The use of bioethanol is beneficial for several reasons. One is that it can be easily integrated into the current fuel distribution system, and another is that it will reduce the production of greenhouse gases as the raw material used is biomass. Bioethanol can be produced from lignocellulosic biomass in three main steps: pretreatment, enzymatic hydrolysis, and fermentation. Enzymatic hydrolysis and fermentation can be carried out either separately, using separate hydrolysis and fermentation (SHF), or simultaneously, using simultaneous saccharification and fermentation (SSF).
Over the past decade SSF has become the preferred process, since end-product inhibition of the enzymes can be avoided by performing fermentation in the same vessel at the same time as hydrolysis [1
]. The capital investment cost of the plant is also reduced as fewer tanks are required [2
]. However, a disadvantage of SSF is that the operating temperature must be a compromise between the optimal temperatures for hydrolysis and fermentation, whereas these can be optimized independently in SHF. Furthermore, the yeast produced during the SHF process can be recycled after fermentation of the hydrolysate, which is not possible in SSF. The yeast thus represents a yield loss as it is difficult to separate it from the solid residue (lignin) [3
Wheat straw and other agricultural residues generally consist of high amounts of hemicelluloses [4
]. Xylose fermentation is therefore very important for these lignocellulosic raw materials in order to effectively convert all the sugars into ethanol, to increase the concentration in the fermentation broth. However, wild-type Saccharomyces cerevisiae
, which is the most commonly used yeast in ethanol fermentation due to its attractive properties, such as high yield of ethanol, high specific rate of fermentation [5
], and its high tolerance to the end product, ethanol [6
], is not able to metabolize xylose. Therefore, naturally xylose-fermenting yeasts, such as Candida shehatae
or Pichia stipitis
] have been widely studied since their ability to ferment xylose was discovered the early 1980s. Unfortunately, their tolerance to inhibitors [9
] and ethanol [10
] is limited, and they also require a very low and well-controlled supply of oxygen for effective xylose-fermentation [11
], which makes them difficult to use in large-scale production.
The ideal solution, combining the robustness and tolerance of S. cerevisiae
with the ability to ferment xylose has been sought by metabolic engineering. In principle, genes from bacteria and fungi encoding xylose isomerase (XI) [13
], or genes from fungi encoding xylose reductase (XR) and xylitol dehydrogenase (XDH) [15
] can be introduced into S. cerevisiae
. The endogenous gene XKS1
encoding xylulokinase (XK) must also be overexpressed for the strain to be able to utilize xylose for growth and ethanol production [16
]. TMB3400 [17
] is an industrial strain of S. cerevisiae
containing genes that encode for XR/XDH/XK, which is able to co-ferment xylose and glucose in non-detoxified lignocellulose hydrolysate of spruce [18
], as well as various pretreated raw materials [20
] in simultaneous saccharification and co-fermentation (SSCF).
In S. cerevisiae
TMB3400, xylose and glucose are competitively transported by the same transport protein [24
], but xylose has an approximately 200-fold lower affinity [26
]. To avoid the inhibition of xylose uptake by glucose, the glucose concentration in the medium must be low. It has been reported that low, but non-zero, concentration of glucose enhances xylose utilization [25
], which means that a slow release or feeding of glucose is required. This is one of the reasons why SSCF has become an interesting process option, as glucose is released during hydrolysis.
Co-fermentation of glucose and xylose in wheat straw hydrolysate has been investigated by Olofsson et al.
]. Their results showed that almost complete xylose fermentation can be obtained if a controlled glucose feed is applied during fermentation. However, during SSF the release of sugar is not controlled, as all the cellulase enzymes are added at once. Thus prefermentation [20
] and enzyme feeding strategies combined with fed-batch fermentation have recently been studied [27
] as a means of controlled glucose release. The best result so far, 80% xylose uptake, has been achieved by Olofsson et al.
using a yeast concentration of 4 g/L and controlled feeding of cellulases giving a glucose release rate of 2 g/L h [28
]. Simultaneous glucose and xylose uptake has been modelled in a study by Bertilsson et al
., indicating that a glucose feed rate between 5 and 10 g/L h would be suitable to obtain maximum xylose uptake rate, with a yeast cell concentration of 5 g/L [29
]. The effect of controlled glucose feeding has also been studied on barley straw hydrolysate by Linde et al.
], however, only 74% and 51% of the xylose was consumed by the yeast TMB3400 at glucose feed rates of 0.21 g/L h and 0.45 g/L h, respectively.
Wheat hydrolysate is derived from wheat meal, following the first generation ethanol production process through liquefaction and saccharification. The hydrolysate obtained is a glucose-rich solution as well as a potential source of nutrients, which has several advantages in the process. The use of wheat-starch hydrolysate, as a complex nutrient source, has been shown to be a potential supplement for lignocellulosic hydrolysates [31
]. It also has a positive effect on glucose fermentation in SSF, increasing both the ethanol yield and concentration [32
], which is important for the process economy [33
]. Therefore including wheat-starch hydrolysate in the feed in SHF, which is a first-generation glucose source, provides considerable possibilities for improvement of xylose uptake and hence increase the ethanol yield and concentration.
To the best of our knowledge, no study has yet been performed to evaluate separate hydrolysis and co-fermentation (SHCF) of steam-pretreated wheat straw combined with wheat-starch hydrolysate feed as a means of improving the co-fermentation of glucose and xylose. In the present work, two process configurations for SHCF were investigated using steam-pretreated wheat straw combined with wheat-starch hydrolysate feed, employing the xylose-fermenting yeast TMB3400.