## 6 Intermediators-Inhibitory By-Products  
### Furfurral and 5-HydroxyMethyl Furfurral Production  
Furfurals are produced during thermo-chemical processing of lignocellulosic biomass. 5-HydroxyMethyl Furfurral (HMF) and furfurals are the most important members of furfurals. HMFis formed from hexoses and their polymers, including cellulose. Furfurals are produced from pentoses, which are obtained from hemicelluloses [87]. Acids inhibit cell growth, specifically weak non-dissociated acids cross the cell wall and energy is required to be exported out of the cell [88]. Furfural decreases specific growth rate, and HMF has a mechanism similar to furfural but produces a longer delay phase during growth. Phenolics interact with the cell membrane causing a loss of the integrity of the membrane and decreasing its permeability. However, methanogenic bacteria are able to adapt to such compounds in a certain period of time, up to a certain concentration [89].  
In cellulose, the p-glucose is dehydrated to 5-Hydroxymethylfurfural (HMF) by hydrothermal pre-treatment, and then decomposed to formic acid and levulinic acid. In supercritical water, the 99% conversion of glucose to HMF and then to formic and levulinic acids occurred at 0.01 s without the use of catalyst [52]. According to [PERSON] et al. [90], when switchgrass was pre-treated at 200 \({}^{\circ}\)C for 10 min, production of furfural was observed at a rate of 0.72 g/100 g switchgrass. It was observed in several studies that when temperature was varied between 200-220 \({}^{\circ}\)C for 5-15 min, the furfural concentration was between 0.2 and 3.1 g/100 g switchgrass [91; 92; 93; 83]. The hydrothermal pre-treatment of sugar cane press mud for the production of methane was studied. The methane concentration decreased at temperature \(>\)200 \({}^{\circ}\)C, accounting to inhibition to methanogenesis due to recalcitrant compounds (furfural) formation at high temperatures. The furfural production was observed at the rate of 0.73 g/100 g of press mud. [PERSON] et al. [94], reported that Maillard reaction was responsible for the formation of recalcitrant compounds at high temperature. This was due to the polymerisation between the carbohydrates and amino acids. These recalcitrant compounds inhibited the methanogenesis and reduced the overall process efficiency.  
Some soluble sugars like xylose, glucose, cell-oligomers, and xylo-oligomers, weak acids like formic, levulinic and acetic acid, furan derivatives like furfurals and HMF and phenol compounds like ferulic acid and vanillin are formed as a result of hydrothermal pre-treatment of lignocellulosic biomass. These compounds are inhibitory for the metabolic activities of substrate degrading microbes and enzymes [95].  
The concentration of inhibitory compounds vary as per the type of pre-treatment applied on the lignocellulosic biomass, and the solid loading of lignocellulosic on the substrate. of pre-treatment [96; 97; 98]. [PERSON] et al. [99] studied that hydrothermal pre-treatment of maple wood. Pre-treatment of maple wood at 230 g/L with hot water at 200 \({}^{\circ}\)C for 20 min led to the formation of xylooligomers and xylose with a concentration greater than 11.2 and 9.2 g/L, respectively. The concentration of HMF and furfurals were 4.1 g/L, while as the phenol compounds were present in 1.3 g/L concentration. [PERSON] et al. [100], stated that hydrothermal pre-treatment of barley star at a temperature of 210 \({}^{\circ}\)C for 5 min, generated inhibitors like furfural (0.7 g/L), acetic acid (2.1 g/L), phenolic compounds (0.2 g/L) and HMF (0.2 g/L), which affected the enzymatic hydrolysis of the substrate.  
Therefore, hydrothermal pre-treatment of ligno-cellulosic biomass is sometimes accompanied by the formation of inhibitors like furfurals and 5-HMF, which act as recalcitrant leading to inhibition of the biological process.