Keratinous animal by-products such as chicken feathers, horns, wool, hairs, bristles etc. are naturally occurring polymeric materials that are normally synthesized within the epithelial cells of higher vertebrates and humans as well, and constitute after cellulose and chitin the third most abundant renewable biomass present on the surface of our planet. Keratin waste materials occur abundantly in slaughterhouses and meat & poultry processing plants, and according to the EU (Regulation (EC) No. 1096/2009) they are classified under the category of the low-risk animal by-products. As a con-sequence, this type of solid residue is not suitable for human consumption and needs to be treated before its disposal into the environment. In this PhD thesis we have focused our attention primarily towards a particularly underestimated and therefore underexploited keratin-rich by-product, namely pig bristles (also known as hog hairs). As a matter of fact, the protein content of porcine bristle can be even larger than 80% of its dry weight. Moreover, considering that in 2014 about 250 million pigs were reared and slaughtered in the EU alone (EUROSTAT, 2016), it means that potentially approximately 60 k-tons of dry proteins could be recovered from this solid waste and then be employed as a renewable and more sustainable source of animal feed proteins. Nevertheless, the conventional methods employed for converting pig bristles into a more digestible dietary protein are normally based on the hydrothermal cooking with or without the addition of acid or alkali. The resulting product, namely hog hair meal, is usually lacking sufficient essential amino acids, (methio-nine, histidine, tryptophan and lysine among others) and still characterized by a relatively low digestibility. Therefore, it would be very beneficial if one could develop a milder technology, which could be utilized to effectively recover the soluble proteins, peptides and amino acids entrapped within this hair-type keratin waste and which, at the same time, could preserve an unaltered nutritional value of the extracted macronutrients. A solution to this problem can be found in the biotechnological route where instead microorganisms and microbial keratinolytic enzymes are employed to bring about the decomposition of keratin-rich substrates. The microbial hydrolysis of the keratinous biomass is a conversion process in which a biocatalytic cocktail of keratin-specific proteolytic enzymes is secreted by bacteria, actinomycetes and keratinophilic fungi that are capable of using keratin as their only source of C, N and energy. The soluble proteins, peptides and free amino acids released during the biological degradation process could constitute a viable alternative protein source to fish meal for aquaculture.
Therefore the main objectives of this PhD thesis are an investigation of the capability of a keratinolytic microorganism, i.e., the filamentous bacterium Amycolatopsis keratiniphila D2, to direct the efficient biodegradation of thermally pretreated pig bristles and the development of a biotechnological process to bring about the cost-effective microbial conversion of porcine bristles into a protein-rich keratin hydrolysate which could then be included in fish feed formulations as an alternative protein source for aquaculture.
In Chapter 2 the effect of culture conditions on bristle protein hydrolysate production during microbial decomposition of thermally pretreated porcine bristles by A. keratiniphila D2 was investigated. The maximum concentration of total extracted proteins (i.e. crude soluble proteins + NH2-free amino groups) in a 3-L aerobic fermenter was obtained for a degradation process carried out at a cultivation temperature of 35 ºC, an initial pH of 6.9, a pig bristle concentration equal to 7% (w/w) and an inoculum grown in GYM (glucose, yeast extract, malt extract) medium for 48 hours. The microbial single-stage hydrolysis process resulted in a final concentration of total extracted proteins of 27 g∙L-1 (45.8 % overall yield of extraction). In Chapter 3, it was demonstrated that a novel two-stage fermentation process, which was additionally run at high solids loadings (15% w/v), could result in a very significant improvement in the amount of total extracted proteins (98 g∙L-1; overall extraction yield of 84.9%) when hydrolyzing thermally pretreated porcine bristles with A. keratiniphila D2. Afterwards, in Chapter 4, the use of cell-free crude keratinases extract obtained from A. keratiniphi-la D2 was tested as an alternative to direct the enzymatic degradation at high solids loadings (15% w/w) of thermally pretreated porcine bristles in the absence of microbial cells. Moreover, two different fed-batch keratinous waste hydrolysis strategies were tested. In addition, the nutritional quality of the obtained keratin protein hydrolysates was also evaluated which, indeed, was confirmed to be improved considerably with respect to the original keratinous material. Finally, Chapter 5 pro-vided a more detailed investigation of some of the proteolytic enzymes which were synthesized by A. keratiniphila D2 when grown on keratinous by-products. In particular, two different proteases were purified from the culture supernatant and were further characterized: both proteolytic enzymes were shown to belong to the S1 family of proteases and to have higher specificity towards keratin-rich substrates.
In conclusion, this PhD thesis provides for the first time, a systematic insight into the development of a biotechnological process of industrial relevance for the recovery of valuable proteins from an exceptionally recalcitrant hair-type keratinous by-product, that is porcine bristles.