Robustness of fermenting strains to lignocellulose derived inhibitors is critical for efficient biofuel and biochemical productions. In this study, the industrial fermenting strain Corynebacterium glutamicum S9114 was evolved for improved inhibitor tolerance using long-term adaptive evolution by continuously transferring into the inhibitors containing corn stover hydrolysate every 24 h, and finally a stably evolved C. glutamicum was obtained after 128 days of serial transfers. The evolved strain exhibited the highly increased conversion rate to the typical lignocellulose derived inhibitors including furfural, 5-hydroxymethylfurfural, vanillin, syringaldehyde, 4-hydroxybenzaldehyde, and acetic acid. Glucose consumption was obviously accelerated, and 22.4 g/L of glutamic acid was achieved in the corn stover hydrolysate, approximately 68.4% greater than that by the original strain. Whole genome re-sequencing revealed various mutations with the potential connection to the improved performance of the evolved strain. Transcriptional analysis further demonstrated that the glucose-PTS transport and the pentose phosphate pathway were significantly upregulated in the evolved strain, which very likely contributed to the accelerated glucose consumption, as well as sufficient NAD(P)H supply for aldehyde inhibitors reduction conversion and thus enhanced the inhibitor tolerance. This study provided important experimental evidences and valuable genetic information for robust strain construction and modification in lignocellulose biorefining processes.

BackgroundLignocellulose is one of the most promising alternative feedstocks for glutamic acid production as commodity building block chemical, but the efforts by the dominant industrial fermentation strain Corynebacterium glutamicum failed for accumulating glutamic acid using lignocellulose feedstock.ResultsWe identified the existence of surprisingly high biotin concentration in corn stover hydrolysate as the determining factor for the failure of glutamic acid accumulation by Corynebacterium glutamicum. Under excessive biotin content, induction by penicillin resulted in 41.7 ± 0.1 g/L of glutamic acid with the yield of 0.50 g glutamic acid/g glucose. Our further investigation revealed that corn stover contained 353 ± 16 μg of biotin per kg dry solids, approximately one order of magnitude greater than the biotin in corn grain. Most of the biotin remained stable during the biorefining chain and the rich biotin content in corn stover hydrolysate almost completely blocked the glutamic acid accumulation. This rich biotin existence was found to be a common phenomenon in the wide range of lignocellulose biomass and this may be the key reason why the previous studies failed in cellulosic glutamic acid fermentation from lignocellulose biomass. The extended recording of the complete members of all eight vitamin B compounds in lignocellulose biomass further reveals that the major vitamin B members were also under the high concentration levels even after harsh pretreatment.ConclusionsThe high content of biotin in wide range of lignocellulose biomass feedstocks and the corresponding hydrolysates was discovered and it was found to be the key factor in determining the cellulosic glutamic acid accumulation. The highly reserved biotin and the high content of their other vitamin B compounds in biorefining process might act as the potential nutrients to biorefining fermentations. This study creates a new insight that lignocellulose biorefining not only generates inhibitors, but also keeps nutrients for cellulosic fermentations.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1132-x) contains supplementary material, which is available to authorized users.