Food As a Substrate for Microorganisms: Definition, Sources and Growth. And also learn about the substrates and uptake of nutrients by various microorganisms.
The nutritional requirements of various microorganisms may differ appreciably but all of them require carbon, nitrogen, and phosphorus sources, as well as other minerals and frequently vitamins. Sources of nitrogen (ammonia, ammonium salts, and nitrates) and of phosphorus are well known.
These as well as sources for other minerals such as calcium, magnesium, potassium and trace elements are infrequently mentioned in the technical literature because of the overriding importance of the carbon source for the technical and economic feasibility of the biomass process. In some instances, vitamins such as biotin, thiamin, or others have to be supplied to the growth medium.
For bench scale experiments this is usually done in the form of a yeast extract. For commercial processes the requirements for vitamins and trace elements have to be determined accurately; and they have to be added in the form of soluble mineral compounds and synthetic vitamins, usually with a yeast or yeast extract supplement.
A specific example of the nutrient requirements of Candida utilis grown on ethanol is shown in Table 13.4. Nutritional requirements have been reviewed by Suomalainen and Oura (1971) and fermentation substrates by Ratledge (1977).
Carbon Sources:
The traditional carbon sources for cell mass production are carbohydrates in the form of fermentable sugars. Beet or cane sugar molasses containing about 50% fermentable sugars and sulfite liquors containing 2 to 3% of hexoses and pentoses are the most common substrates. These substrates require a minimum of preparation, generally stripping of SO2 and clarification for the removal of insoluble solids.
Whey containing about 5% lactose, demineralized whey, or ultra-filtered whey also requires little processing. Starches from grains require enzymatic hydrolysis to fermentable sugars, except that some molds, bacteria, and yeasts themselves have sufficient extracellular amylase activity.
The use of malt amylases has often been replaced by the use of fungal and bacterial amylases. Cellulose or cellulose containing wastes require fairly extensive pretreatment by mechanical subdivision (milling) or treatment with alkali or acids. Ethanol (either synthetic or fermentation ethanol) and methanol require no pretreatment.
Methane and n-alkanes are hydrocarbon compounds that may be used for the production of biomass. Methane is a major constituent of natural gas. The n-alkanes occur in gas oils which are commonly marketed as kerosene, diesel fuel, or heating oil. Gas oils with a boiling point between 200° and 380°C may contain from 8 to 20% of n-alkanes.
Gas oil can be used directly as a substrate. Its use requires solvent extraction of the produced biomass. N-alkanes isolated by molecular sieve adsorption methods and with chain lengths from 10 to 23 carbons are most suitable. With shorter chain length compounds, microbial growth rates are too slow, and with longer chain length compounds the viscosity of the substrate is too high.
Figure 13.1 shows a schematic diagram of the range of substrates, microorganisms and end products. Extensive cost figures for fermentation substrates have been provided by Ratledge (1977). Specific uses of these substrates as well as experimental or unusual substrates.
Table 13.5 shows a comparison of the yield of biomass, the oxygen consumed, and the calories evolved for bacterial growth. It is apparent that hydrocarbon substrates which have a higher carbon content than carbohydrates give higher cell yields, require more oxygen per gram of cells grown, and evolve more heat.
Fermentation:
In contrast to the production of antibiotics and enzymes many processes for the production of microbial biomass do not require conditions of absolute sterility. However, the fast growth rates of microorganisms in biomass production impose stringent requirements for efficiency in the supply of oxygen and for the removal of heat evolved in the aerobic process.
Faust (1975) has calculated the cost of producing biomass on paraffin (n-alkanes) in a large plant and finds the following distribution of costs (as a percentage of total costs) – Raw material 49%; energy 17%; labor and overhead 8%; and depreciation 26%. Of the energy costs almost one half was required for cooling. The increasing cost of energy has caused a re-evaluation of the classical, agitated fermentor with its relatively high energy requirement for agitation and the concomitant need for additional heat removal.
The simplest form of a fermentor consists of a cylindrical vessel equipped with air sparger tubes at the bottom of the fermentor and internal or external cooling coils. One can induce directional flow of the fermentor liquid by the use of draft tubes. Such air lift fermentors (also called bubble column fermentors or loop fermentors) have been described by Lehman et al. (1975), Cooper et al. (1975), and Hatch (1975).
