Production of Single-Cell Protein and Mushrooms

Read this article to learn about the various methods of production of single-cell protein and mushrooms.

Single-Cell Protein (SCP) :

Single-cell protein (SCP) refers to the microbial cells or total protein extracted from pure microbial cell culture (monoculture) which can be used as protein supplement for humans or animals. The word SCP is considered to be appropriate, since most of the microorganisms grow as single or filamentous individuals. This is in contrast to complete multicellular plants and animals.

If the SCP is suitable for human consumption, it is considered as food grade. SCP is regarded as feed grade, when it is used as animal feed supplement, but not suitable for human consumption. Single-cell protein broadly refers to the microbial biomass or protein extract used as food or feed additive. Besides high protein content (about 60-80% of dry cell weight), SCP also contains fats, carbohydrates, nucleic acids, vitamins and minerals.

Another advantage with SCP is that it is rich in certain essential amino acids (lysine, methionine) which are usually limiting in most plant and animal foods. Thus, SCP is of high nutritional value for human or animal consumption.

It is estimated that about 25% of the world’s population currently suffers from hunger and malnutrition. Most of these people live in developing countries. Therefore, SCP deserves a serious consideration for its use as food or feed supplement. In addition to its utility as a nutritional supplement, SCP can also be used for the isolation of several compounds e.g. carbohydrates, fats, vitamins, minerals.

Advantages of Using Microorganisms for SCP Production:

The protein-producing capabilities of a 250 kg cow and 250 g of microorganisms are often compared. The cow can produce about 200 g protein per day. On the other hand, microorganisms, theoretically, when grown under ideal conditions, could produce about 20-25 tonnes of protein.

There are many advantages of using microorganisms for SCP production:

1. Microorganisms grow at a very rapid rate under optimal culture conditions. Some microbes double their mass in less than 30 minutes.

2. The quality and quantity of protein content in microorganisms is better compared to higher plants and animals.

3. A wide range of raw materials, which are otherwise wasted, can be fruitfully used for SCP production.

4. The culture conditions and the fermentation processes are very simple.

5. Microorganisms can be easily handled, and subjected to genetic manipulations.

Safety, Acceptability and Toxicology of SCP:

There are many non-technological factors that influence the production of SCP. These include the geographical, social, political and psychological factors. In many countries, there are social and psychological barriers to use microorganisms as food sources. It is desirable to first consider the safety, acceptability and toxicology of SCP, particularly when it is considered for human consumption. There are several limitations for the widespread use of SCP.

1. The nucleic acid content of microbial biomass is very high (4-6% in algae; 10-15% in bacteria; 5-10% in yeast). This is highly hazardous, since humans have a limited capacity to degrade nucleic acids.

2. The presence of carcinogenic and other toxic substances is often observed in association with SCP. These include the hydrocarbons, heavy metals, mycotoxins and some contaminants. The nature and production of these compounds depends on the raw materials, and the type of organism used.

3. There is a possibility of contamination of pathogenic microorganisms in the SCP.

4. The digestion of microbial cells is rather slow. This is frequently associated with indigestion and allergic reactions in individuals.

5. Food grade production of SCP is more expensive than some other sources of proteins e.g. soy meal. Of course, this mainly depends on the cost of raw materials. In general, SCP for human consumption is 10 times more expensive than SCP for animal feed.

For the above said reasons, many countries give low priority for the use of SCP for human consumption. In fact, mass production of SCP using costly raw materials has been discontinued in some countries e.g. Japan, Britain, Italy. However, these countries continue their efforts to produce SCP from cheap raw materials such as organic wastes.

Microorganisms and Substrates Used for Production of SCP:

Several microorganisms that include bacteria, yeasts, fungi, algae and actiomycetes utilizing a wide range of substrates are used for the production of SCP. A selected list is given in Table 29.1.

The selection of microorganisms for SCP production is based on several criteria. These include their nutritive value, non-pathogenic nature, production cost, raw materials used and growth pattern.

Substrates:

The nature of the raw materials supplying substrates is very crucial for SCP production. The cost of raw material significantly influences the final cost of SCP. The most commonly used raw materials may be grouped in the following categories.

