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Bioconversion of vegetable, animal, and industrial wastes by means of Fungi mycelia in an artificial rumen

Atanas Torev
V. Kolarov Higher institute of Agriculture, Plovdiv, Bulgaria

In nature there are several thousand different higher fungi species, but not all of them have mycelia whose characteristics are in optimum combinations. Many of these fungi cannot be subjected to submerged cultivation, do not assimilate a large range of substrates, synthesize only a small amount of protein, and have an unsatisfactory degree of protein assimilation and other limitations.

The V. Kolarov Higher Institute of Agriculture in Plovdiv, Bulgaria, has isolated strains of higher fungi mycelia that combine the most desirable biological, technological, and nutritive characteristics. A strain called PS-64 is especially promising. It was isolated in 1964, patented, and registered in the world collection of micro-organisms. PS-64 is available for license.

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Nature is regulated by biological, physical, and chemical laws that have contributed to the balance and harmony of the life of our planet. However, if we consider natural phenomena from a human viewpoint, we can also find some limitations that prevent our using the full capacity of nature to our advantage.

If we examine the atmosphere, we find that about 80 per cent of its content is nitrogen, but plants living and "bathing" in nitrogen do not assimilate even a small part of it from the air. Human beings have discovered, however, that atmospheric nitrogen can be converted into nitrates by soil bacteria found in the nodules of certain legumes.

Another of nature's limitations is that, of the three fundamental substrates used by human beings, animals, and micro-organisms (carbohydrates, fats and proteins), plants synthesize protein the least. For example, wheat, maize, and rice contain 55 to 70 per cent starch and only 8 to 12 per cent protein. The legumes alone are exceptions to this rule, containing as much as 40 per cent protein, as in the case of soya beans.

For optimal human and animal nutrition 10 to 20 per cent protein is required in the diet. Lower organisms, such as bacteria, yeasts, moulds, and fungi and their mycelia, contain from 40 to 80 per cent protein. Table 1 indicates the advantages of using lower organisms instead of higher ones for protein production. The amino-acid compositions of various protein sources are listed in table 2.

TABLE 1. Advantages of using lower rather than higher organisms for protein production

• Protein content of 40 to 80 per cent.
• Well-balanced amino-acid composition, except for sulphur-containing amino acids (methionine and cystine).
• Protein digestibility of up to 80 per cent or more.
• Rapid growth: the great majority of the lower organisms reproduce themselves from 1,000 to 5,000 times faster than plants and animals.
• Capacity for assimilating and processing a great range of substrates, mainly of carbohydrate origin,
• Ability to assimilate mineral forms of nitrogen and phosphorus.
• Easy to breed and to engineer genetically.

How can protein-producing lower organisms be used in animal or human diets? This problem is not a new one: industrial plants for the production of fodder yeasts have been functioning for many decades. Now bacterial and mycelial proteins are also industrially produced on various types of hydrocarbon substrates. These proteins are added to animal feed or are used as food additives in human nutrition.

The question arises as to whether these methods are the most suitable and efficient ways of using lower organisms for producing protein and for balancing its deficiencies in animal feeds and human foods. If we consider animal biology more carefully, especially that of ruminants, we see that nature suggests another possibility. In the peculiar stomach of ruminants, i.e. their rumen, there exists a specific microflora that grows extremely fast and accumulates a biomass rich in protein. When the micro-organisms die, they disintegrate in the animal's stomach and are used as a protein source.

TABLE 2. Amino-acid composition of various protein sources

a. FAO/WHO Ad Hoc Expert Committee. WHO Technical Report Series. no. 52 /FAO. Rome. 1974).

If some source of nitrogen, e.g. urea, is added to a ruminant's feed, the microflora uses the synthetic nitrogen to grow and accumulate a larger amount of biomass and protein. Thus, a peculiar type of self-enrichment of feeds occurs in the stomachs of some animals, leading to a balancing of the dietary protein: the micro-organisms are a rich protein source.

What can be done to enrich the diet of non-ruminant domestic animals, such as swine and poultry? At the present time their feed is protein-balanced by adding soya and sunflower meals, fodder yeasts, and fish and carcass meals, all of which are either in short supply or rather expensive. A fundamentally new solution to the problem of enrichment would be to create and use artificial rumens for producing self-enriched feeds.

An artificial rumen

Using the principles of natural rumens and other biological laws, we have constructed an apparatus that can be called an "artificial rumen."

The artificial rumen consists of two chambers:

1. A fermentator-mixer with a special device for shaking, sterilizing, cooling, and inoculating a medium with a specific strain.
2. A cultivation chamber in which the inoculated substrate enables the strain-producer to develop in a solid-phase fermentation process, and the carbohydrate substrate and the nitrogen source accumulate a definite amount of biomass and protein respectively.

TABLE 3. Desirable properties of some higher fungi mycelia when added to foodstuffs

1. Fungi are commonly used as foodstuffs for both human beings and animals and can be easily digested by them.
2. Mycelia are relatively easy to grow over a broad range of conditions, including temperature, pH, and aeration.
3. Mycelia can be produced on a great range of substrates, including many varieties of sugars, starch, hemi-cellulose, semi-destroyed cellulose, glycerine, and alcohol, used both independently and in their natural or artificial combinations.
4. The cellular membrane of the higher fungi mycelia is more easily broken down than that of bacteria and yeasts.
5. Protein assimilation in some mycelia is as high as 80 to 85 per cent.
6. The strains tend to be stable and easily kept in both solid and liquid media.
7. Fungi mycelia are less likely to cause adverse reactions in man, such as allergenicity or toxicity, than other single-cell protein sources.
8. The filamentous structure allows the product to be easily added to different types of foodstuffs.

