Many or most organic cellulosic matter, after proper mechanical treat­ment (homogenizing), can be put to microbial conversion for (a) bio — methanation and/or (b) hydrogen production.

1. Biomethanation can utilize human or animal excreta as well as mixed green/organic wastes. This part has been discussed earlier in Secs. 1.12 and 1.13.

2. Hydrogen production is discussed hereafter.

Biohydrogen. Major routes are

1. Enzymatic (partly microbial) through microbial routes

2. Klebsiella and Clostridium groups of microbes

3. Different cyanobacteria (blue-green algae)

4. Various photosynthetic bacteria

5. Many aerobes, i. e., bacilli and alkaligenes

6. Facultative groups, i. e., enterobacters, and coli forms

7. Various anaerobes, i. e., rumens, methanogenic, methylotropes, and clostridia

Enzymatic. Glucose dehydrogenase oxidizes glucose into gluconic acid and NADPH, which helps the reduction of H+ by hydrogenase. Glucose dehydrogenase and hydrogenase are purified from Thermoplasma aci- dophylium and Pyrococcus furiosus (optimal growth at 59°C and 100°C, respectively) (Woodward).

Based on metabolic patterns, the microbial systems may be of four types:

1. Photosynthetic microbes evolving H2 mediated through NADPH (Nicotine Adenine Dinucleotide Phosphate [Coenzyme II-reduced]) by photoenergy.

2. Cytochrome systems operating in facultative anaerobes that convert mainly formates to H2.

3. Cytochrome containing strict anaerobe, Desulfovibrio desulfuricans.

4. Clostridia, micrococci, methanobacteria, and others, without cytochrome, anaerobic heterotrophs.

Klebsiella oxytocae. ATCC (American Type Culture Collection) 13182 can convert formates to H2 (100%), but only 2 moles of H2 for each mole of glucose (5%). C. butyricum can convert glycerol to 1,3-propanediol, butyric acid, 2,3-butanediol, formic acid, and CO2 and H2. Klebsiella pneumoniae can convert glycerol into 1,3-propanediol, acetic acid, formic acid, and CO2 and H2. The presence of acetate enhances the production of butyrates and H2, and less propanediol.

Before discussing cyanobacteria and photosynthetic bacteria, we should review the basic reactions involved in photosynthesis, i. e., steps in so-called photophosporylation:

H2O + NADP+ + PO4 + ADP—^ O2 + NADPH + H+ + ATP

CO2 + NADPH + H+ + ATP———— ► (HCOH)n + NADP+ + ADP + PO4

+hv

Aerobic: 6CO2 + 6H2O ———- * C6H12O6 + 6O2

+hv

Anaerobic: Isopropanol or H2S + CO2————- ► Acetone or S +

(CH2O)n H2O

Cyanobacteria. Popularly known as blue-green algae, and justifiably so (they consume CO2 and evolve O2), they are bacteria (absence of nuclei, mitochondria, chloroplasts, etc.) as well as algae.

Cyanobacteria are oxygenic photoautotrophs, possessing photo I and II systems. Cyanobacteria have been well studied, and the details of their physiology and biochemistry are available in reviews and books. They are held by many scientists as potential sources of chemicals, bio­chemicals, food, feed, and fuel. Most of them are molecular nitrogen fixers and possess a nitrogenase system for H2 production. They are found to be symbiotic to cycads, lichens, and so forth. Some are hetero­cystous, lacking photolysis of water, and produce H2 through the nitro — genase step (when N2 is low). The nonheterocystous species produce H2 at higher efficiency at low N2 and O2 concentrations. Some of the species favor anoxic and dark conditions, but with the presence of organic sub­strates. They may even use sulfides as a source of electrons under an anaerobic environment. They are highly adaptable to a changing envi­ronment and are widely found in salty or sweet water, deserts, hot springs (up to 75°C), as well as Antarctica. Some heterocystous Anabaena exhibit H2 production in an atmosphere of argon and absence of molecular nitrogen. This was the clue to the knowledge that the enzyme nitrogenase, the main biocatalyst for molecular nitrogen fixa­tion, is present in cyanobacteria and is the key route of H2 production:

