In spite of striking advantages, the main challenge encountered with fermentative H2 production processes are low substrate conversion efficiency and residual substrate present in acid-rich wastewater generated from the acidogenic process. Anaerobic bacteria have a theoretical maximum yield of 4 mol H2/mole glucose [3]. In prac­tice, yields are lower, as the NADH oxidation by NFOR is inhibited under standard conditions and only proceeds at very low partial pressures of H2 [11]. Up to 4 moles of H2 can theoretically be produced per mole of glucose through the known fermentative pathways [109]. However, various biological limitations such as H2- end-product inhibition and waste-acid and solvent accumulation limit the molar yield to around 2 moles per mole glucose consumed. Typical H2 yields range from 1 to 2 mol H2/mol glucose and result in 80-90% of the initial carbon remaining in the wastewater [7, 23, 25, 51, 109, 76, 110, 111]. Even under optimum conditions about 60-70% of the original organic matter remains as residue in the wastew­ater. Also a maximum yield of 4 mol H2/mole glucose is still low for practical applications [3].

The generation and accumulation of soluble acid metabolites causes a sharp drop in the system pH and inhibits H2 production. H2 yield is lower when more reduced organic compounds, such as lactic acid, propionic acid, and ethanol, are produced as fermentation products, because these represent end products of metabolic path­ways that bypass the major H2-producing reaction [11]. The undissociated soluble metabolites can permeate the cell membrane of H2-producing bacteria and then dissociate in the cell leading to physiological balance disruption [91]. Thus, some maintenance energy should be used to restore the physiological balance in the cell, which reduces the energy used for bacteria growth and inhibit the bacterial growth on the other hand. If the dissociated soluble metabolites is present in the system at a high concentration, the ionic strength will increase, which may result in cell lysis [91]. High concentrations of soluble metabolites can inhibit H2-producing bacterial growth thereby reducing H2 production [91, 78, 112]. The fermentation metabolic end-products and the resultant H2 yields vary based on the environmental conditions even within the same bacterium [3, 86].

H2 production is limited by the thermodynamics of the hydrogenase reaction, which involves the enzyme-catalyzed transfer of e — from an intracellular electron carrier molecule to H+ [11]. The partial pressure of H2 is one of the important fac­tors, as the pressure increases, H2 production decreases [7]. H2 production becomes thermodynamically unfavourable at H2 partial pressures greater than 60 Pa [11].

Operating bioreactors at low H2 partial pressure by stripping H2 from the solu­tion is as it is generated [57, 102], accomplishes both efforts simultaneously [11]. Conceptually, efforts are to be made in optimizing operational conditions to prevent consumption of H2 by propionic acid-producing bacteria, ethanol-producing bacte­ria and homoacetogens and those that channel more reducing equivalents towards reduction of H+ by hydrogenases to maximize H2 production [11]. The physiolog­ical and physicochemical conditions under which the microorganisms give optimal H2 yields is important and needs to be established. Optimization of process param­eters is one of the vital steps as to enhance H2 yield as well as to enhance substrate degradation efficiency and assumes significance prior to up-scaling the process.