Biological cellulose saccharification using a coculture of Clostridium thermocellum and Thermobrachium celere strain A9

Sreyneang Nhim,Chakrit Tachaapaikoon,Ayaka Uke,Ya-Jun Liu,Sirilak Baramee,Patthra Pason,Khanok Ratanakhanokchai,Rattiya Waeonukul,Akihiko Kosugi

doi:10.1007/s00253-022-11818-0

Abstract

An anaerobic thermophilic bacterial strain, A9 (NITE P-03545), that secretes β-glucosidase was newly isolated from wastewater sediments by screening using esculin. The 16S rRNA gene sequence of strain A9 had 100% identity with that of Thermobrachium celere type strain JW/YL-NZ35. The complete genome sequence of strain A9 showed 98.4% average nucleotide identity with strain JW/YL-NZ35. However, strain A9 had different physiological properties from strain JW/YL-NZ35, which cannot secrete β-glucosidases or grow on cellobiose as the sole carbon source. The key β-glucosidase gene (TcBG1) of strain A9, which belongs to glycoside hydrolase family 1, was characterized. Recombinant β-glucosidase (rTcBG1) hydrolyzed cellooligosaccharides to glucose effectively. Furthermore, rTcBG1 showed high thermostability (at 60°C for 2 days) and high glucose tolerance (IC50 = 0.75 M glucose), suggesting that rTcBG1 could be used for biological cellulose saccharification in cocultures with Clostridium thermocellum. High cellulose degradation was observed when strain A9 was cocultured with C. thermocellum in a medium containing 50 g/l crystalline cellulose, and glucose accumulation in the culture supernatant reached 35.2 g/l. In contrast, neither a monoculture of C. thermocellum nor coculture of C. thermocellum with strain JW/YL-NZ35 realized efficient cellulose degradation or high glucose accumulation. These results show that the β-glucosidase secreted by strain A9 degrades cellulose effectively in combination with C. thermocellum cellulosomes and has the potential to be used in a new biological cellulose saccharification process that does not require supplementation with β-glucosidases.Key points• Strain A9 can secrete a thermostable β-glucosidase that has high glucose tolerance• A coculture of strain A9 and C. thermocellum showed high cellulose degradation• Strain A9 achieves biological saccharification without addition of β-glucosidase

Highlights

The bioconversion of cellulosic biomass to sugars is a major bottleneck in the development of methods for the use of cellulosic feedstocks and the commercialization of bio-based chemicals and biofuels
To relieve the feedback inhibition and promote cellulose saccharification, we have demonstrated previously that glucose can be produced from cellulosic biomass by C. thermocellum cultures supplemented with thermostable β-glucosidases in a process called biological simultaneous enzyme production and saccharification (Prawitwong et al 2013)
Single colonies were separated by the anaerobic Hungate roll tube technique using cellobiose as the sole carbon source in an agar medium supplemented with esculin and ferric ammonium citrate

Summary

Introduction

The bioconversion of cellulosic biomass to sugars is a major bottleneck in the development of methods for the use of cellulosic feedstocks and the commercialization of bio-based chemicals and biofuels This bottleneck occurs because cellulosic biomass is resistant to enzymatic degradation and the hydrolytic enzymes required are expensive. Clostridium thermocellum (Ruminiclostridium thermocellum, Hungateiclostridium thermocellum, Acetivibrio thermocellus)—an anaerobic thermophilic bacterium—is the most potent cellulose-degrading bacterium known to produce “cellulosomes” (Bayer et al 2004; Lynd et al 2002) These cellulosomes contain a large variety of enzymes, including enzymes with the potential to degrade cellulosic biomass (Bayer et al 2004). Biosafety issues must be considered when using genetically modified microbes in industrial applications (Friehs 2004; Kumar 2014)

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Conclusion