Table 1 Protein and nucleic acid content of microorganisms

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1 Single cell protein (SCP) production Microbial biomass is produced commercially as single cell protein (SCP) for human food or animal feed and as viable yeast cells to be used in the baking industry. Rapid developments in microbial protein production occurred during the 1960s and 1970s. Extensive research was conducted on a wide range of microorganisms as possible alternate protein sources, motivated by large increases in the price of conventional animal feed. SCP is not pure protein, but refers to the whole cells of bacteria, yeasts, filamentous fungi or algae, and also contains carbohydrates, lipids, nucleic acids, mineral salts and vitamins. It has several advantages over conventional plant and animal protein sources, which include: 1 rapid growth rate and high productivity; 2 high protein content, 30 80% on a dry weight basis; 3 the ability to utilize a wide range of low cost carbon sources, including waste materials; 4 strain selection and further development are relatively straight forward, as these organisms are amenable to genetic modification; 5 the processes occupy little land area; 6 production is independent of seasonal and climatic variations; and 7 consistent product quality. Table 1 Protein and nucleic acid content of microorganisms 1

2 The protein content and quality is largely dependent on the specific microorganism utilized and the fermentation process. Fast-growing aerobic microorganisms are primarily used due to their high yields and high productivity. Bacteria generally have faster growth rates and can grow at higher temperatures than yeasts or filamentous fungi, and normally contain more protein. Yeasts grow relatively rapidly and, like bacteria, their unicellular character gives somewhat fewer fermentation problems than do filamentous organisms. However, many filamentous fungi have a capacity to degrade a wide range of materials and, like yeasts, can tolerate a low ph, which reduces the risk of microbial contamination. They are also more easily harvested at the end of fermentation than yeasts or bacteria. Selection of a suitable microbial strain for SCP production must take several characteristics into account,including: 1- growth rate, productivity and yields on the specific, preferably low-cost, substrates to be used; 2- temperature and ph tolerance; 3- oxygen requirements, heat generation during fermentation and foaming characteristics; 4- growth morphology and genetic stability in the fermentation; 5- ease of recovery of SCP and requirements for further downstream processing; 6- structure and composition of the final product, in terms of protein content, amino acid profile, RNA level, flavour, aroma, colour and texture. 7- Other major factors are safety and acceptability. Most SCP products are currently used as animal feed and not for human consumption. Nevertheless, these products must meet stringent safety requirements. Obtaining regulatory approval for the production of proteins for human consumption is an even lengthier and more expensive process, and obviously influences the choice of production organism. A safety aspect that must be considered for all SCP products is nucleic acid content. Many microorganisms have naturally high levels and the problem is further exacerbated because fermentation conditions favouring rapid growth rates and high protein content also promote elevated RNA levels. This can be problematic as the digestion of nucleic acids by humans and animals leads to the generation of purine compounds. Their further metabolism results in elevated plasma levels of uric acid, which may crystallize in the joints to give 2

3 gout-like symptoms or forms kidney stones. Slow digestion or indigestion of some microbial cells within the gut and any sensitivity or allergic reactions to the microbial protein must also be examined. For filamentous fungi, the possibility of aflatoxin production must be eliminated. An additional concern is the absorption of toxic or carcinogenic substances, such as polycyclic aromatic compounds, which may be derived from certain growth substrates. The major substrates that have been used in commercial SCP production are alcohols, n-alkanes, molasses, sulphite liquor and whey. Table 3: Microorganisms used for SCP production using various carbon sources. 3