Some of the details of the effect of turbines on the rate of mixing and oxygen transfer by impellers have been worked out by McManamey et al. (1973). Moser and Lafferty (1975) have dealt with the theoretical problems of fermentor design, particularly with regard to the optimization of the oxygen transfer rate.
Nutritional Value of Microbial Biomass and SCP:
The principal value of microbial biomass as a component of feeds or foods is its contribution to protein nutrition. The protein content is highest in bacteria and lowest in the filamentous fungi. It is intermediate in yeasts and algae. For use in feeds the high Nucleic Acid (NA) content of microorganisms causes no problems since mammals, with the exception of man and some primates, convert uric acid to allantoin, which is very soluble and readily excreted in urine.
Man does not possess urate oxidase (uricase), and the ingestion of purine compounds leads to increased plasma levels of uric acid and may lead to metabolic disturbances, specifically gout. A figure of 2 g of NA per day per adult is used as the acceptable upper limit. This would permit the consumption of up to 10 g of yeast or bacterial protein (in the form of cell biomass) per day.
The quality of various proteins may be evaluated by feeding tests with experimental animals of which the rat is the most widely used species. The determination of the coefficient of digestibility, the biological value, the Net Protein Utilization (NPU), nitrogen balance studies, and the Protein Efficiency Ratio (PER) are widely used.
In the United States and Canada the PER method is accepted by regulatory agencies as the method of choice. It is relatively simple and gives fairly reproducible results if a reference protein, such as casein, is used as a standard of comparison. In general the method reflects the deficiency of the most limiting amino acid of a protein.
Unfortunately, the PER method is the least appropriate method for determining the contribution of a protein to a diet containing several animal and vegetable proteins. But this is exactly the proposed use of microbial protein in the human diet, and it is also its most likely practical application.
Experiments with mixed protein diets are costly and time consuming. One such noteworthy experiment is illustrated in Fig. 13.4. The quality of the protein is expressed as the minimum amount required for the maintenance of nitrogen balance in humans. Mixtures of egg protein with wheat, corn, potato, or algal proteins were used. A mixture of 60% egg protein and 40% algal protein was optimal and nitrogen balance was maintained at levels of less than 0.5 g N per kg body weight in humans.
The potential nutritional value of a protein can be estimated from the percentages of essential amino acids. This is indeed no more than an estimate because digestibility and specific amino acid imbalances affect the nutritional value. Table 13.7 shows the amino acid composition of commercially available biomass preparations.
Extensive data on the amino acid composition of microcial biomass are available in the following reviews:
Such compilations of individual data are not shown here because the amino acid composition of biomass preparations varies with the strain and the conditions of growth, and data obtained with a single species often show greater variation than between species. For instance, Wolf et al. (1975) emphasize the differences in amino acid composition of C. utilis grown by them on sulfite liquor and values given in the literature for the same yeast grown on the same substrate.
In general microbial proteins are rich in lysine and relatively poor in sulfur-containing amino acids. In this respect they resemble soybean proteins. But each preparation of microbial biomass or SCP must be analyzed separately to determine its amino acid composition.
The danger of generalizing from the literature is shown by the work of Erdman et al. (1977A), who determined the amino acid concentrations in the proteins of Lacto- bacillus acidophilus, L. bulgaricus, L. casei, L. fermenti, L. plantarum, and L. thermophilus. They found 3-fold variations in isoleucine content and a 2-fold variation in lysine content between the highest and the lowest percentages of these amino acids.
In assessing the nutritional value of proteins one must be aware of the distinction between whole cells and isolated SCP which differ somewhat in their amino acid composition. For SCP the method of extraction and recovery of the protein influences amino acid composition as Vananuvat and Kinsella (1975A.B) have demonstrated for Kluyveromyces fragilis.
Charatyan and Wolnowa (1975) have shown the differences in biological value between the biomass and the isolated SCP of various yeasts with Tetrahymena pyriformis as the test, organism. The yeasts were Candida guilliermondii grown on hydrocarbon, C. scottii grown on wood hydrolysate, Saccharomyces cerevisiae grown on a carbohydrate, and Mycoderma vini grown on alcohol.
Methionine is the limiting amino acid in most preparations of microbial biomass. Synthetic D, L-methionine is rather inexpensive and may be used to improve the quality of the protein. For instance, the PER of primary grown bakers’ yeast was 2.02. It could be increased to 2.27 with the addition of 0.16% of D,L-methionine and to 2.77 with the addition of 0.50% of D,L-methionine based on the weight of the yeast solids. Yeast protein was added to the diet of rats based on true protein, not on “crude” protein. Seely (1975) reports a PER of 2.2 for a protein fraction from bakers’ yeast essentially free from nucleic acids.
