolation and cloning of the two genes for the homologous iron hydrogenases in the green alga Chlamydomonas reinhardtiV1 Random mutagenesis of hydrogenase genes could rapidly isolate novel forms retaining activity in the pres­ence of O2 and/or improved hydrogenase kinetics. Screening mutants of Chlamydo — monas reinhardtii has, however, revealed unexpected biochemical complexities, in particular the requirement for functional starch metabolism in H2 photoproduction.72 Several changes were indeed identified during the successful improvement of H2 photoproduction by this alga:73

• There was rational selection of mutants with altered electron transport activities with maximized electron flow to hydrogenase.

• Isolates were then screened for increased H2 production rates, leading to a mutant with reduced cellular O2 concentrations, thus having less inhibition of hydrogenase activity.

• The most productive mutant also had large starch reserves.

Using the conventional representation of electron transport inside chloroplast membrane systems, the possible interactions of photohydrogen production and other photosynthetic activities can be visualized (figure 7.7).74 Active endogenous metabolism could remove photoproduced O2 by using O2 as the terminal electron

acceptor in mitochondria; the problem of different spatial sites for O2 production and O2 utilization still, however, requires a reduced sensitivity of hydrogenase to O2 as the gas cannot be removed instantaneously — only in “test-tube” systems can O2-removing chemicals be supplied, for example, as glucose plus glucose oxidase to form gluconic acid by reaction between glucose and O2.65

The obvious implication of the redox chemistry of figure 7.7 is that the normal pro­cesses of photosynthesis, involving reduction of NADP for the subsequent reduction of CO2 to sugars, can be separated in time, with light-dependent O2 evolution and dark H2 production or if H2 production can proceed with inhibited O2 evolution, that is, “indirect biophotolysis.”64 The particular advantage of this arrangement is that the light-dependent stage can be operated in open pools to maximize productivity at mini­mal cost. Sustained H2 production could be achieved over approximately 100 hours after transfer of light-grown C. reinhardtii cells to a medium deficient in sulfur; these conditions reversibly inactivated Photosystem II and O2 evolution, whereas oxidative respiration in the continued light depleted O2, thus inducing hydrogenase.75 The subse­quent H2 production only occurred in the light and was probably a means of generating energy by Photosystem I activity (figure 7.7). Starch and protein were consumed while a small amount of acetic acid was accumulated.

This was the first reported account of a single-organism, two-stage photobiological production process for H2, although a prototype light/dark device using three stages (one light and two dark) with a marine microalga and a marine photosynthetic bacterium was tested in Japan in the 1990s.76 How much H2 could a microalgae-based approach produce? With C. reinhardtii cells given an average irradiance of 50 mol photons/m2/ day (a possible value in temperate latitudes, although highly variable on a day-to-day and seasonal basis), the maximum H2 production would be 20 g/m2/day, equivalent to 80 kg/acre/day (or 200 kg/hectare/day) — but the likely value, allowing for low yields of H2 production measured under laboratory conditions, the far from complete absorp­tion of incident light, and other factors, is only 10% of this.74 In a further refinement of this approach, the sulfate-limited microalgae were shown to form a stable process for 4000 hours: two automated photobioreactors were coupled to first grow the cells aero­bically before being continuously delivered to the second, anaerobic stage.77 Until all the biological and physical limitations can be overcome, however, large infrastructural investments in high and predictable sunlight regions would be required, and the capital costs for such solar power stations would be high, but the technical complexity may only approximate that of installing extensive photovoltaic cell banks for the direct produc­tion of electricity, an option vigorously advocated by critics of biofuels programs.78,79

Cyanobacteria (“blue-green algae”) are prokaryotes but share with higher pho­tosynthetic organisms the basic electron transport chains of Photosystems I and II (figure 7.7). The molecular biology and biochemistry of hydrogenases in cyanobac­teria is well understood, the complete genomes of several such organisms have been sequenced, and interspecies gene transfer is established.80 Much of the research has unfortunately concentrated on nitrogenase as a source of H2, but many cyanobacteria contain hydrogenases catalyzing the reversible formation of H2, a route with far more biotechnological potential for commercial H2 generation, and protein engineering has begun to reduce the O2 sensitivities of cyanobacterial hydrogenases.81 The physi­ological role of hydrogenase in cyanobacteria has been debated for decades; recent results suggest some kind of safety valve function under low O2 condition when a light-to-dark transition occurs, and inactivating quinol oxidase (an enzyme with a similar hypothetical function) and nitrate reductase (a third electron “sink”) increase photohydrogen evolution rates.82