We have out into the artificial rumen carbohydrate meals such as roughage of maize, barley, or a mixture of them in various ratios, as well as other kinds of cereals and wastes from the agricultural and food industries, including cornstalks, beet slices, pressed wastes of apple and other fruits, coffee pulps, molasses, alcoholic industry by-products, and pig and poultry excrement. Wastes can be used in different amounts and combinations depending on their availability in a region, the time of year, and the kind of animal for which the feed is intended. A nitrogen source is also included in the artificial rumen, after which the medium is moistened to some extent. Then it is sterilized to destroy unwanted microflora. The sterilized nutrient medium is cooled and then inoculated with a pure culture of fungus mycelium, whose major desirable characteristics in terms of protein production are listed in table 3. Technologies for the production and use of higher fungi mycelia have been intensively developed.

In the artificial rumen, parameters including temperature, aeration, pH, and pressure are maintained at specific levels. The fungus mycelium develops in the substrate and, using the carbohydrate and the nitrogen sources, it accumulates a definite amount of biomass, i.e. protein. Other valuable biological substances are also produced by the fungus mycelium.

Despite the similarities between the artificial and natural rumens, the artificial rumen differs in some essential aspects from the natural one. In the latter, the feed disintegration is more thorough: assimilation can reach as much as 80 per cent or even more, the remaining 20 per cent being lost as excrement. In the artificial rumen, the fungi mycelia partly disintegrate the carbohydrates in the substrate, assimilating only 30 to 40 per cent of the substrate; at the expense of the substrate and the nitrogen, the mycelia accumulate an amount of biomass, containing mainly protein plus the whole complex of biologically active substances present in the mycelia cells.

From the 30 to 40 per cent of the carbohydrate substrate they use, the mycelia accumulate about 15 to 20 per cent biomass, i.e. the coefficient of performance is 50 per cent. The net loss of organic matter in the artificial rumen is 15 to 20 per cent.

In a substrate containing 50 to 55 per cent crude protein and 40 to 45 per cent pure protein, a 15 to 20 per cent increase in mycelial biomass enriches the nutrient medium by approximately 10 to 12 per cent crude protein and 6 to 8 per cent pure protein (corrected for nucleic acid N). Crude protein was determined by the Kjeldah method as total nitrogen and multiplied by 6.25; pure protein was determined by the Bernstein method as nitrogen and multiplied by 6.25.

If the initial raw material is maize containing 8 to 10 per cent pure protein, from the self-enriched substrate a total of 18 to 20 per cent protein can be obtained, of which 14 to 16 per cent is pure protein.

The balanced fodder mixtures fed to animals contain from 14 to 20 per cent raw protein and from 12 to 16 per cent pure protein. By means of the artificial rumen we have obtained balanced fodder mixtures without adding any protein sources in contrast to the traditional practice

Advantages of the artificial rumen method

The artificial rumen method has many advantages over the classical methods of producing balanced fodder mixtures:

TABLE 4. Self-enriched fodder-protein mixtures obtained by means of an artificial rumen (percentages)

1. Protein self-enriched fodder mixtures can be obtained without the addition of soya bean, sunflower and cotton groats, fodder yeasts, fish and carcass meals, or other protein sources.
2. In the self-enriched fodder mixtures, in addition to proteins, other valuable substances are accumulated, such as vitamins, enzymes, and other physiologically active substances that help in the digestion and assimilation of animal feeds.
3. For the production of self-enriched fodder mixtures, not only concentrated feeds such as maize, barley, and wheat, but also many agricultural and industrial wastes can be used. High-protein feeds can be obtained exclusively from wastes and by-products, such as beet slices, leaves and stalks of sugar cane and maize, pressed fruit wastes, coffee pulp, pig and poultry manure, molasses, etc. Each country can find appropriate waste materials for use in the self-enrichment of fodder.
4. The artificial rumen creates the potential for producing

TABLE 5. Self-enriched fodder-protein mixtures obtained by means of an artificial rumen (percentages)

TABLE 6. Self-enriched fodder-protein mixtures obtained by means of an artificial rumen (percentages)

TABLE 7. Self-enriched fodder-protein mixtures obtained by means of an artificial rumen (percentages)

Some of the results obtained in the production of self-enriched fodder mixtures are given in tables 4 to 7. From these tables it can be seen that, for the production of protein-enriched fodder mixtures, various vegetable, animal and industrial wastes can be used. Depending on the character of these wastes and, above all, on their cellulose content, fodder mixtures containing from 14 to 25 per cent raw protein can be obtained by means of solid-phase fermentation.

In our experiments with white rats, we have fed them either with protein-balanced mixtures produced by an artificial rumen or with conventional meals, including soya bean groats, fodder yeasts, and fish and carcass meals. The rats fed rumen-enriched mixtures have shown weight gains similar to or, in some cases, even greater than those fed conventional mixtures (unpublished data).

Our economic estimates indicate that a ton of fodder mixture produced through an artificial rumen will cost about 15 to 20 per cent less than conventionally balanced fodder-protein mixtures, depending on the sources of the basic materials.

Conclusions

Protein enrichment can be achieved in many ways. Some of the methods have been in use for a number of years, and others have been developed but are not Yet in use. With the exception of those using soya beans and sunflower and cotton groats, all have two disadvantages: a raw material deficiency and a high initial cost. We think that using waste products from agriculture, animal husbandry, and the food industry, and transforming them into valuable protein-enriched feeds by means of the artificial rumen, can contribute effectively to meeting world protein needs.

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