N2 + 8H+ + 8e~ + 12ATP ^ 2NH3 + H2 + 12ADP + 12Pi

A “reversible hydrogenase” (in photolysis of water, 2H2O ^ 2H2 + O2), is present in both heterocyst and vegetative cells and produces H2 at a lower rate than a nitrogenase. An “uptake hydrogenase” also operates (minor) connected to cytochrome chain, providing both H+ and elec­trons. H2 evolution is common, but the photolytic O2 is inhibitory to nitrogenases, which is protected by other biochemical and structural alternatives existing in heterocysts.

Large amounts of ATP, which is required for the reaction are gener­ated in the event of photosynthesis and respiration. The electron (reduc — tant) supply in the nitrogenase equation comes from metabolites, i. e., amino acids, mainly from carbohydrates (maltose, glucose, fructose, other pentoses, tetroses, etc.), produced and stored in the vegetative cells through photo I and II systems.

Nitrogenase Co II, i. e., NADPH (gained through the pentose phosphate route) happens to be an electron donor through NADP oxidoreductase/ ferredoxin or flovodoxin. Other electron-supplying batteries are also envisaged.

1. Through uptake hydrogenase-ferredoxin (photoactivated)

2. Through pyruvate-ferredoxin oxidoreductase

3. Reduced ferredoxin from isocitrate dehydrogenase

4. NADH generated in the glycolytic route

Under anaerobic or low aerobic conditions, nitrogenase activity may exist in vegetative cells, but H2 generation is of poor order.

Photosynthetic bacteria. Hydrogen production is guided by the surplus of ATP and reductant organic metabolites (carbon sources from the

Krebs cycle) and reduced nitrogen sources (glutamate/aspartate). Interactions of hydrogenase and nitrogenase may be complementary or competitive in different species or mutants. Nitrogenase (Mo, Ni, or Fe) also with mixed isozymes are reported. Some mutants liberate H2 more efficiently, utilizing DL-malate, D-malate, and L-lactate. Photoautotrophic growth is found to be less efficient in producing H2 than photoheterotrophic growth with limited nitrogen in nutrients. Normally, in photosynthetic bacteria, hydrogenase utilizes the hydrogen as a reductant for CO2 fix­ation and also for fixing molecular nitrogen. Nitrogenase reduces molec­ular nitrogen, along with the production of molecular hydrogen at the expense of almost six stoichiometric equivalents of ATP. This means that concurrent nitrogenase activity during photosynthesis competitively con­sumes the ATP that is produced and lowers the CO2-fixing efficiency.

Rhodospirillum and Rhodopseudomonas grow aerobically in the dark. But Rhodospirillum rubrum growing on glutamate (a nitrogen source) exhibit good hydrogen release during photosynthesis. Quantitative pro­duction of hydrogen has also been observed, growing on acetate, succi­nate, fumarate, and malate, by photosynthesis, initially in the presence of limited ammonium salts.

In Rhodopsuedomonas acidophilla, hydrogenase and nitrogenase are genetically linked. Several species of Rhodospirillaceae can perform nonnitrogenase-mediated hydrogen production in the absence of light, using glucose and organic acids including formates. Different strains of Rhodopseudomonas gelatinus and Rhodobacter sphaerolides exhibit highly efficient production of hydrogen [90 pL/(h • mg) cell] grown in a glutamate—malate medium.

In some cultures of Rhodopseudomonas capsulata, R. rubrum, and Rhodomicrobium vannielli, replacement of glutamate by N2 gas improved productivity of H2 (760 mL/d, 10 days) decreasing a little on aging. The model of a nozzle loop bioreactor, with immobilized R. rubrum KS—301 in calcium alginate, initial glucose concentration of 5.4 g/L, 70 h at 30°C, showed production of hydrogen 91 mL/h (dilu­tion rate of 0.4 mL/h). Improvement was suggested by using an agar gel for immobilization.