1. High-energy sources e.g. alkanes, methane, methanol, ethanol, gas oil.

2. Waste products e.g. molasses, whey, sewage, animal manures, straw, bagasse.

3. Agricultural and forestry sources e.g. cellulose, lignin.

4. Carbon dioxide, the simplest carbon source.

Production of SCP from High Energy Sources:

There are a large number of energy-rich carbon compounds or their derivatives which serve as raw materials for SCP production. These include alkanes, methane, methanol, and ethanol and gas oil. Bacteria and yeasts are mostly employed for SCP production from high energy sources. Some scientists question the wisdom of using (rather misusing) high-energy compounds for the production of food, since they regard it as a wasteful exercise.

Production of SCP from alkanes:

Alkanes can be degraded by many yeasts, certain bacteria and fungi. The major limitation of alkanes is that they are not easily soluble, hence they cannot enter the cells rapidly. It is believed that the cells produce emulsifying substances which convert insoluble alkanes into small droplets (0.01-0.5 pm) that can enter the cells by passive diffusion.

It is observed that when cells are grown on a medium of alkanes enriched with lipids, the diffusion of alkanes into the cells is enhanced. Certain yeasts have been successfully used for producing SCP from alkanes e.g. Saccharomycopsis lipolytica, Candida tropicalis, Candida oleophila.

Petroleum products for SCP production:

Several oil companies have developed fermentation systems, employing petroleum products for large scale manufacture of SCP by yeasts. Two types of petroleum products are mainly used for this purpose.

1. Gas oil or diesel oil containing 10-25% of alkanes with carbon length C₁₅-C₃₀ (i.e. long chain alkanes).

2. Short chain alkanes with carbon length in the range of C₁₀-C₁₇, isolated from gas oil by use of molecular sieves.

Airlift bioreactor system with continuous operation was once used (in France and Britain) to produce SCP from gas oil employing the organism Saccharomycopsis lipolytica. But this is now discontinued for political reasons.

Degradation of alkanes:

Alkanes have to be first broken down to appropriate metabolites for their utilization to form SCP. The most important step in this direction is the introduction of oxygen into alkanes which can be brought out by two pathways-terminal oxidation and sub-terminal oxidation (Fig. 29.1).

In terminal oxidation, the terminal carbon gets oxidized to the corresponding monocarboxylic acid. The latter then undergoes β -oxidation to form acetic acid. In some microorganisms, the oxidation may occur at both the terminal carbon atoms (by a process referred to as co-oxidation) to form a dicarboxylic acid. This can be further broken down to acetate and succinate by β-oxidation. Terminal oxidation is the predominant pathway occurring in majority of yeasts and bacteria.

Sub-terminal oxidation involves the oxidation of interminal carbon atoms (any carbon other than terminal i.e. C₂, C₃, C₄, and so on). The corresponding ketone produced undergoes a-oxidation, decarboxylation, and finally β-oxidation to form acetate and propionate. The individual enzymes responsible for terminal oxidation or sub-terminal oxidation have not been fully identified.

Limitations of SCP production from alkanes:

The production of SCP from alkanes is a very complex biotechnological process and has been extensively studied. The major drawback of alkanes as substrates is the formation of carcinogens, along with SCP which are highly harmful. For this reason, many countries have discontinued alkane-based production of SCP.

Production of SCP from methane:

Methane is the chief constituent of natural gas in many regions. Although methane can be isolated in pure gas form, it cannot be liquefied. The handling and transportation of methane (an explosive gas) are very difficult and expensive. Certain bacteria that can utilize methane for SCP production have been identified e.g. Methylococcus capsulatus, Methylomonas methanica, Methylovibrio soehngenii. So far, yeasts that can utilize methane have not been identified.

The bacterial enzyme methane oxygenase oxidizes methane to methanol, which can be converted to formaldehyde and then to formic acid. Although methane was extensively researched for its use as a source of SCP, it is not widely used due to technical difficulties.

Production of SCP from methanol:

Methanol is a good substrate for producing SCP. Methanol as a carbon source for SCP has several advantages over alkanes and methane. Methanol is easily soluble in aqueous phase at all concentrations, and no residue of it remains in the harvested biomass. Technically, methanol can be easily handled. The sources for methanol are natural gas, coal, oil and methane.

Many species of bacteria (Methylobacter, Arthrobacter, Bacillus, Pseudomonas, Vibrio) yeasts (Candida biodinii, Hansenula sp, Torulopsis sp) and fungi (Trichoderma lignorum, Gliocladium delinquescens) are capable of producing SCP from methanol. Bacteria are mostly preferred because they require simple fermentation conditions, grow rapidly and possess high content of protein.

Oxidation of methanol:

Methanol gets oxidized to formaldehyde, then to formic acid and finally to carbon dioxide, as depicted in Fig. 29.2.