4 Single cell protein production processes Many pilot plants have been developed over the last 30 years that utilize a range of substrates and microorganisms. However, relatively few have operated commercially, due to obstacles encountered on scale-up or for economic reasons. The physiological problems that are often encountered on scale-up include difficulties with: 1 oxygen requirements and oxygen transfer rates; 2 nutrient and temperature gradients; 3 effects of CO2, as high levels may inhibit respiration in certain microorganisms; 4 hydraulic pressure in deep fermenters. The SCP production processes essentially contain the same basic stages irrespective of the carbon substrate or microorganism used. 1 Medium preparation. The main carbon source may require physical or chemical pretreatment prior to use. Polymeric substrates are often hydrolysed before being incorporated with sources of nitrogen, phosphorus and other essential nutrients. 2 Fermentation. The fermentation may be aseptic or run as a clean operation depending upon the particular objectives. Continuous fermentations are generally used, which are operated at close to the organism s maximum growth rate, to fully exploit the superior productivity of continuous culture. 3 Separation and downstream processing. The cells are separated from the spent medium by filtration or centrifugation and may be processed in order to reduce the level of nucleic acids. This often involves a thermal shock to inactivate cellular proteases. RNase activity is retained and degrades RNA to nucleotides that diffuse out of the cells. Depending upon the growth medium used, further purification may be required, such as a solvent wash, prior to pasteurization, dehydration and packaging. The various processes described below have been relatively successful in commercial terms, and/or involve notable technological developments. THE BEL PROCESS Whey is byproduct of cheese contains approximately 45 g/l lactose and 10 g/l protein. It is particularly suitable for the production of SCP using lactoseutilizing yeast, although attempts have also been made to grow other 4

5 organisms, including Penicillium cyclopium. Several processes have been developed for the utilization of lactose in milk whey. Some of the more successful have been those operated by Bel Industries in France. The Bel process was developed with the aim of reducing the pollution load of dairy industry waste, while simultaneously producing a marketable protein product. A number of plants are operated using Kluyveromyces lactis or K.marxianus (formerly K. fragilis) to produce a protein, which is used for both human and animal consumption. These processes initially involve whey pasteurization, during which 75% of whey proteins are precipitated.the lactose concentration is adjusted to 34 g/l and mineral salts are also added. This supplemented whey is introduced into a 22m 3 continuous fermenter, maintained at 38 C and ph 3.5, with an aeration rate of 1700m 3 /h. The yeasts utilize the lactose and attain biomass concentrations of 25 g/l, with a biomass yield of g/g lactose. Yeast cells are recovered by centrifugation, then resuspended in water, recentrifuged and finally roller-dried to 95% solids. Levels of residual sugar remaining in the spent medium are less than 1g/L. THE SYMBA PROCESS The Symba process was developed in Sweden to produce SCP for animal feed from potato processing wastes. It is not economically attractive as a stand-alone operation. However, alternative routes for the purification of these waste-waters are difficult and expensive. A high proportion of the available substrate is starch, which many microbes cannot directly utilize. To overcome this problem the process was developed with two microorganisms that grow in a symbiotic association. They are the yeasts Saccharomycopsis fibuligera, which produces the hydrolytic enzymes necessary for starch degradation, and Candida utilis. The process is operated in two stages. In the first stage, S. fibuligera is grown in a small reactor on the sterilized waste, supplemented with a nitrogen source and phosphate. At this point, the starch is hydrolysed,which is the rate-limiting step of the whole process. The resulting broth is then pumped into a second larger fermenter of 300m 3 capacity where both organisms are present. However, C. utilis comes to dominate the second stage and constitutes up to 90% of the final product. The Symba process operates continuously and after 10 days the pollution load of the waste is reduced by 90%. Resultant protein-rich biomass (45% protein) is concentrated by centrifugation and finally spray or drum dried. 5

6 Fig. 1: The Symba process. THE BIOPROTEIN PROCESS There has been a considerable amount of research into the production of SCP using alkanes as carbon sources, notably methane and liquid straight chain hydrocarbons. These compounds present certain technical problems. They are not miscible with water, and methane, in particular, is explosive when mixed with oxygen. Some of these substrates also require purification, or the protein product derived from them needs to be treated to remove adsorbed toxic compounds. In addition, these fermentations present cooling and aeration problems, as these highly exothermic processes require substantially more oxygen than when carbohydrates are used. The Bioprotein process, developed in the 1990s by Norferm, uses methanerich natural gas as a sole carbon and energy source for the growth of Methylococcus capsulatus. A mixture of heterotrophic bacteria is also present, which helps to stabilize the process. This highly aerobic continuous fermentation is performed in a loop-fermenter,with medium containing ammonia, minerals and methane. Biomass is continuously harvested by centrifugation and ultrafiltration, prior to heat inactivation and spray drying. The final product contains 70% protein and is currently marketed as Pronin. 6

