FOOD AND INDUSTRIAL MICROBIOLOGY

Module 9. Microbial process strategies

Lesson 27
SINGLE CELL PROTEIN AND INDUSTRIAL ALCOHOL

27.1 Introduction

Microbial primary metabolites used in the food and feed industries include: alcohols (ethanol), amino acids (monosodium glutamate, lysine, threonine, phenylalanine, and tryptophan), flavor nucleotides, organic acids (acetic, propionic, succinic, fumaric, and lactic), polyols (glycerol, mannitol, erythritol, and xylitol), polysaccharides (xanthan and gelan), sugars (fructose, ribose, and sorbose), and vitamins (riboflavin, cyanocobalamin, and biotin). Over the last three decades, traditional industrial microbiology, using the tools of molecular biology, has led to the development of recombinant organisms aimed at production of high value biopharmaceutical products such as erythropoietin, human growth hormones and interferons. The major microbial hosts for the production of recombinant proteins are E. coli, B. subtilis, S. cerevisiae, Pichia pastoris, Hansenula polymorpha and Aspergillus niger.

27.2 Single Cell Protein

In the 1950s and 1960s concern grew about the ‘food gap’ between the industrialized and the less industrialized parts of the world. As a result of this concern, alternate and unconventional sources of food were sought. It was recognized that protein malnutrition is usually far more severe than that of other foods. The hope was that microorganisms would help meet this world protein deficiency.

However, the limitations of conventional sources of proteins were recognized. These include: (a) possible crop failure due to unfavorable climatic conditions in the case of plants; (b) the need to allow a time lapse for the replenishment of stock in the case of fish; (c) the limited land available for farming in the case of plant production.

On the other hand the production of Single cell protein (SCP) has a number of attractive features: (a) it was not subject to the vicissitudes of the weather and can be produced every minute of the year. (b) Microorganisms have a much more rapid growth than plants or animals.

SCP is itself not entirely lacking in disadvantages. One of the most obvious is that many developing countries, where protein malnutrition actually exists, lack the expertise and/or the financial resources to develop the highly capital intensive fermentation industries involved. But this short-coming can be bridged by the use of improvised fermentors and recovery methods which do not require sophisticated equipments. Other criticisms of SCP are that microorganisms contain high levels of RNA and that its consumption could lead to uric acid accumulation, kidney stone formation and gout.

Organisms to be used in SCP production should have the following properties:

(a) Absence of pathogenicity and toxicity: It is obvious that the large-scale cultivation of organisms which are pathogenic to animals or plants could pose a great threat to health and therefore, should be avoided. The organisms should also not contain or produce toxic or carcinogenic materials.

(b) Protein quality and content: The amount of protein in the organisms should not only be high but should contain as much as possible of the amino acids required by man.

(c) Digestibility and organoleptic qualities: The organism should not only be digestible, but it should possess acceptable taste and aroma.

(d) Growth rate: It must grow rapidly in a cheap, easily available medium.

(e) Adaptability to unusual environmental conditions.

In order to eliminate contaminants and hence reduce the cost of production, environmental conditions which are antagonistic to possible contaminants are often advantageous. Thus, strains which grow at low pH conditions or at high temperature are often chosen. The heterotrophic microorganisms currently used are bacteria (and actinomycetes and fungi (moulds and yeasts); protozoa have not been used in SCP production. Of the substrates currently in use, the gaseous hydrocarbons (methane, propane, butane) are almost exclusively attacked by bacteria. Liquid hydrocarbons (n-paraffins, gas oil, diesel oil) and alcohols are utilized by both bacteria and yeasts. Fungi have the advantage that they are lower in RNA content and are easily harvested.

Table 27.1 Organisms and substrates which have been used for single cell protein production

27.2.1 Nucleic acids and their removal from SCP

Apart from the fears of carcinogenicity and toxicity from petroleum derivatives mentioned above, both of which fears have been allayed in extensive studies, another area of concern in SCP feeding is the consumption of high levels of nucleic acid. Man has lost the enzyme uricase which oxidizes uric acid to the soluble and excretable allantoin. When nucleic acid is eaten by man, it is broken up by nucleases present in the pancreatic juice, and converted into nucleosides by intestinal juices before absorption. Guanine and adenine are converted to uric acid, which as had been pointed out earlier cannot be converted to the soluble and excretable allantoin. As a result when foods rich in nucleic acid are consumed in large amounts, an unusually high level of uric acid occurs in the blood plasma. Owing to the low solubility of uric acid, uricates may be deposited in various tissues in the body including the kidneys and the joints when the diseases known as kidney stones and gout may respectively result.

