Enhanced ethanol production from lignocellulosic hydrolysates by inhibiting the hydrogen synthesis in Thermoanaerobacterium aotearoense SCUT27(Δldh)

Abstract

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 (H2) 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 H2 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 H2 production by gene knockout. Deletion of hyd or ech in SCUT27(Δldh) showed a decrease in substrate consumption and ethanol production, while hfsB gene knockout resulted in significant improvement in overall fermentation performance. Compared with SCUT27(Δldh), the ethanol concentration, yield and productivity of SCUT27(Δldh/ΔhfsB) 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(Δldh/ΔhfsB) 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(Δldh/ΔhfsB) has great potential in the production of ethanol from lignocellulosic hydrolysates. © 2019 Society of Chemical Industry

Metabolic engineering of Thermoanaerobacterium aotearoense strain SCUT27 for biofuels production from sucrose and molasses

“…In addition, fructose metabolism might be accelerated by overexpression of fructose metabolic genes or deletion of the potential transcriptional repressor [ 60 ]. As T. aotearoense SCUT27 has the great ability to utilize pentose [ 23 , 24 , 61 ], the waste of sugar refinery molasses and sugarcane bagasse hydrolysate (containing sucrose, glucose, fructose, xylose, arabinose and so on) could be co-utilized for biofuels production. Furthermore, co-cultured with other cellulolytic strains, such as C. thermocellum , T. aotearoense SCUT27 may make full use of molasses, bagasse and straw to achieve the maximum benefit.…”

Thermoanaerobacter Species: The Promising Candidates for Lig-nocellulosic Biofuel Production

“…Strains with a deletion of the hfs genes exhibited a 95% decrease in both hydrogen and acetic acid production, and hfsB seemed as the most important gene of the operon hfsABCD [110]. The genes hyd, ech and hfsABCD, highly homologic to that of T. saccharolyticum and T. tengcongensis, were also found in strain SCUT27 and proved to be related to H2 production by gene knockout [112].…”

“…Shaw et al investigated three putative hydrogenase enzyme systems in T. saccharolyticum and found that the strain with specified hfs and ldh deletions exhibited an increased ethanol yield from consumed carbohydrates [110,111]. The deletion of hfsB was also performed in strain SCUT27 and results indicated the enhanced ethanol production was obtained, due to the increased NADH supply and alcohol dehydrogenase activity [112]. Pyruvate formate lyase-activating protein (PflA) could assist the formation of pyruvate formate lyase (Pfl), which catalyzed pyruvate into acetyl-CoA and formate.…”

“…A unique combination of hydrogenases, a ferredoxin-dependent [NiFe] hydrogenase and an NADH-dependent Fe-only hydrogenase, was found to be responsible for H2 formation in T. tengcongensis [120]. Three putative hydrogenase systems were identified in T. saccharolyticum: A four-gene operon containing two [FeFe]hydrogenase genes (hfsBD), provisionally termed hfs (hydrogenase-Fe-S), of which the deletion resulted in a growth defect [111], was found to be the main enzymatic catalyst of hydrogen production; A second hydrogenase gene cluster, a bifurcating NAD(P)Hlinked [FeFe]-hydrogenase (hyd), exhibited methyl viologen-linked hydrogenase enzymatic activity and was specific to the transfer of electrons from NAD(P)H to H + ; A third gene cluster, a putative [NiFe]-hydrogenase with homology to the ech genes, did not exhibit the hydrogenase activity [110,112]. Strains with a deletion of the hfs genes exhibited a 95% decrease in both hydrogen and acetic acid production, and hfsB seemed as the most important gene of the operon hfsABCD [110].…”