The products obtained from methanol have to form C₃ compounds (such as pyruvate) for final production of SCP. Carbon dioxide formed from methanol can be utilized by photosynthetic organisms for the formation of ribulose diphosphate. Alternately, formaldehyde may condense with ribulose 5-phosphate to form 3-keto 6-phosphohexulose which then gives fructose 6-phosphate and finally pyruvate. This pathway is referred to as ribulose monophosphate (or Quayle) cycle.

Formaldehyde can condense with glycine to form serine which in a series of reactions forms phosphoenol pyruvate. This is referred to as serine pathway.

Production process:

Imperial Chemical Industries (ICI), U.K. was the first company to develop a process for continuous methanol fermentation for large scale production of SCP. Later, Hoechst (Germany) and Mitsubishi (Japan) also developed similar fermentation systems.

ICI employed Methylophilus methylotrophus (formerly called Pseudomonas methylotrophus) for producing SCP from methanol. A bioreactor, referred to as ICI pressure cycle fermenter was used for this purpose (Fig. 29.3). This fermenter has three components-airlift column, down-flow tube and gas release space. The operation was carried out at temperature 35-37°C and pH 6.5-7.0. The cells were subjected to disruption by heat or acid treatment. The nutrient solution can be clarified by decanting.

ICI Pruteen:

The single-cell protein produced by ICI from methanol and ammonia using M. methylotrophus was referred to as ICI pruteen. This SCP was exclusively used for animal feeding. ICI invested a huge amount (around £40 million) in 1979 and installed a continuous culture system for SCP production. This was the world’s largest continuous airlift fermenter. Unfortunately, the plant could not be operated for long due to economic reasons.

For instance, in 1984 the cost of soy meal was around $125-200 per ton while ICI pruteen was sold at $600 per ton! This is mainly because of the high cost of methanol which represents approximately half of the production cost expenses. In the Middle East, due to high availability and low cost of methanol, the production of SCP appeared to be attractive. In the erstwhile Russia, there were several plants producing SCP from methanol which were later closed.

Genetic engineering for improved SCP production from methanol:

The efficiency of SCP production has been improved by using genetic engineering. The assimilation of ammonia by M. methylotrophus is an essential step for cellular growth. This organism lacks glutamate dehydrogenase. It possesses glutamine synthase and glutamine ketoglutarate transaminase to utilize ammonia for the formation of glutamate (Fig. 29.4A).

This is an energy (ATP) dependent reaction’. By employing recombinant DNA technology, the gene for the enzyme glutamate dehydrogenase from E. coli was cloned and expressed in M. methylotrophus. These genetically transformed organisms were more efficient in assimilating ammonia. They could grow rapidly and convert more methanol to SCP. However, the overall increase in the production of SCP did not exceed 10%.

Production of SCP from ethanol:

Ethanol is a good substrate for the production of SCP for human consumption (feed grade SCP). However, this process, as such, is not economically feasible. However, several factors-local raw materials, innovative fermentation technology, political decisions and foreign trade balances influence production of SCP. It may not be surprising if large scale production of SCP commences, on one day, from ethanol for a variety of reasons.

Production of SCP from Wastes:

There are several materials that serve no useful purpose and they are collectively referred to as wastes e.g. molasses, whey, animal manures, sewage, straw, date wastes. These waste products, formed in various industries and other biological processes, largely contribute to environmental pollution. There are several advantages of utilizing wastes for the production of SCP.

These include the conversion of low-cost organic wastes to useful products, and reduction in environmental pollution. However, there has been very limited success for the large scale production of SCP from wastes. This is mainly because of transportation cost and technical difficulties. The technology adopted and the organism employed for SCP production depends on the waste being used as the substrate. Thus, Saccharomyces cerevisiae is used for molasses, Kluyveromyces fragile for cheese whey.

Symba process:

Symba process is a novel technology developed in Sweden to produce SCP by utilizing starchy wastes by employing two yeasts, Endomycopsis fibuligira and Candida utilis. The Symba process is carried out in three phases.

Phase I:

The waste material containing starch is sterilized by passing through a heat exchanger.

Phase II:

The sterilized material is passed through two bioreactors. The first reactor contains E. fibuligira which hydrolyses starch. When this hydrolysate is passed to the second bioreactor, the organism, C. utilis grows to form biomass.

Phase III:

The microbial biomass can be separated by centrifugation. The samples of SCP can be dried, packaged and stored.

