Continuous fermentative hydrogen and methane production from Laminaria japonica using a two-stage fermentation system with recycling of methane fermented effluent

In this study, a two-stage fermentation system to produce H₂ and CH₄ from Laminaria japonica was developed. In the first stage (dark fermentative H₂ production, DFHP), response surface methodology (RSM) with a Box-Behnken design (BBD) was applied for optimization of operational parameters, including cycle-frequency, HRT, and substrate concentration, using an intermittent-continuously stirred tank reactor (i-CSTR). Overall performance revealed that the degree of importance of the three variables in terms of H₂ yield is as follows: cycle-frequency > substrate concentration > HRT. In the confirmation test, H₂ yield of 113.1 mL H₂/g dry cell weight (dcw) was recorded, corresponding with 96.3% of the predicted response value under desirable operational conditions (cycle-frequency of 17 hr, HRT of 2.7 days, and substrate concentration of 31.1 g COD/L). In the second stage, an anaerobic sequencing batch reactor (ASBR) and an up-flow anaerobic sludge blanket reactor (UASBr) were employed for CH₄ production from H₂ fermented solid state (HFSS) and H₂ fermented liquid state (HFLS), respectively. The CH₄ producing ASBR and UASBr showed a stable CH₄ yield and COD removal until a HRT of 12 days and OLR of 3.5 g COD/L/d, respectively. Subsequently, for recycling of CH₄ fermented effluent from the UASBr (MFE_UASBr) as diluting water in DFHP, the tap water and MFE_UASBr mixing ratio (T/M ratio) was optimized (a T/M ratio of 5:5) in a batch test using heat pretreated MFE_UASBr at 90 °C for 20 min, resulting in the best performance. Although slight decreases of H₂ yield (7.6%) and H₂ production rate (3.5%) were recorded, 100% reduction of alkali addition was possible, indicating potential to maximize economic benefits. However, a drastic decrease of H₂ productivity and a change of liquid-state metabolites were observed with the use of non-heat pretreated MFE_UASBr. These results coincided with those of the microbial analysis, where non-H₂ producing bacteria, such as Selenomonas sp., were detected. The results indicate that pretreatment of MFE_UASBr may be required in order to recycle it in DFHP.