Production of protozoan protein from sugarcane pomace in solid state fermentation in a tray bioreactor

Master's thesis in the field of chemical-biotechnology engineering

Abstract

Protozoan protein refers to the protein obtained from the cultivation of bacteria, yeast, filamentous fungi or algae, which can be used as human food or animal feed. In fact, single-cell protein is the dried cells of microorganisms that are obtained by their fermentation on different substrates. Lignocellulosic materials, including agricultural wastes such as sugarcane bagasse, are among the most abundant and cheapest substrates for the production of single-cell protein in the world. In the present study, protozoan protein was produced in solid state fermentation from sugarcane bagasse using the microorganism Saccharomyces servicii. Tray bioreactor was used for solid state fermentation in this research. For protein extraction, a suitable buffer was selected from different buffers (citrate, phosphate, carbonate-bicarbonate and distilled water). In the following, the effect of operational parameters including: initial humidity of the solid substrate, duration of fermentation, internal temperature of the bioreactor, internal humidity of the bioreactor, as well as enrichment of the substrate with different nitrogen sources and alkaline pretreatment of the substrate to optimize single cell protein production in solid state fermentation were investigated. Finally, an amino acid test was performed on the sample produced under optimal conditions in order to evaluate the nutritional value. Among the buffers used, the highest amount of protein extraction was from the carbonate-bicarbonate buffer. Fermentation time and sugar consumption were checked at temperatures of 25, 30 and 35 degrees Celsius with 85% humidity. The maximum amount of protein production in 72 hours and the amount of sugar consumption at the mentioned temperatures were 58.58, 59.62 and 71.45% for the upper tray and 53.24, 56.52 and 66.44% for the middle tray respectively. At the temperature of 35°C, the highest amount of cellular activity was obtained by 43.43% for the upper tray and 71.71% for the middle tray. The best result was obtained in enriching the substrate with nitrogen sources using sodium nitrate. Among the different concentrations of sodium hydroxide solution, 2% solution (weight-volume) led to the production of the most protein at the rate of 3.612 g/liter for the upper tray and 3.530 g/liter for the lower tray. Finally, by optimizing all the parameters (after 72 hours, at a bioreactor temperature of 35°C, 85% bioreactor humidity, 70% initial humidity of the solid substrate, enrichment of the substrate with a carbon source of sodium nitrate and pretreatment with a 2% sodium hydroxide solution), the amount of protein for the upper and middle trays was 3.929 and 3.813 g/liter, respectively, and in this case, the protein production efficiency for the upper tray was 0.132 and for The bottom tray was 0.128 g/g substrate. The produced product also contained essential amino acids leucine, isoleucine, valine, methionine, phenylalanine, and many non-essential amino acids. rtl;">1-1- Introduction

Today in the world, lack of food, especially in the third world countries, is a very important issue, and most societies are somehow trying to find a way to solve this problem. With the increase in world population, the need for food, especially protein, is increasing day by day. In countries that are considered to be poor, the increase in population leads people to hunger and severe malnutrition [1.] Since proteins, along with carbohydrates and fats, form the three main groups of food, finding their various and new sources has provided a suitable platform for research in this field. The inadequacy of traditional protein sources such as meat, fish, and eggs compared to population growth led scientists to search for cheap sources of protein for people. On the other hand, increasing concern about pollution caused by agricultural and industrial wastes and efforts to transform waste materials into products with high commercial value have also received much attention in recent decades [2]. Microorganisms such as yeasts and bacteria have been used in human homemade food for thousands of years. In recent decades, new methods have been developed for the use of microbial fermentation products in human food and animal feed [3].Considering the lack of protein sources on the one hand, and the high amount of microbial cell protein on the other hand, it is very important to use microbial protein sources as human and livestock food (instead of common protein sources). This microbial protein is called protozoan protein[1]. Currently, protozoan protein is used in livestock and poultry feed. Due to the high amount of nucleic acids in this type of protein and its low digestibility, there are doubts about substituting microbial protein instead of animal protein sources. They are also considered as a foreign factor for the body that can cause allergic reactions. Of course, if this protein is used as human food, it must be extracted and concentrated before use, and its nucleic acid should be reduced, because high nucleic acid causes the production of uric acid and eventually causes gout.

Protozoan protein, as mentioned, is a microbial protein that is not pure and refers to complete cells of bacteria, yeasts, filamentous fungi, or algae, which contain a variety of acids. It also contains amino acids, carbohydrates, fats, nucleic acids, mineral salts and vitamins and has a very high nutritional value. In fact, this type of protein is a product that is fermented by the activity of various microorganisms on inexpensive substrates such as agricultural waste, factory effluents, and cellulose waste from paper mills. It is obtained [4].

Using microorganisms to produce protozoan protein instead of using animal and vegetable protein sources has advantages, and according to these cases, a clear prospect for the production of this type of protein is considered. These advantages include [5]:

         1- The possibility of genetic modification of microorganisms

         2- The growth speed and high protein content of microorganisms       

          3- The use of a wide range of methods, substrates and microorganisms for this purpose

          4- No dependence of production on climate changes

          5- No need for a lot of space for production

          6- Speed ??and high production efficiency.

However, the production of single-cell protein with high quality and efficiency requires the selection of the appropriate microorganism and substrate and the selection of the appropriate method for fermentation.

1-2- History

The term protozoan protein was first coined by Professor Carl Wilson[2] in 1944 at the Massachusetts Institute of Technology[3] [6]. This term is not appropriate for a material with a protein content of less than 65% and they recommend the term "single cell mass" for such cases. It is also more appropriate to use the term "fungal protein", which has recently been used in many sources, for the protein-containing cell mass obtained from fungi.

The production of single-celled protein from yeast was first carried out by the Germans during World War I. Protein[4] was the first commercial protozoan protein that was used as an animal feed additive. In the middle of 1930 and World War II, it received more attention and its production reached 15,000 tons per year.

The first international conference on single-cell protein was held in Massachusetts, USA in 1968, and in that conference, the British Petroleum Company [5] was the only presenter of its industrial production. In the second conference that was held in the same place in 1973; Many companies presented their industrial products. Factories that industrially produced single-cell protein until 1990; They mainly used two sources of hydrocarbon or sugar substrate. Sources of sugar substrate mainly included molasses, agricultural waste, wastewater from food industry and wood and paper industries, while hydrocarbon substrates mainly included petroleum derivatives, ethanol and methanol.