In the search for a replacement for fossil fuels bioethanol comes out as one of the most promising alternatives. For sustainable production without interference with food production it is necessary to use lignocellulosic sources such as agricultural or forestry residues as raw materials 
. However, the inherent recalcitrance of these materials makes extensive pretreatment and hydrolysis necessary for efficient release of fermentable sugars 
. This often creates significant amounts of by-products that act as inhibitors of the subsequent fermentation, lowering the efficiency and feasibility of the process 
The most widely used microorganism for production of fuel ethanol, be it 1st
generation, is Saccharomyces cerevisiae
. This yeast is capable of in situ
detoxification of toxic hydrolysates, however, the inhibitor to cell ratio has to be low 
. A low ratio can be achieved by increasing the local cell concentration, by cell immobilization or flocculation and cell recirculation 
. With a high local cell concentration, the ratio of inhibitors to cells becomes smaller locally and thus the cells can better handle the toxicity of a hydrolysate 
Cell immobilization can be done in a number of ways, but the one giving the highest local cell density is undoubtedly encapsulation in a semi permeable membrane. Local cell densities of several hundred grams dry weight per litre of capsule volume have been achieved 
. Encapsulation of yeast cells has been shown to improve the fermentative performance in toxic lignocellulosic hydrolysates 
, as long as the inhibitors can be converted at a high rate 
. Increased thermotolerance has also been observed upon encapsulation 
. It is obvious that encapsulating cells will affect their growth and metabolism due to the close contact with other cells and due to the increased diffusion resistances that may lead to nutrient-limited conditions in the core of the capsule. It has been shown, that encapsulation leads to significantly lower cellular contents of RNA and protein as well as a lower RNA/protein ratio, and to higher cellular contents of trehalose, glycogen and total carbohydrates 
. However, it is not clear how encapsulation affects the cells on a more molecular level, and how the responses facilitate increased tolerance to inhibitors or elevated temperatures. Furthermore, genome-wide investigations, on e.g. transcriptome or proteome level, of the physiological changes in yeast encapsulated in liquid core capsules have not yet been performed. A better understanding of the biochemical background of the improved tolerance may be used to design superior yeast strains, able to ferment toxic hydrolysates at high rates even without the need of an enclosing membrane.
Quantitative proteomics is of utmost importance for the understanding of changes in cellular physiology arising from different treatments of the cells, as different proteins, including post translationally modified variants, are directly linked to metabolic fluxes and cellular structure, and therefore ultimately determine the physiology. There are a number of different quantitative proteomic methods available, with two major types of protein separation, namely two dimensional gel electrophoresis (2-DE) and multidimensional liquid chromatography (multidimensional protein identification, MudPIT), often called nLC-MS/MS. For identification of proteins both methods depend on mass spectrometry in combination with database searches. For 2-DE one of the currently most popular methods is 2-D difference gel electrophoresis (2-D DIGE), where proteins from different samples are labelled with different fluorescent probes, enabling quantification of proteins from different samples in the same gel 
. In MudPIT the most common in vitro labelling method is by isobaric mass tags, such as iTRAQ® or TMT®, that are applied after enzyme digestion of the protein samples to covalently label the peptides of different samples 
. The isobaric mass tags have different isotopic substitutions that, as the tags are cleaved off the peptides in the MS/MS mode, result in reporter ions of different weight, thus enabling quantification of proteins from different samples.
In this study, we compare the protein expression levels in yeast cells growing anaerobically either in liquid core capsules enclosed by alginate-chitosan membranes or in suspension, using both the 2-D DIGE approach and a MudPIT approach with TMT®. In addition to the overall elucidation of physiological changes in the cells due to encapsulation, the aim was to find possible reasons for the increased tolerance towards lignocellulosic derived inhibitors as well as for the enhanced yield of ethanol and lower glycerol yield of encapsulated yeast 
. We show that encapsulation leads to general down-regulation of proteins involved in growth and protein synthesis, and up-regulation of proteins involved in both specific responses to nutrient limitation, as well as general stress responses. We conclude that the triggering of general stress responses is the underlying mechanism of the improved inhibitor tolerance of encapsulated yeast.