Inactive dried brewers’ or bakers’ yeasts are widely used as dietary supplements. They contribute some protein and vitamins (except for vitamin C, vitamin B12, and fat soluble vitamins) as well as trace minerals. There are frequent allusions to unidentified, nutritional factors of yeast in the trade literature.
These should not be dismissed lightly because some of these factors have actually been identified. For instance, the significance of selenium for animal and human nutrition was first discovered in experiments with brewers’ yeast and C. utilis yeast.
Also, yeast is currently the best source of the glucose tolerance factor, a chromium-containing organic compound, which mediates the action of insulin. The factor is essential for the aged who have lost the ability to synthesize the glucose tolerance factor from inorganic chromium of the diet.
Robbins and Seeley (1977) showed that the isolated, comminuted cell walls of bakers’ yeast had a strong effect in reducing cholesterol levels in rats fed a hypercholesterolemic diet. The effect appeared to be larger than that of other non-digestible polymeric carbohydrates which can serve as sources of fiber.
Reduction of the Nucleic Acid Content of Microbial Biomass:
There is no need for the removal of nucleic acid (NA) from biomass for use in feeds. However, for use in food and with the purpose of supplying a major portion of the required protein from biomass, the presence of NA presents a serious obstacle. This is expressed well in a blunt statement by Viikari and Linko (1977) – “As long as safe processes for the removal of nucleic acids continue to be economically prohibitive, the nucleic acid content constitutes a universal and major limitation to the use of SCP as human food.”
This accounts for the voluminous literature which deals with this important question. It should be kept in mind that the high costs to which these authors refer are due to the poor yield of isolated protein and rarely to the specific treatment for NA removal. As a matter of fact all methods for isolating protein from microbial biomass suffer from the same high costs due to poor yields whether they are coupled with a specific attempt to reduce NA or not. [A recently announced process for treating the dried cell mass with anhydrous NH3 and methanol may well present a more promising approach.]
The NA content of microbial biomass shows considerable variation, not only in its relation to total cell mass but also in relation to its protein content. For instance, Sinskey and Tannenbaum (1975) demonstrated the great effect of growth rate on NA concentration.
Aerobacter aerogenes grown at a dilution rate of 0.25/hr had a protein to NA ratio of about 5 to 7, while at a dilution rate of 0.7/hr, the ratio of protein to NA was about 3. Apart from such variations the problems caused by high NA concentrations are similar for fungi, algae, yeasts, and bacteria.
Bacteria, which have the highest protein content, also have the highest concentration of NA. In general about 15 to 20% of the total nitrogen of biomass preparations is NA nitrogen. The content of other nitrogenous fractions is usually quite small – although there are notable exceptions. For instance, Fusarium graminearum contains about 73% of its nitrogen in the protein and amino acid fraction, 15% in the NA fraction, and 10% as n-acetyl glucosamine.
The nitrogen content of the free amino acid pool accounted for 7% of the total nitrogen. It must be added that almost all authors include free amino acid nitrogen in the protein fraction when dealing with microbial biomass. For SCP preparations which have undergone precipitation this is, of course, not the case.
Several processes have been developed to reduce the NA content of biomass preparations. These can be divided into processes which aim at the production of biomass with a low NA content and those which aim at the production of isolated SCP. Sinskey and Tannenbaum (1975), Hedenskog and Mogren (1973), Kihlberg (1972), and others have reviewed the various methods which have been proposed.
For the treatment of whole cells, heat shock or alkaline extraction or a combination of both methods seems to be the simplest procedure. For instance, Viikari and Linko (1977) achieved a reduction of NA from 9 to 3% by a 30 min treatment of cells of Paecilomyces varioti with 0.125 N NaOH at 50°C.
The use of heat shock is described in a recent patent i.e. Imp. Chem. Ind. 1977 in which cells of Pseudomonas methylotropha are heat shocked at a pH below 5 and at a temperature exceeding 60°C followed by raising of the pH to between 6 and 10. Similar results have been reported for Fusarium graminearum. Trevelyan (1976) obtained almost complete removal of NA by extraction with 5% NaCl solution at 120°C.