Thermophilic cyanobacteria are known to be capable of H2 photoproduction at up to 50°C in open-air cultures maintained for more than 3 weeks.8384 If a fermentable carbon source is supplied, a sustained photoevolution of H2 can be achieved, with photolysis of water (a Photosystem II activity — see figure 7.7), whereas carbohy­drate-mediated reduction of the plastoquinone pool continues independently.85 This H2 production system has been termed “photofermentation”; in principle, relatively little light energy is required to drive the reaction because of the energy input from the fermentable substrate.64 The green alga C. reinhardtii shares this pattern of metabo­lism with cyanobacteria, behaving under photofermentative conditions much like an enteric bacterium such as E. coli, exhibiting pyruvate formate lyase activity and accu­mulating formate, ethanol, acetate, CO2, and H2 as well as glycerol and lactate.86

The overlapping molecular structures of cyanobacteria and nonphotosynthetic bacteria were exemplified by the coupling (both in vivo and in vitro) between cyano — bacterial photosynthetic electron transport components with clostridial hydrogenase; even more remarkable was the expression in a Synechococcus strain of the hydrogenase gene from C. pasteurianum, the enzyme being active in the cyanobacterial host.87 As a possible pointer to the future of designing an improved photosynthetic organism for H2 production, the “hard wiring” of a bacterial hydrogenase with a peripheral subunit of a Photosystem I subunit of the cyanobacterium Thermosynechococcus elongatus resulted in a fusion protein that could associate functionally with the rest of the Pho­tosystem I complex in the cyanobacterium and display light-driven H2 evolution.88

Photosynthetic bacteria differ from other photosynthetic organisms in using bacteriochlorophyll rather than chlorophyll as the central pigment for light-induced electron transport; they also lack Photosystem II (figure 7.7) and perform anoxy — genic photosynthesis and require electron donors more reduced than water, includ­ing reduced sulfur and organic compounds.89 Being able to fix gaseous nitrogen, the photosynthetic bacteria contain nitrogenase in addition to hydrogenase and occur globally in widely different habitats, including fresh, brackish, and sea waters, hot sulfur springs, paddy fields, wastewaters, and even in Antarctica. Hydrogen can be photoproduced in the presence of an organic substrate, sometimes with high effi­ciencies deduced from the maximum theoretical H2 production on a molar basis (table 7.3). Both free and immobilized cells have been used to produce H2 during extended periods (table 7.4). All photosynthetic bacteria can use H2 as a reductant for the fixation of CO2 into organic carbon, and considerable reengineering of the molecular biochemistry is unavoidable if the cells are to be evolved into biological H2 producers.27 Photofermentations are also known, and Rhodobacter capsulatus has been used as a test organism to evaluate photobioreactor designs potentially reaching 3.7% conversion efficiency of absorbed light energy into H2 fuel energy.90

Photosynthetic bacteria may have the additional capability of catalyzing the “water shift” reaction (chapter 6, section 6.2.1):

CO + H2O ^ CO2 + H2

Photofermentative Hydrogen Production by Photosynthetic Bacteria

TABLE 7.4

Photofermentative Hydrogen Production by Immobilized Cells

H2 Evolution rate

Species Electron donor Immobilization method (ml/hr/g dry weight)

but, unlike the thermochemical process, at moderate temperatures and without multiple passages of gases through the reaction vessel.64 A continuous process was devised for Rhodospirillum rubrum with illumination supplied by a tungsten light.91 With biomass as the substrate for gasification, a substantially (if not entirely) biological process for H2 production can be envisaged. A National Renewable Energy Laboratory report concluded that a biological reactor would be larger and slower but could achieve comparable efficiencies of heat recovery in integrated systems; the most likely niche market use would occur in facilities where the water gas shift was an option occasionally (but gainfully) employed but where the start-up time for a thermal catalytic step would be undesirable.92

Patents describing processes for H2 production using photosynthetic microbes cover at least 23 years, and include topics as diverse as their basic biology, molecular and enzymic components, and analytical methodologies. In the last 5 years, however, several patents have also appeared focusing on biohydrogen production by fermentative organisms (table 7.5).