Pilot Plant Production of Lysine Background: Lysine has been recognized as an essential component of feeds.

. In the Philippines, lysine requirements are being met by importation since there is no commercial plant for its local production. For this reason, PCIERD has initiated a project to locally develop the technology for lysine production. BIOTECH had undertaken project implementation with PCIERD assistance. The project had completed bench-scale studies with conditions optimized for a 4-liter and 30-liter fermentors as well as the downstream recovery. In the development of technologies, it is necessary that prior to commercial operations, pilot studies be done to provide the basis for the design of the commercial plant. PCIERD and BIOTECH had initial discussions with two (2) companies interested in participating in the pilot plant studies. However, the company, Pacific Biotechnology Corporation, (PBC) pursued further discussions. However, the private cooperator would participate provided results of the bench-scale studies are duplicated. It was willing to shoulder the costs of raw materials (sugarcane juice and molasses) amounting to P80,000.00 for the piloting. However, the PCIERD Governing Council ruled that there should be a 50:50 share in project expenses with PBC. But in view of PGBs nonconcurrence with proposed cost-sharing, the project is now to be funded solely by PCIERD. In order to obtain the data sufficient for the techno-economic studies for commercial plant, at least 8 runs in the 250-l fermentor and 12 runs in the 1000-li fermentor are needed. While results from the bench-scale studies indicate good yields (52 gm/li), there is need to undertake piloting in order to verify viability at higher substrate volume.

 SIGNIFICANCE: Lysine is one of the essential amino acid present in very small amounts of cereals. The deficiency of lysine in cereal proteins has become a major problem in human nutrition particularly in oriental countries due to their dependence on cereal foods. Similarly, the lysine content of feedstuff is at sub-optimal levels so that amino acid fortification has been widely practiced in the manufacture of formula feed for animal production. Over 34,000 tons of L-Lysine are used annually as additives in the manufacture of nutritionally balanced food. Lysine is added to improve the nutritional value of cereal-based diets and it is currently sold at approximately US$ 3-4/kg. in every competitive market dominated by a few Japanese companies. Presently, 80% of lysine in the world market is made by microbial fermentation and the remaining 20% by chemical synthesis. The commercial production of 40,000 tons of lysine per year by microbial fermentation is a success story in the production of industrial chemicals. Lysine is currently being used in the pharmaceutical, food, feed milling and cosmetics industries thus, the outlook for this amino acid is high because of expanding market demand. In the Philippines, amino acid requirements are met only through importation since there are no industries that produce them. The prohibitive price and insufficient supply of amino acids add to problems of malnutrition and poor livestock production in the country. If the production of amino acid can be done locally using available agricultural substrate, our feed industry would not only be assured of supply of much-needed imported animal feed supplements but also save as much needed dollars because of reduced dependence on imported feed additives.

On the other hand, cost of labor and raw materials in the country are much cheaper than US, France, and Japan. This makes local production of lysine likewise cheaper. OBJECTIVES: The overall objective of this project is to scale up fermentation and the downstream processes for lysine production with the utilization of sugarcane juice and molasses substrates. The specific objectives are as follows: 1. To optimize the fermentation process for lysine production in 250-li and 1000-li fermentors. 2. To optimize the downstream processing for lysine separation 3. To prepare the techno-economic feasibility study of commercial lysine plant. REVIEW OF LITERATURE All proteinaceous amino acids are L-X-amino acids and are important as nutrients, seasoning, flavoring and precursors for pharmaceutical, cosmetics and other chemicals. They may be produced either by isolation from natural materials or by chemical, microbial or enzymatic synthesis. Chemical synthesis produces a racemic product which may be require additional resolution. The first two procedures give rise to optically pure amino acids. The commercialization of biological process for the production of amino acids ahs led to new approaches to the generation of the correct metabolic imbalance within the producer organism, in order to produce large quantities of metabolic intermediate that are otherwise subject to strict metabolic regulation. Whenever the

intracellular metabolic pathways governing amino acid production are understood and not to complex, amino acid can be made more efficiently by fermentation than by chemical synthesis. In other countries, many researches have been focused in the production of amino acid by fermentation methods and many processes have been developed for various amino acids. The production of L-Lysine by fermentation began with a technique developed by Pfizer and Co. Laboratories in 1956 (Casida, 1969). At about the same time, Kinoshita and Associates (1958) found 5 strains of microorganisms capable of accumulating large amounts of L-Lysine directly. This direct microbial production method has been developed into a commercial process and it has been widely used in Japan, USA and France. Although wild strains of microorganisms normally produce limited quantities of amino acids to meet growth requirements, their wasteful overproduction is prevented by feedback inhibition and repression. Until now, production of amino acids by direct fermentation often involves the use of auxotropic or regulatory mutants which are capable of over-producing amino acids for industrial purposes. (Tech 1986). In the case of lysine production, auxotropic and regulatory mutants of Brevibacterium flavum and Corynebacterium glutamicum have been used (Sano and Shiio, 1970; Shiio and Sano, 1969; Nakayama et al., 1961). Strains with a combination of auxotropic and regulation deficiency have also been widely used (Nakayama and Arami, 1981; Kubota et al., 1976 and Tosaka et al., 1978). Highest lysine accumulation reported by a homoserine auxotropic of Brevibacterium lactofermentum was about 60 g/liter . At the National Institute of Biotechnology and Applied Microbiology (BIOTECH), the technologies for upstream and downstream process have been developed at laboratory and benchscales. Lysine production of 50 to 55 g/li has been achieved under

optimum conditions. The downstream process scheme can be modified to produce the desired product for food and feed uses which include lysine-rich feed concentrate, lysine-HCl and lysine. METHODOLOGY: The following activities are included in this project: 1. Sugarcane juice and molasses will be used to conduct studies on lysine production. Sugarcane will be purchased from a farm nearby BIOTECH. Sugarcane juice will be extracted using the facilities available in the pilot plant at BIOTECH. Molasses will be purchased from sugar industries in nearby Calamba. 2. A 200 and 1000 liter stirred tank fermentors will be used to study process variables for lysine production at pilot scale. 3. Downstream processing for lysine powder will be carried out. Product recovery involving separation, purification, concentration, precipitation and drying will be studied. 4. The mass and energy balance for the process will be calculated and will be used to evaluate the economic feasibility of the processes.