Aerobes.

1. Bacillus licheniformis isolated from cattle dung showed production of H2 in mixed culture media. Immobilized on brick dust, the aerobe maintained H2 production for about 2 months in a continuous system, with an average bioconversion ratio of 1.5 mole of H2 per mol of glucose.

2. Alcaligenes eutrophus, when grown on gluconates or fructose anaer­obically, produces H2. Hydrogenase directly reduces the coenzyme using hydrogen, and the excess hydrogen is spilled out. Higher con­centration of formate reduced hydrogen production.

HCOOH ^ CO2 + H2

Facultative anaerobes.

1. Enterobacter: Enterobacter aerogenes, as an example, can use varied and mixed nutrients, i. e., glucose, fructose, galactose, mannose, pep­tones, and salts (pH 4.0, 40°C); and may show activity for about a month in a continuous culture; evolution of hydrogen was about 120 mL/h/L of medium; 0.8 mol/mol of glucose. Accumulation of acetic, lactic, or succinic acids is likely to cause antimetabolic suppression in older cultures.

2. Escherichia coli: Anaerobically, it can use formate to produce CO2 and H2. Carbohydrates as nutrient sources usually end up with mixed products, i. e., ethanol, acetate, hydrogen, formate, carbon dioxide and succinate.

Various anaerobes.

1. Ruminococcus albus mostly converts cellulose to CO2, H2, HCOOH, C2H5OH, CH3, and COOH. Pyruvatelyase may be functional in the production of H2 (237 mol/mol of glucose). Further details are not available.

2. P. furiosus (thermophilic archeon) possesses nickel-containing hydro — genase and produces hydrogen using carbohydrate and peptone, at 100°C. The metabolic system seems to be uncommon to those of non — thermophiles.

3. Methanobacterium (Methanotrix) soehngenii (methanogens) can grow on acetate and salts media, but can split formate into hydrogen and carbon dioxide. M. barkeri, in the presence of bromoethane sulphonate, has suppressed methane production; instead, hydrogen, carbon dioxide, carbon monoxide, and water were produced.

4. Methylomonas albus BG8 and Methylosinus trichosporium OB3b (methylotrophs) used various substrates, i. e., methane, methanol, formaldehyde, formate, pyruvate, and so forth. But formate was found to be most useful for production of hydrogen under anaerobic conditions.

5. C. butyricum, C. welchii, C. pasturianum, C. beljerinscki, and so forth are very efficient in utilizing different carbohydrate sources and even effluents to produce hydrogen (see Fig. 1.14). Immobilization of these cells has also been successful

Removal of or reducing concentrations of either CO2 or H2 or the com­bination of both is likely to favor a forward reaction, i. e., to improve pro­duction of H2. Attempts to remove CO2 by collecting the evolved gases through 25% (w) NaOH solution, using E. aerogenes (E 82005), showed better production of H2, which improved further by enriching nitroge­nous nutrients in the culture media—from 0.52 moles of H2 per mole of glucose, increased to 1.58 moles [9].

Similar attempts are made using E. cloacae and reducing the partial pressure of H2 during the production of gases, by reducing the operat­ing pressure of the reactor and simultaneous removal of CO2 [through 30% (w/v) KOH], maintaining an anoxic condition by flushing Ar at the onset [10]; by reducing the operating pressure to 0.5 atm, the molar ratio of H2 yield per mole of substrate doubled (1.9-3.9). Other technical and economic benefits were also cited. There are other similar claims of improved biohydrogen production [11], using altered nutrients (20 g of glu­cose, 5 g of yeast extract, and 5 g/L of tryptone) and different mutants of E. aerogenes HU-101. HU-101 and mutants A1, HZ3, and AAY, respec­tively yielded 52.5, 78, 80, and 101.5 mmol of hydrogen per liter of media.