Depending on organisms and growth conditions, changes in external pH can bring about subsequent alterations in several primary physiological parameters, includ­ing internal pH, concentration of other ions, membrane potential and proton-motive force [77]. pH also influences the efficiency of substrate metabolism, protein syn­thesis, synthesis of storage material and metabolic by-product release. This is especially important for fermentative H2 production where the activity of acidogenic bacteria is considered to be crucial and rate limiting [24, 58]. The restricted nature of specific groups of bacteria at particular pH values helps to maintain the bioreactor in an acidogenic microenvironment. Maintaining pH in the acidic range (5.5—6.0) is ideal for effective H2 production due to repression of MB, thus indirectly pro­moting H2 producers within the system [21, 30, 72]. The activity of hydrogenase is observed to be inhibited by maintaining low or high pH in fermentation [58]. Most methanogens are limited to a narrow pH range (6.8-7.2), while most H2-producing acidogenic bacteria can grow over a broader pH range. AB function well below pH 6, while for MB optimum range is between 6.0 and 7.5 [78, 79]. The pH range of

5.5-6.0 is reported to be ideal to avoid both methanogenesis and solventogenesis [21, 79], which is important for good H2 production. Effective H2 production was observed by maintaining operating pH in and around 6 compared to near neutral pH [21, 75]. Increase in initial/feeding pH (from acidic to neutral) has resulted in suppressed H2 production [21, 26, 31, 32]. However, highly acidic pH (<4.5) is detrimental to H2 production as it inactivates H2 producing bacteria [72, 80].

Cyclic voltammograms (CV) obtained at acidic and neutral pH conditions visu­alized well — defined redox pairs both in forward and reverse scans and the signal corresponded to intracellular electron carriers, NADH/NAD+ (Eo, -0.32 V) [24] (Fig. 3). Shuttling of H+ between metabolic intermediates can be correlated to the e — discharge observed in CV. At acidic pH, the e — discharge was almost similar at

12, 20 and 24 h suggesting the effective H+ shuttling throughout the cycle oper­ation. This helps to maintain the system under acidogenic conditions for longer periods leading to higher H2 production. At neutral operation, the e — discharge var­ied with time and approached maximum at 12 h prior to decrease suggesting the neutralization/reduction behaviour of H+ by MB.

Acidic pH (below 6) showed less substrate degradation efficiency than the corre­sponding neutral operation due to reduced methanogenic activity [24]. Neutral pH illustrated effective substrate removal efficiency over the corresponding acidic oper­ation. Maintenance of acidic conditions in association with pre-treatment has also been observed to be effective in H2 production during treatment of various types of wastewater [30, 31, 38].

VFA (soluble acid metabolites generated from acidogenic fermentation) and pH are integral expressions of acid-base conditions of anaerobic microenvironments which provides information pertaining to the balance between two of the most important microbial groups (AB and MB). Production of acids gradually reduces the buffering capacity of system, which, in turn, results in a decline in the system pH due to accumulation of organic acids leading to process inhibition [23, 81]. If pH is not maintained in the optimum range, cessation of H2 production will result along with a marked shift in microbial population [75]. Relatively higher levels of soluble metabolite production were observed under acidic operation over the cor­responding neutral microenvironment, which corroborated well with H2 production data [26, 31, 38, 82]. Therefore, pH can be considered as a manipulable variable for process control. Among the two process variables viz., influent pH and reactor pH, the later is more difficult to control. Bicarbonate-alkalinity is an important pro­cess parameter which indicates the system buffering capacity in association with pH microenvironment and VFA concentrations.

Sulfate, if present in wastes will be converted into hydrogen sulfide by sulfate — reducing bacteria (SRB) in the anaerobic microenvironment, resulting in toxicity to other anaerobes [83]. SRB are reported to have H2 utilization hydrogenase and can readily use H2 as the electron donor [84]. pH of the system microenvironment has a direct influence on the sulfate reduction linked to H2 production. At acid pH, the SRB activity gets inhibited wherein H2 production is unaffected. H2 production has markedly recovered and increased when pH was reduced to 5.5, even in the presence of higher sulfate concentration (3 g SO42- /l) [85].