Toxicity and Other Safety Aspects:
The most important aspect of the safety of microbial biomass preparations for humans is the presence of NA. It limits the daily consumption to about 10 g of microbial protein (in the form of whole cell preparations). A reduction of the NA content is technically feasible but costly, and isolated microbial proteins (SCP) have not appeared on the market in commercial quantities.
A major part of the presently available microbial biomass is produced with S. cerevisiae grown on ale wort or molasses, S. uvarum grown on beer wort, Candida utilis grown on sulfite liquor or molasses, and Kluyveromyces sp. grown on whey. These yeasts have been generally recognized as safe (GRAS) for human consumption.
On the other hand, processes for microbial biomass developed during the past 15 years have encountered vigorous opposition by portions of the public and by regulatory agencies in Western countries, an opposition which does not appear to be justified on the basis of the available evidence. This opposition has led to the abandonment of planned installations, for instance in Italy and Japan, and in the closing of some facilities.
At present the prospect for acceptance of petroleum grown yeast for human consumption is not good even if all the conditions of the guidelines of the Protein Advisory Group of the United Nations were to be fulfilled. However, extensive research on novel processes continues, and it is likely that such processes will result in the commercial production of microbial biomass for feeds.
Nucleic Acids:
High uric acid levels in human plasma cause gout. Sources of NA in diets are principally organ meats and secondarily beer and possibly other fermented beverages. Careful studies by Edozien et al. (1970) and Waslien et al. (1970) have established the relationship between the feeding of yeast and algae and the elevation of the plasma uric acid level in humans. On the basis of their work an upper limit of 2 g of NA per day has been generally accepted. Some caution is indicated for persons whose normal diet contains a considerable amount of organ meats (liver, lungs, brain, and spleen).
Polycyclic Aromatic Hydrocarbons:
The carcinogenic activity of some polycyclic hydrocarbons is well established. Typical carcinogens are benzo (a) pyrene and benzo (a) anthracene. Grimmer (1974) has determined the presence of 13 polycyclic hydrocarbons including benzo (a) pyrene and benzo (a) anthracene in bakers’ yeast from France, Britain, Germany, and the U.S.S.R., in various European food yeasts, and in yeasts grown on gas oil.
The yeasts grown on gas oil generally showed lower levels of polycyclic hydrocarbons than the yeasts currently used in the production of bread or as food supplements. Wolf et al. (1975) have reported on the concentration of various benzpyren compounds in food yeasts and algae in comparison with that in common vegetables.
Lysinoalanine:
Some proposed processes for the isolation of microbial protein make use of alkaline extraction as the first step. A nephrotoxic factor has been found in alkali treated soybean protein and identified as lysinoalanine. It appears that treatment of a protein at a pH above 10.5 at 25°C or treatment at a pH above 8 at 100°C leads to the formation of some lysinoalanine, but small quantities of lysinoalanine can be found in most heat treated protein foods.
If the problem has any practical significance it is of a general nature and not specifically a problem associated with the production of SCP, but it is obvious that some attention has to be paid in any process which employs alkaline extraction at a high temperature.
Other Factors:
Yeast autolysates may contain small concentrations of histamine and tyramine which result from the decarboxylation of the corresponding amino acids. Between 0.1 and 1.6 mg of tyramine and between 0.2 and 2.8 mg of histamine have been reported per g of yeast autolysate. The use of yeast autolysates as condiments limits the actual amount of histidine and tyramine consumed.
Microorganisms assimilate some heavy metals and other toxic materials from nutrient media. Payer et al. (1975) investigated the contamination of algae with toxicants derived from their environment. They concluded that the level of contaminants is smaller or within the range of that of other food products.
Human Feeding Studies:
Several reports in the literature indicate that feeding of microbial bio-mass to human’s results in discomfort to some individuals and occasionally in stronger gastrointestinal disturbances, desquamation of the skin, and other symptoms, particularly at high levels of feeding.
This literature has been reviewed by Scrimshaw (1975) and Calloway and Waslien (1971). Some of the reactions appear to have been the result of sensitization of individuals and suggest allergic reactions. Feldheim (1975) reviewed the results of several reports of mass feeding trials with humans (with species of algae) and found generally good acceptance.
It is important to note that reactions to microbial biomass from various species of organisms may vary as much as reactions to various species of edible plants. Hence, biomass preparations have to be judged on the basis of specific tests and on the basis of practical experience by inclusion in the human diet.