BACKGROUND Thermophilic anaerobic bacteria have gained increased interest as a desirable biological catalyst for bioethanol production from lignocellulosic biomass. However, by-products such as lactic acid, acetic acid and hydrogen (H-2) not only lead to low ethanol yield but also increase the difficulty of ethanol purification. Considering that ethanol production is dependent on the supply of NADH, blocking or inhibiting lactic acid (by knocking out ldh) and H-2 formation should be a reliable strategy to further increase the ethanol production. RESULTS Comparative genome analysis indicated that three hydrogenase gene clusters (hyd, ech and hfs) were identified in Thermoanaerobacterium aotearoense SCUT27, and they were proved to be related to H-2 production by gene knockout. Deletion of hyd or ech in SCUT27(Delta ldh) showed a decrease in substrate consumption and ethanol production, while hfsB gene knockout resulted in significant improvement in overall fermentation performance. Compared with SCUT27(Delta ldh), the ethanol concentration, yield and productivity of SCUT27(Delta ldh/Delta hfsB) were increased from 25.15 +/- 0.45 g L-1, 0.33 +/- 0.01 g g(-1) and 0.30 +/- 0.01 g L-1 h(-1) to 39.64 +/- 0.16 g L-1, 0.36 +/- 0.00 g g(-1) and 0.41 +/- 0.01 g L-1 h(-1) respectively using glucose in fed-batch fermentation. The enhanced ethanol production was due to the increased NADH supply and alcohol dehydrogenase activity. Finally, the feasibility of ethanol production by SCUT27(Delta ldh/Delta hfsB) from hydrolysates of soybean hull, wheat straw, corn cob and corn straw was demonstrated. CONCLUSION Knocking out the ldh and hfsB in SCUT27 is an efficient strategy for diverting carbon flux and NADH towards ethanol production, and the resulting strain SCUT27(Delta ldh/Delta hfsB) has great potential in the production of ethanol from lignocellulosic hydrolysates. (c) 2019 Society of Chemical Industry