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