In this study, a two-stage fermentation system to produce H-2 and CH4 from Laminaria japonica was developed. In the first stage (dark fermentative H-2 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 H-2 yield is as follows: cycle-frequency > substrate concentration > HRT. In the confirmation test, H-2 yield of 113.1 mL H-2/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 H-2 fermented solid state (HFSS) and H-2 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 (MFEUASAr) as diluting water in DFHP, the tap water and MFEUASBr mixing ratio (T/M ratio) was optimized (a T/M ratio of 5:5) in a batch test using heat pretreated MFEUASBr at 90 degrees C for 20 min, resulting in the best performance. Although slight decreases of H-2 yield (7.6%) and H-2 production rate (3.5%) were recorded, 100% reduction of alkali addition was possible, indicating potential to maximize economic benefits. However, a drastic decrease of H-2 productivity and a change of liquid-state metabolites were observed with the use of non-heat pretreated MFEUASBr. These results coincided with those of the microbial analysis, where non-H-2 producing bacteria, such as Selenomonas sp., were detected. The results indicate that pretreatment of MFEUASBr may be required in order to recycle it in DFHP. Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.