## 3 Major Routes of Xylose Transport and Metabolism in Bacteria
### Metabolic Pathways to Xylitol and Xylonic Acid
#### 3.3.1 Xylitol
If the cofactor NAD\({}^{+}\) is abundant in the medium, the enzyme X\({}_{\text{OH}}\)DH (Figure 1) converts xylitol into xylulose, which enters in the PPP pathway after being phosphorylated. The accumulation of xylitol in the cultivation media is believed to be a consequence of co-factor imbalance. This can be attributed to the co-factor preference of xylose reductase towards NADPH and xylitol dehydrogenase towards NAD (Figure 1). Furthermore, it has been reported that NADH accumulation inhibits the NAD linked xylitol dehydrogenase [107]. Many pentose-utilizing microorganisms suffer from bottlenecks, resulting in low xylitol productivity. For this reason, the genetic manipulation of wild species by adaptation, mutation, and recombinant techniques has been proposed. Selective gene manipulation to improve productivity has been targeted after gradual enrichment of the genetic database and increased knowledge about the metabolic mapping of many of these microorganisms. With this tool, it is possible to optimize selective xylose transporter systems to increase the xylose uptake rate, as previously referred. Improvements for xylitol production have included the overexpression of XR to maximize the reduction in xylose into xylitol or deletion of the X\({}_{\text{OH}}\)DH gene to stop the oxidation of xylitol into xylulose, besides the optimization of the supply of cofactors involved in xylitol production (as NADH or NADPH) [104, 112, 113, 114]. XR genes from yeasts such as _C. tenuis_ or _C. tropicalis_ were successfully expressed in _E. coli_ and the conversion of xylose into xylitol was then achieved [115, 116, 117]. It is also reported that xylitol production from xylose can be improved through the heterologous expression of xylose transporters [116]. Currently, most studies on xylitol production in bioreactors focus on yeast-based processes [105, 118]. To improve productivities, fed batch or continuous reactor operation modes as well as high cell density strategies that include cell immobilization or retention inside membranes should be preferred. The use of immobilized cells during a continuous fermentation mode is often attractive. This option allows for cell re-use, ensuring high productivities during an extended period, compared with freely suspended cells. However,most bioreactor studies regarding xylitol production are reported to be operated under batch mode. For xylitol production, non-growing cells in a viable state (\"resting cells\") require an additional co-substrate to carry out the reduction reaction. Hence, a fed-batch bioreactor system with intermediate co-substrate addition at repeated intervals may be a solution to improving the productivity and therefore the xylitol yield [104]. Fine-tuning concerning operational parameters control is paramount [60]. Regarding microbial xylitol production, a number of cultivation parameters need to be considered, such as the pH of the culture, initial substrate concentration, medium composition, inoculum level, temperature, and aeration conditions [119]. With this many variables at play, the search for the optimal conditions for each microorganism has been an area extensively addressed. As reported by [PERSON] and [PERSON] (1998), both XR and XOHOH (Figure 1) enzyme activities are dependent on the concentration of xylose in the culture medium, since xylitol formation does not occur in the absence of this pentose [120]. Moreover, the xylose concentration in the lignocellulosic hydrolysate should be as high as possible to ensure an economically viable process, although it should be borne in mind that, when concentrating the hydrolysates, the inhibitors present (e.g., furfural and hydroxymethylfurfural) may reach levels that adversely affect the behavior of the micro-organisms [119,121]. Another key factor on the xylitol production is the aeration rate in the culture since this is directly related to the regeneration of the cofactors NAD\({}^{+}\)/NADH or NADP\({}^{+}\)/NADPH.