There are several types of microorganisms that can produce hydrogen by dark fermentation. Every organism has different requirements such as; substrate pref­erence, pH and temperature. These parameters can greatly influence the hydrogen yield by affecting microbial metabolism. Fermentative hydrogen production by pure microorganisms has been studied by many researchers. Pure cultures have some advantages. In particular, they can be easily and reliably manipulated to determine the optimal growth conditions. However, there are some clear disad­vantages to using pure cultures since they can easily be affected by contamination and therefore their use requires aseptic conditions which could greatly increase overall system costs [7]. Generally pure cultures used in dark fermentation can be divided into two general groups:

1. Anaerobic bacteria (e. g. Rumen bacteria, Clostridium and other Firmicutes)

2. Facultative anaerobic bacteria (e. g. Escherichia coli, Enterobacter, Citro- bacter) [10].

Clostridium and E. coli are the two most widely used bacteria for two-stage hydrogen production. Various Clostridium species can produce hydrogen and Clostridium butyricum, a mesophilic and strict anaerobic bacteria, is perhaps the most commonly employed. As early as the 1960s, Clostridium butyricum and

Clostiridium welchii were reported to produce fermentative hydrogen in a 10 L fermenter by Magna Corporation [11]. As discussed above, the theoretical yield from glucose by Clostridium butyricum is 4 mol H2/mol glucose. A few studies have reported yields of more than 2 mol H2/mol glucose [12, 13], with the max­imum yield of 3.26 mol H2/mol glucose [14]. Hydrogen production from glucose using C. acetobutylicum [15], C. butyricum [16], C. paraputrificum [17] C. bei — jirincki AM21B [10], C. cellobioparum [18], C. pasteurianum [10] resulted between 0.42 and 2.73 mol H2/mol glucose. Clostridium species can sporulate under the proper conditions and generally produce acetate and butyrate as by-products. It is important to adjust system operation conditions to avoid spor — ulation [19]. Although they are generally known as mesophilic microorganisms, some thermophilic species have been isolated and these are generally capable of higher hydrogen yields [20]. C. thermolacticum can use lactose to produce hydrogen with a yield of 1.5 mol H2/mol lactose. C. thermoalcaliphilum [21], C. thermobutyricum [22] C. thermohydrosulphuricum [23], C. thermosaccharo — lyticum [24], C. thermosuccinogenes [25] are other thermophilic Clostridial species that have been used.

Enteric bacteria are generally not capable of metabolizing complex carbohy­drates, but the necessary genes can be introduced [26]. E. coli is one of the most studied facultative anaerobes for hydrogen production, and has been subject to a variety of genetic engineering including mutagenesis and the introduction of for­eign genes. Organisms have been genetically modified to consume pentoses, or to increase lactate and succinate activities [7]. E. coli has long been known to pro­duce hydrogen under anaerobic conditions, but with low yields if there is a large amount of residual formate [27].

Enterobacter species are gram negative motile facultative anaerobes. Hydrogen production by this organism is influenced by many process parameters such as initial substrate concentration, initial medium pH, temperature and iron concen­tration. Enterobacter aerogenes HU-101 [28], immobilized Enterobacter cloacae IITBT08 [29], immobilized Enterobacter cloacae DM11 [30] gave 1.17, 2.3 and

3.8 mol H2/mol glucose, respectively. Molasses has been used as carbon source to produce hydrogen by Enterobacter aerogenes with hydrogen yields between 0.52 and 2.2 mol H2/mol sucrose [31, 32].