27.2.2 Various ways have been devised for the removal of nucleic acids from SCP

(a) Growth and cell physiology method: The RNA content of cell is dependent on growth rate: the higher the dilution rate (in continuous cultures) the higher the RNA/ protein ratio. In other words the higher the growth rate the higher the RNA content. The growth rate is therefore reduced as a means of reducing nucleic acid. It must however be borne in mind that high growth is one of the requirements of reducing costs in SCP, hence the method may have only limited usefulness.

(b) Extraction with chemicals: Dilute bases such as NaOH or KOH will hydrolyze RNA easily. Hot 10% sodium chloride may also be used to extract RNA. The cells usually have to be disrupted before using these methods. In some cases the protein may then be extracted, purified and concentrated. Use of pancreatic juice: RNAase from bovine pancreatic juice, which is heat-stable, has been used to hydrolyze yeast RNA at 80°C at which temperature the cells are more permeable.

(c) Activation of endogenous RNA: The RNAase of the organism itself may be activated by heat-shock or by chemicals. The RNA content of yeasts have been reduced in this way.

27.2.3 The fermentation process

The fermentation process requires a pure culture of the chosen organism that is in the correct physiological state, sterilization of the growth medium which is used for the organism, a production fermenter which is the equipment used for drawing the culture medium in the steady state, cell separation, collection of cell free supernatant, product purification and effluent treatment.

Fermenters can vary in size from laboratory experimental models of one or two litres capacity, to industrial models of several hundred litres capacity. The most commonly used principle has been the chemostat: a perfectly mixed suspension of biomass into which medium is fed at a constant rate and the culture is harvested at the same rate so that the culture volume remains constant. Among the various designs which have been put to effect, air-lift has enjoyed the greatest success as the configuration of choice for continuous SCP production. The control of key process variables is a critical element of SCP production, from oxygen transfer, substrate and product concentration, to the appearance of minimal amounts of toxic compounds through undesired metabolic processes, which may compromise the quality of the final product. The biomass from yeast fermentation processes is harvested normally by continuous centrifugation. Filamentous fungi are harvested by filtration. The biomass is then treated for RNA reduction and dried in steam drums of spray driers. Drying is expensive, but results in stabilized product with shelf lives of years. Generally, under combined conditions of low water activity and presence of intractable solid substrate, fungi show luxuriant growth. Hence, proper growth of fungi in Solid state fermentation gives much higher concentration of the biomass and higher yield when compared to submerged fermentation .

The advantage in SSF process is the unique possibility of efficient utilization of waste as the substrate to produce commercially viable products. The process does not need elaborate prearrangements for media preparation. The process of SSF initially concentrated on enzyme production. But presently, there is worldwide interest for (SCP) production due to the dwindling conventional food resources.

27.3 Industrial Alcohol

Ethyl alcohol, CH₃CH₂OH (synonyms: ethanol, methyl carbinol, grain alcohol, molasses alcohol, grain neutral spirits, cologne spirit, wine spirit), is a colorless, neutral, mobile flammable liquid. It is rarely found in nature, being found only in the unripe seeds of Heracleum giganteun and H. Spondylium.

27.3.1 Uses of ethanol

(i) Use as a chemical feed stock: In the chemical industry, ethanol is an intermediate in many chemical processes because of its great reactivity as shown above. It is thus a very important chemical feed stock.

(ii) Solvent use: Ethanol is widely used in industry as a solvent for dyes, oils, waxes, explosives, cosmetics etc.

(iii) General utility: Alcohol is used as a disinfectant in hospitals, for cleaning and lighting in the home, and in the laboratory second only to water as a solvent.

(iv) Fuel: Ethanol is mixed with petrol or gasoline up to 10% and known as gasohol and used in automobiles.

27.3.2 Manufacture of ethanol

Ethanol may be produced by either synthetic chemical method or by fermentation. Due to the increase in price of crude petroleum, the source of ethylene used for alcohol production, attention has turned worldwide to the production of alcohol by fermentation. Fermentation alcohol has the potential to replace two important needs currently satisfied by petroleum, namely the provision of fuel and that of feedstock in the chemical industry.

The production of gasohol (gasoline – alcohol blend) appears to have received more attention than alcohol use as a feed stock.

27.3.3 Substrates

a). Fermentable substrates

Following are the types of substrates used for alcohol production:

Sugary Materials: Examples of sugary materials are sugarcane and its by products/wastes (molasses, bagasse) and sugar beet, tapioca, sweet potatoes, fruit juice, sweet sorghum, etc. Sugar cane molasses is largely being used in many countries for alcohol production.