Applications of Symba product:

The yeast biomass produced in Symba process is of good nutritive value. It is widely used as an animal feed for pigs, calves and chicken. The animals grow quite well and no adverse effects have been reported.

Pekilo — a fungal protein rich product:

A filamentous fungus, Paecilomyces variotii, with good fibrous structure was used for the production of Pekilo. This protein, rich in fungal biomass, was produced by fermentation of wastes such as molasses, whey, sulfite liquor and agricultural wastes. It can be produced by a continuous fermentation process. Pekilo is rich in proteins (containing essential amino acids), vitamins and minerals.

It was used as an animal feed in supplementing the diets of calves, pigs, chickens and hens without any adverse effects. It is unfortunate that the production of Pekilo has been discontinued at most places due to economic and commercial considerations.

Quorn-the mycoprotein for humans:

The protein Quorn is the mycoprotein produced by the fungus Fusarium graminearum. Many companies in the developed countries are engaged in the production of fungal proteins for human consumption. Quorn is the trade name for Fusarium mycoprotein produced in Britain by Marlow Foods (ICI in association with Bank-Hovis-McDougall).

The fungus Fusarium can be grown continuously on simple carbohydrate sources (like glucose). Ammonium ions supply nitrogen. Mineral salts and vitamins are also added. The fermentation is carried out at pH 6.0 and temperature 30°C. At the end of fermentation, the culture is heated to 65°C to activate RNases. This is necessary to degrade RNA and reduce the content from 10% to around 1%.

The breakdown products of RNA namely the nucleotides diffuse out from the cells and can be easily removed. (Reduction in RNA content is desirable to make the product acceptable for human consumption. This is because humans have a very limited capacity to digest nucleic acids). It is possible to produce 1 kg of fungal biomass with a protein content of about 135 g from 1 kg glucose utilized in the culture medium.

The dried Fusarium product is artificially flavoured and marketed in pieces that resemble beef, pork and chicken. The nutritional composition of mycoprotein when compared to beef is given in Table 29.2. Besides being rich in essential nutrients, mycoprotein has a good content of dietary fiber. There are several advantages of fiber consumption- prevents constipation, decreases intestinal cancers, improves glucose tolerance and reduces serum cholesterol.

Production of SCP from Wood:

The natural waste wood sources containing cellulose, hemicellulose and lignin are attractive natural sources for the production of SCP. It is however, essential to breakdown these cellulosic compounds into fermentable sugars. For this purpose, extracellular celluloses can be used. Certain bacteria (Cellulomonas sp) and fungi (Trichoderma sp, Penicillium sp) are good sources for celluloses.

Techniques for the production of celluloses have been well standardized from several organisms. The cost of production of celluloses is a critical factor in determining the ultimate production cost of SCP. In some instances, the cellulosic materials can be directly used for biomass production. The resultant SCP is used as animal feed.

Production of SCP from CO₂:

Certain algae grown in open ponds require only CO₂ as the carbon source. In the presence of sunlight, they can effectively carry out photosynthesis, and produce SCP. The examples of these algae are Chlorella sp, Senedesmus sp and Spirulina sp. Chlorella is used as a protein and vitamin supplement for enriching ice-creams, breads and yoghurts in some countries. In some parts of the world, the algae in ponds are used for the removal of organic pollutants. The resultant algae biomass can be harvested, dried and powdered. Algae SCP are very useful as animal supplements.

Nutritive value of Spirulina SCP:

Traditionally Spirulina sp have been eaten by people in some parts of Africa and Mexico. SCP of Spirulina is of high nutritive value (protein-65%, carbohydrate- 20%, fat-4%, fibre-3%, chlorophyll-5%, ash-3%). Spirulina is a good source of protein for human consumption, particularly in developing countries.

Production of SCP from Sewage:

Domestic sewage is normally used for large scale production of methane, which in turn may be utilized for the production of SCP. The sewage obtained from industrial wastes in cellulose processing, starch production and food processing can be utilized for the production of SCP.

The organism Candida utilis is used to produce SCP by using effluent formed during the course of paper manufacture. Other microorganisms namely Candida tropicalis, Paecilomyces varioti are employed to use sulfite waste liquor for the production of SCP.

Genetically Engineered Artificial Protein As Animal Feed:

Rumen bacteria can synthesize amino acids. Some workers have developed genetically engineered strains of rumen bacteria that can produce a protein rich in methionine, threonine, lysine and leucine. This artificial protein has a total of 100 amino acids, of which 57 are essential.

