Hydrogen production by mixed (non-sterile) cultures would probably have lower production costs and in addition has many other advantages including continuous hydrogen production without light input, a large variety of carbon substrates, including organic wastewaters can be used as carbon sources, hydrogen can be produced at ambient temperatures, and sterile conditions are not required. Innocula of mixed anaerobic bacteria can be derived from a variety of sources including;

sewage sludge, cattle dung compost, river sediment [19], anaerobically digested sludge, acclimated sludge and animal manure [7].

The presence of non-hydrogen producing organisms can be a disadvantage for a mixed culture since they can consume a proportion of the substrate and can also use H2 as an electron donor. As well, the product of dissimulatory sulfate reduction, H2S, can be a potential catalyst poison. Thus, the selection of hydrogen producing organisms can be very important for establishing efficient and clean hydrogen production. The ability of some hydrogen producing bacteria to form spores can be used as an advantage to eliminate non-spore forming methanogens using pre-treatment methods based on this ability. Using chemical inhibitors such as acid and base, or operating the continuous culture under low HRT (hydraulic retention time) and pH conditions can also eliminate methanogens. Nevertheless, heat treatment between 75 and 121 °C is the mostly used method to select spore forming Clostridia. However, heat treatment can also eliminate non-spore forming H2 producers such as Enterobacter species, and can select some spore forming hydrogen consumers like acetogens. As an alternative, hydrogen consumption can be reduced by sparging with N2 or releasing the produced H2 from the headspace [3].

Many studies have been conducted on dark fermentative hydrogen production by using mixed cultures. Generally these studies can be divided into three groups; batch, fed-batch and continuous cultures. Batch studies with a variety of mixed cultures have demonstrated reasonable hydrogen yields. A mixed culture carrying out fermentation of synthetic wastewaters gave a hydrogen yield of 2.48 mol H2/mol glucose [33]. A mixture of aerobic and anaerobic sludges derived from lake mud was reported to give 1.4 mol H2/mol glucose [34]. Sludges subjected to different pretreatments were effective in producing hydrogen; heat conditioned aerobic sludge, 2 mol H2/mol glucose [35]; heat treated anaerobic sludge, 1.75 mol H2/mol glucose [36]; acid treated anaerobic sludge, 1 mol H2/mol glu­cose [37]; treated microflora from cow dung, 2.27 mol H2/h TSS; heat shocked microflora from soil, 0.92 mol H2/mol glucose [38]. Using sucrose as a carbon source with heat treated anaerobic sludge resulted in 1.9 and 3.4 mol H2/mol sucrose in two different studies [39, 40]. Using glucose with heat treated anaerobic sludge resulted in hydrogen yields of 0.98 and 1 mol H2/mol glucose [41, 42].

Fed-batch cultures can be useful for industrial processes since this mode of operation can help prevent product inhibition. Therefore, fed-batch operation could show improved yields over batch cultures since it could prevent the pH drop associated with accumulation of volatile fatty acids. Fed-batch cultures have successfully been employed in a number of studies; olive mill wastewater was treated by a mixed culture and gave 14.7 mmol H2/gVSS degraded [43], it was also used for hydrogen production (17.82 mmol H2/l-reactor-h) using anaerobic POME sludge [44], and a mixed culture from windrow yard compost gave 7.44 mmol H2/L-reactor-h from glucose [45].

However, using continuous cultures may have important advantages for industrial applications. CSTR (continuously stirred tank reactor) and UASB (up- flow anaerobic sludge blanket) reactors are the two main types of continuous

Fig. 10.2 Dark fermentation system

reactors that have been used for hydrogen production. Glucose has been widely used as substrate for dark fermentative hydrogen production by mixed cultures in continuously operated CSTRs. Different studies have reported hydrogen yields between 1.1 and 1.9 mol H2/mol glucose [42, 46-48]. Similarly, using pure sucrose in CSTRs gave yields of 3.3 and 3.6 mol H2/mol sucrose [49-51]. However, the main disadvantage of these suspended culture systems is cell washout at high dilution rates. Therefore, immobilized systems have been devel­oped which not only prevent washout of cells, but also improve production rates since higher biomass concentrations are possible. Various techniques are possible, including the use of inert supports and self-immobilization. Cultures immobilized on activated carbon using sucrose as a sole carbon source gave 0.24-6.08 l H2/g VSS/h [52] or yields of 0.57 and 1.59 mol H2/mol sucrose [52, 53]. Granules formed by self-immobilization resulted in 0.86-2.2 mol H2/mol glucose in dif­ferent studies [54-58]. Other studies using mixed cultures in UASB reactors gave hydrogen yields between 0.84 and 2.47 mol H2/mol glucose [57, 59-61].