Starchy materials: Starchy materials used in ethanol production are tapioca, maize, wheat, barley, oat, sorghum, rice and potatoes. But tapioca and corns are the two major substrates of the interest. It has been estimated that 11.7 kg of corn starch can be converted into about 7 liters of ethanol.

Lignocellulosic materials: The sources of cellulosic and lignocellulosic materials are the agricultural wastes and wood. However, yield of ethanol from lignocellulose is low because of lack of suitable technology and failure of conversion of pentoses into ethanol. On the basis of technology available today about 409 liters of ethanol can be produced from one tonne of lignocelluloses. Production of ethanol from lignocelluloses follows the following steps: (i) hydrolysis, (ii) fermentation, and (iii) recovery.

27.3.4 Fermentation

Alcohol-resistant yeasts, strains of Saccharomyces cerevisiae are used, and nutrients such as nitrogen and phosphate lacking in the broth are added.

a). Distillation

After fermentation the fermented liquor or ‘beer’ contains alcohol as well as low boiling point volatile compounds such as acetaldeydes, esters and the higher boiling, fuel oils. The alcohol is obtained by several operations. First, steam is passed through the beer which is said to be steam-stripped. The result is a dilute alcohol solution which still contains part of the undesirable volatile compounds. Secondly, the dilute alcohol solution is passed into the center of a multi-plate aldehyde column in which the following fractions are separated: esters and aldehydes, fusel oil, water, and an ethanol solution containing about 25% ethanol. Thirdly, the dilute alcohol solution is passed into a rectifying column where a constant boiling mixture, an azeotrope, distils off at 95.6% alcohol concentration.

To obtain 200°C proof alcohol, such as is used in gasohol blending, the 96.58% alcohol is obtained by azeotropic distillation. The principle of this method is to add an organic solvent which will form a ternary (three-membered) azeotrope with most of the water, but

with only a small proportion of the alcohol. Benzene, carbon tetrachloride, chloroform, and cyclohezane may be used, but in practice, benzene is used. Azeotropes usually have lower boiling point than their individual components and that of benzene-ethanol-water is 64.6°C. On condensation, it separates into two layers. The upper layer, which has about 84% of the condensate, has the following percentage composition: benzene 85%, ethanol 18%, water 1%. The heavier, lower portion, constituting 16% of the condensate, has the following composition: benzene 11%, ethanol 53%, and water 36%. In practice, the condensate is not allowed to separate out, but the arrangement of plates within the columns enable separation of the alcohol. Four columns are usually used. The first and second columns remove aldehydes and fusel oils, respectively, while the last two towers are for the concentration of the alcohol.

A flow diagram of conventional absolute alcohol production from molasses is given in the Figure 27.1.

Fig. 27.1 Flow diagram of alcohol production from molasses (Okofer, 2007)

27.3.5 Developments in alcohol production

Due to the current interest in the potential of ethanol as a fuel and a chemical feedstock, research aimed at improving the conventional method of production has been undertaken, and more will, most certainly, be undertaken. Some of the techniques aimed at improving productivity are the following:

(i) Developments of new strains of yeast of Saccharomyces uvarum able to ferment sugar rapidly, to tolerate high alcohol concentrations, flocculate rapidly, and whose regulatory system permits it to produce alcohol during growth.

(ii) The use of continuous fermentation with recycle using the rapidly flocculating yeasts.

(iii) Continuous vacuum fermentation in which alcohol is continuously evaporated under low pressure from the fermentation broth.

(iv) The use of immobilized Saccharomyces cerevisiae in a packed column, instead of in a conventional stirred tank fermentor. Higher productivity consequent on a higher cell concentration was said to be the advantage.

(v) In the ‘Ex-ferm’ process sugar cane chips are fermented directly with a yeast without first expressing the cane juice. The chips may be dried and used in the off season period of cane production. It is claimed that there is no need to add nutrients as would be the case with molasses, since these are derived from the cane itself.

(vi) The use of Zymomonas mobilis, a Gram-negative bacterium which is found in some tropical alcoholic beverages, rather than yeast is advocated. The advantages claimed for the use of Zymomonas are the following:

(a) Higher specific rates of glucose uptake and ethanol production than reported for yeasts.

(b) Higher ethanol yields and lower biomass than with yeasts.

(c) Ethanol tolerance is at least as high or even higher than with yeast.

(d) Tolerates high glucose concentration

(e) Grows anaerobically and, unlike yeasts, does not require the controlled addition of oxygen for viability at the high cell concentrations used in cell recycle.

(f) The many techniques for genetic engineering already worked out in bacteria can be easily applied to Zymomonas spp. for greater productivity