7 It is approved in the EU for use as a fish and animal feed, but may be used in human foods in the future. Production of Amino acids: Fermenter Procedure Starting from shake flasks the inoculum culture is grown in shake flasks and transferred to the first seed tank (1,000 2,000 liters) in size. After suitable growth the inoculum is transferred to the second seed tank (10,000 20,000 liters), which serves as inoculum for the production tank (50, ,000 liters). The fermentation is usually batch or fed-batch. In batch cultivation all the nutrients are added at once at the beginning of the fermentation, except for ammonia which is added intermittently to help adjust the ph, and fermentation continues until sugar is exhausted. In a fed-batch process, the fermenter is only partially filled with medium and additional nutrients added either intermittently or continuously until an optimum yield is obtained. The fed-batch appears preferable for the following reasons: (a) Most amino acid production requires high sugar concentrations of up to 10%. If all were added immediately, acid would be quickly produced which will inhibit the growth of the microorganisms and hence reduce yield. (b) Where auxotrophic mutants are used, excess supply of nutrients leads to reduced production due to overgrowth of cells or feedback regulation by the nutrient. (c) During the lag phase of growth, the oxgen demand of the organism may exceed that of the organism leading to reduced growth. The main raw materials used are cane or beet molasses and starch hydro lysates from corn or cassava as glucose. As nitrogen source, inorganic sources such as ammonia or ammonium sulfate is generally used. Phosphates, vitamins and other necessary supplements are usually provided with corn steep liquor. The production of amino acids by fermentation was stimulated by the discovery of an efficient L- glutamic acid producer Corynebacterium glutamincum. Many microorganisms have been reported to produce amino acids. They are mainly bacteria, but they also include some molds and yeasts. 7

8 The four most widely reported bacteria belong to the following four genera, Corynebacterium spp., Brevibacterium spp., Microbacterium spp. and Arthrobacter spp. Production strains for amino acids are generally classified as wild-type, capable of producing amino acids under defined conditions, but generally low-yielding in quantity, auxotrophic or regulatory mutants. Down Stream Processing: After fermentation, the cells may be filtered using a rotary vacuum filter. The extraction method of the amino acid from the filtrate, depends on the level of purity desired in the product. However two methods are generally used: the chromatographic (ion exchange) method or the concentrationcrystallization method. - Production of L-glumatic acid: Industrial-scale fermenters are normally stainless steel stirred tank reactors of up to 450m 3. These are batch processes, operated aerobically at C, the specific temperature depending on the microorganisms used. Apart from carbon and nitrogen sources, the fermentation medium normally contains inorganic salts, providing magnesium, manganese, phosphate and potassium, and limiting levels of biotin. Corynebacteria are nutritionally fastidious and may also require other vitamins, amino acids, purines and pyrimidines. The preferred carbon sources are carbohydrates, preferably glucose or sucrose. Cane or beet molasses can be used, but the medium requires further modification as their biotin levels tend to be too high. This can be overcome by the addition of saturated fatty acids, penicillin or surfactants which promote excretion. The nitrogen source (ammonium salts, urea or ammonia) is fed slowly to prevent inhibition of L-glutamate production. Medium ph is maintained at 7 8 by the addition of alkali, otherwise the ph progressively falls as the L-glutamate is excreted into the medium. Product recovery involves separation of the cells from the culture medium. The L-glutamic acid is then crystallized from the spent medium by lowering the ph to its isoelectric point of ph 3.2 using hydrochloric acid. Crystals of L-glutamic acid are then filtered off and washed. 8