# Biorefinery: The Production of Isobutanol from Biomass Feedstocks
## 3 Biomass Isobutanol Production
### Isobutanol Production from Lignocellulose
#### 3.1.1 Cellulosic Isobutanol Produced by Natural Cellulose-Degrading Microorganisms  
Degrading cellulose with microorganisms that naturally utilize lignocellulose and convert the resulting C5 and C6 sugars into isobutanol is the simplest way to obtain cellulosic isobutanol. To minimize the lignocellulosic glycation process and production costs, the consolidated bioprocessing(CBP) strategy was developed. The first cellulosic isobutanol was synthesized by introducing the engineered isobutanol pathway into _Clostridium cellulolyticum_[41], which utilizes cellulose naturally. The strain produced a final isobutanol titer of 0.66 g/L. The simple introduction of the isobutanol synthesis pathway resulted in very low amounts of isobutanol titer, which is not sufficient for industrial scale production. The inability of _C. cellulolyticum_ to process large amounts of substrate has been reported [42]. Destroying the ability of _C. cellulolyticum_ to form spores by knocking out the _spo0A_ gene improved cellulose utilization, but there was little variation in isobutanol titer (even lower than the wild type when the cellulose substrate reaches 50 g/L) [43]. The biosynthesis of cellulose isobutanol was also attempted in _Geobacillus thermoglucosidasius_[44], which produced isobutanol at 0.6 g/L from cellobiose in 60 h. While unsuccessful at producing large volumes of cellulosic isobutanol, this experiment demonstrated the strong thermal stability of ALAS and KIVD, which are widely used for isobutanol synthesis. \begin{table}
\begin{tabular}{l l l l l l l l l} \hline
**Microorganism** & **Carbon Source** & **Strategy** & **Genes Involved** & **Titer** & **Time** & **Reactor** & **Reference** \\ \hline _Crystallization_ & Cellulose & Engineered isobutanol pathway & \(\begin{array}{l  
\begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline
**Microorganism** & **Carbon Source** & **Strategy** & **Genes Involved** & **Titer** & **Time** & **Reactor** & **Reference** \\ \hline \hline \multirow{4}{*}{_Sachrumscapes cerevisiae_} & \multirow{4}{*}{Xylose} & \multirow{4}{*}{Xylose} & \multirow{4}{*}{Xylose XI pathway} & _All2C_, _All6C_, _Xls  
\begin{table}
\begin{tabular}{l l l l l l l l l} \hline \hline
**Microorganism** & **Carbon Source** & **Strategy** & **Genes Involved** & **Titer** & **Time** & **Reactor** & **Reference** \\ \hline \multirow{6}{*}{Cellobose} & \multirow{6}{*}{Cellobose} & Inhibition competition pathway & _Aadfile_, _ArtifBC_, _Afur_, _AldaA_, _Apta_, _Apta_, _Apta_, _Apta_, _Apta_, _Apta_, _Apta_  
\begin{table}
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**Microorganism** & **Carbon Source** & **Strategy** & **Genes Involved** & **Titer** & **Time** & **Reactor** & **Reference** \\ \hline  & & & & _E. coli_ BLF2: & & & & & \\  & & & _Allah_ & & & & & \\  & & & _lib_CC\({}_{\text{ICB}}\), _lib_DC\({}_{\text{YqhD}}\), _als_S\({}_{\text{BS}}\), & & & & & \\  & & Chemical mutagenesis & & & & & & \\  & & Protein conversion & & _E. coli_ AY3: & & & & & \\  & _E. coli_ AY3 & & & & & & & \\  & & Cofactor engineering & _AgInA_, _AgInA_, _AlahS_, _AlaxS_, _AlAxA_, _I_-1.5 & & & & & \\  & & Engineered isobutanual pathway & _J_Acc/N\({}_{\text{IS}}\), \(S\)0.870, \(S\)0.101A, & & & & & \\  & & Microbial consortium &  
The cellulose-utilizing _Clostridium thermocellum_ is considered a promising producer of cellulosic biofuels, so additional experiments have attempted cellulosic isobutanol synthesis in this bacterial species [69]. Refinement gene expression systems [70; 71; 72] and gene editing techniques [73] have already been developed for _C. thermocellum_, and its basic metabolic network has been explored. Introducing the KIVD into _C. thermocellum_, optimizing the isobutanol synthesis pathway, and limiting the urea content of the medium enabled the engineered strain to ferment 5.4 g/L of isobutanol in 75 h [45]. Experiments have also identified a new pathway in _C. thermocellum_ that converts KIV to isobutanol.