Two shock loads of a commercial detergent (I-150 mg chemical oxygen demand (COD)/L, fed for 56 h; II-300 mg COD/L fed for 222 h) were applied in a lab-scale Expanded Granular Sludge Blanket (EGSB) reactor, fed with 1,500 mg COD/L of ethanol. The impact of the surfactant was assessed in terms of granular sludge morphology, specific methanogenic activity (SMA) in the presence of individual substrates, and reactor performance. COD removal efficiency remained unaffected in the shock I, but 80 h after starting exposure to the shock II, the COD removal efficiency decreased drastically from 75 to 17%. In the first 8 h of operation of shock I, the SMA was stimulated and decreased afterwards, being recovered 5 days after the end of exposure time. Concerning to shock II, the SMA was immediately and persistently reduced during the exposure time, although, the inhibition of SMA in presence of H(2)/CO(2) showed a trend to increase after the exposure time. Acetoclastic bacteria were observed as the most sensitive to the toxic effects of surfactant whereas the hydrogenotrophic bacteria were less affected. The inhibitory effects were dependent on surfactant concentration and exposure time. The ratio filaments length per total aggregates area (LfA) was an early-warning indicator of biomass washout, since it increased 3 and 5 days before effluent volatile suspended solids (VSS) rise, respectively, in shocks I and II.

Proof of principle of biohythane and potential energy production from garden waste (GW) is demonstrated in this study in a two-step process coupling dark fermentation and anaerobic digestion. The synergistic effect of using co-cultures of extreme thermophiles to intensify biohydrogen dark fermentation is demonstrated using xylose, cellobiose and GW. Co-culture of Caldicellulosiruptor saccharolyticus and Thermotoga maritima showed higher hydrogen production yields from xylose (2.7±0.1molmol(-1) total sugar) and cellobiose (4.8±0.3molmol(-1) total sugar) compared to individual cultures. Co-culture of extreme thermophiles C. saccharolyticus and Caldicellulosiruptor bescii increased synergistically the hydrogen production yield from GW (98.3±6.9Lkg(-1) (VS)) compared to individual cultures and co-culture of T. maritima and C. saccharolyticus. The biochemical methane potential of the fermentation end-products was 322±10Lkg(-1) (CODt). Biohythane, a biogas enriched with 15% hydrogen could be obtained from GW, yielding a potential energy generation of 22.2MJkg(-1) (VS).

In the present study, two granular systems were compared in terms of hydrogen production rate, stability and bacterial diversity under extreme thermophilic conditions (70 degrees C). Two EGSB reactors were individually inoculated with heat treated methanogenic granules (HTG) and HTG amended with enrichment culture with high capacity of hydrogen production (engineered heat treated methanogenic granules - EHTG), respectively. The reactor inoculated with EHTG (R(EHTG)) attained a maximum production rate of 2.7l H(2)l(-1)day(-1) in steady state. In comparison, the R(HTG) containing the HTG granules was very unstable, with low hydrogen productions and only two peaks of hydrogen (0.8 and 1.5l H(2)l(-1)day(-1)). The presence of active hydrogen producers in the R(EHTG) system during the reactor start-up resulted in the development of an efficient H(2)-producing bacterial community. The results showed that "engineered inocula" where known hydrogen producers are co-inoculated with HTG is an efficient way to start up biohydrogen-producing reactors.