Lignocellulosic biomass is hydrolyzed by explained treatments (mostly by acid treatment) (chapter "Hydrolysis of lignocellulosic biomass). The obtained hydrolysate (mixed sugar-solution) is then fermented by microorganisms (e.g. bacteria, yeast). This hydrolysate (mixed sugar-solution) often contains not only glucose, but also other sugars e.g. xylose, mannose, galactose, arabinose and also oligosaccharides. Therefore, in the fermentations process, microorganisms are required, which are able to efficiently ferment these sugars for the successful industrial production of bioethanol. For calculation of the theoretical maximum yield, have a look at the following reaction equations:
Referring to these equations, the theoretical maximum yield is 0.51 kg bioethanol and 0.49 kg carbon dioxide per kg of xylose and glucose.
During the fermentation process, microorganisms (e.g. bacteria, yeast) can use the fermentable sugars for food and in the process, they produce ethyl alcohol and other by-products. The microorgansims are typically able to use the 6-carbon sugars (C6-sugars) – generally glucose. That means, that cellulosic biomass, containing a huge amount of glucose or precursors to glucose can be converted (fermented) easily to bioethanol.
One of the most effective producer of bioethanol is the yeast Saccharomyces cerevisiae, also having a high tolerance to bioethanol and other inhibitory compounds in the acid hydrolysates of lignocellulosic biomass. As most wild-type strains of Saccharomyces cerevisiae can only ferment hexoses (e.g. glucose), but not pentoses (e.g. xylose and arabinose), the bioethanol production from a lignocellulose hydrolysate is inadequate. For bioethanol production of lignocellulose hydrolysates, a metabolic engineered strain of S.cerevisiae is required, in order to enhance the xylose flux.
Other natural yeast strains, capable of fermenting xylose are Pichia stipitis, Candida shehatae, and Candida parapsilosis. They can metabolize xylose by means of the xylose reductase (XR), which converts xylose to xylitol and the xylitol dehydrogenase (XDH), which converts xylitol to xylulose. In the last few yeast, several studies have been conducted in order to produce a recombinant S. cerevisiae, capable of converting xylose, because of carrying heterologous XR and XDH from P. stipitis and xylulokinase (XK) from S. cerevisiae.
Other xylose-fermenting microorganisms are found among bacteria, other yeast strains and filamentous fungi. Most of the xylose-fermenting bacteria include both native and genetically engineered organisms.
In table 1, some native and genetically engineered bacterial species, capable of fermenting xylose to bioethanol are listed up.
Table 1: List of native and genetically engineered bacterial species, capable of fermenting xylose to bioethanol (source: Jeffries and Jin, 2000)
The bacteria, currently most promising for industrial exploitation are Escherichia coli, Klebsiella oxytoca and Zymomonas mobilis. Zymomonas has the ability to produce bioethanol from glucose-based biomass very fast and efficient. Studies with Z. mobilis have shown a 5 % higher ethanol yield and up to five-fold higher volumetric productivity, compared to traditional yeast fermentations. In detail, Z. mobilis can produce bioethanol yields up to 97 % of theoretical and bioethanol concentrations up to 12 % (w/v) in glucose fermentations.
AsZ. mobilis is capable of fermenting hexose sugars (e.g. glucose and fructose), but not pentose sugars (e.g. xylose), xylose-fementing Z. mobilisstrains have been successfully produced by introducing a xylose-metabolizing pathway from E. coli (Department of Energy,United States).
K. oxytoca, an enteric bacterium, found in paper, pulp streams and other sources of wood, is capable of growing at a pH at least as low as 5.0 and temperatures of up to 308 K. This bacterium is able to grow on numerous sugars e.g. hexoses and pentoses, as well as on cellobiose and cellotriose.
Microorganisms, which are used in fermentation processes are often described in terms of their performance parameters. These are:
- Temperature range
- pH range
- Alcohol tolerance
- Growth rate
- Osmotic tolerance
- Genetic stability
- Inhibitor tolerance
The fermentation process can be performed as a batch, fed batch or continuous process, depending on kinetic properties of microorganimsms and type of lignocellulosic hydrolysate, additionally on process economics aspects.
Fed-batch reactors are mostly used in industrial applications, as they combine the advantages from both batch and continuous processes. Most advantageous is the ability to increase maximum viable cell concentrations, prolong culture lifetime and allow product accumulation to a higher concentration.
During this process, used microorganisms are maintained in culture in the exponential growth phase. Fresh medium is added continuously, while cell suspension is removed exactly from the bioreactor.