The gene for artificial protein was synthesized by 14 overlapping oligonucleotides held to maltose binding protein gene. This gene was expressed in E. coli under the transcriptional control of tac promoter. The production of this artificial protein accounts to around 12% of the intracellular proteins. However, the large scale production of artificial protein by rumen bacteria is yet to be clearly established and commercialized.

Mushrooms:

Mushrooms are fungi belonging to the classes basidiomycetes (Agaricus sp, Auricularia sp, Tremella sp) and ascomycetes (Morchella sp, Tuber sp). Majority of edible mushrooms are the species of basidomyces. It is estimated that there are around 4,000 species of basidiomyces. Of these, around 200 are edible, and a dozen of them are cultivated on large scale. Some of the most important edible mushrooms, their common names and the substrates used are given in Table 29.3.

The cultivation of edible mushrooms is one of the rare examples of a microbial culture wherein the cultivated macroscopic organism itself is directly used as human food. Mushroom growing is one of the fastest developing biotechnological industries world over. Further growth of mushroom industry is expected for the production of enzymes, and pharmaceutical compounds, including antitumor agents and antibiotics.

Poisonous mushrooms:

There are certain poisonous mushrooms also. They usually possess unpleasant taste and odour. These mushrooms produce some poisonous substances like phallin and muscarine. The examples of poisonous mushrooms are Amanita phalloides, A. muscaria, A. viraosa, Lepiota morgani and Boletus satanas.

Nutritive value of edible mushrooms:

Some people regard edible mushrooms as vegetable meat. Mushrooms contain 80-90% water, depending on the growth conditions (temperature, humidity). Edible mushrooms are rich sources of protein (35-45% of dry weight). However, all these proteins are not easily digestible by humans. Mushrooms also contain fats and free fatty acids (7-10%), carbohydrates (5-15%) and minerals in good concentration. Certain undesirable substances may also be present in edible mushrooms e.g. cadmium, chromium.

Many delicious recipes of edible mushrooms can be prepared. This actually depends on the dietary habits of the people. Some of the common recipes are mushroom soup, mushroom paneer, mushroom pulao, and mushroom omelets.

Advantages of edible mushroom biotechnology:

1. Mushrooms can be produced by utilizing cheap and often waste substrates (industrial and wood wastes).

2. They are of high nutritive value being rich in proteins, vitamins and minerals.

3. Many delicious recipes can be prepared from mushrooms.

4. Due to low carbohydrate content, consumption of mushrooms is advocated to diabetic patients.

Production of Edible Mushrooms:

Mushroom production is basically a fermentation process. This is mostly carried out by solid-substrate fermentation. A wide range of substrates (straw, saw dust, compost, wooden logs) depending the organism can be used (Refer Table 29.3). Mushroom production is a good example of a low technology utilization in an otherwise sophisticated modern biotechnology.

The most common edible mushroom cultivated world over (that may constitute about 20% world mushroom produce) is the white button mushroom, Agaricus bisporus. Lentinula edodes is the second most cultivated mushroom in the world. The substrates straw, compost or horse manure can be used. The substrate selection depends on the local factors.

A schematic representation of mushroom production is depicted in Fig. 29.5. The compost with desired formulation is prepared and sterilized. It is spread into the trays which are then transferred to production room and inoculated with spawn. Spawn is the term used for the mushroom inoculum containing spores and/or small pieces of fruiting body.

After inoculation (spawning), the culture is maintained at optimal growth conditions. The trays are regularly watered to maintain 70-80% humidity. The ideal temperature is about 15°C, and pH about 7.0. It takes about 7-10 days for each crop of mushroom production. It is possible to have 3-4 crops, before terminating the production process. The mushrooms can be harvested and marketed.

Mushrooms have a very short life 8-12 hours, unless stored at low temperature (refrigerator 2-5°C). Therefore, they should be immediately consumed, stored or canned. Variations in culturing mushrooms: The production of mushrooms is highly variable and mostly depends on the organism and the substrate used, besides several other local factors. There are distinct differences in the mushroom cultivation methods between different countries. For instance, garden and field cultivation methods are used in Europe, while in USA, cave and house cultivation techniques are employed.

Some mushrooms (e.g. Volirariella sp) are suitable for cultivation in summer and rainy reason while others grow well in winter (Agaricus bisporus, Pleurotus sp). It is however, possible to grow these mushrooms any time in a year with appropriate temperature and humidity control arrangements.