Hydrocarbons And Their Derivatives

There has been considerable interest in hydrocarbons. Development work has been done using n-al-kanes for production of organic acids, amino acids, vitamins and co-factors, nucleic acids, antibiotics, enzymes and proteins (Fukui and Tanaka, 1980). Methane, methanol and n-alkanes have all been used as substrates for biomass production (Hamer, 1979; Levi et al., 1979; Drozd, 1987; Sharp. 1989).

Drozd (1987) discussed the advantages and disadvantages of hydrocarbons and their derivatives as fermentation substrates, particularly with reference to cost, process aspects and purity. In processes where the feedstock costs are an appreciable fraction of the total manufacturing cost, cheap carbon sources are important. In the 1960s and early 1970s there was an incentive to consider using oil or natural gas derivatives as carbon substrates as costs were low and sugar prices were high. On a weight basis n-alkanes have approximately twice the carbon and three times the energy content of the same weight of sugar. Although petroleum-type products are initially impure they can be refined to obtain very pure products in bulk quantities which would would reduce the amount of effluent treatment and downstream processing. At this time the view was also held that hydrocarbons would not be subject to the same fluctuations in cost as agriculturally derived feedstocks because it would be a stable priced commodity and might be used to provide a substrate

Table 4.7. Partial analysis of corn-steep liquor (Belik et a!., 1957. Misecka and Zelinka, 1959; Rhodes and Fletcher, 1966)

Total solids 51%w/v

Acidity as lactic acid 15%w/v

Free reducing sugars 5.6%w/v

Free reducing sugars after hydrolysis 6.8%w/y

Total nitrogen 4%w/v Amino acids as % of nitrogen

Alanine 25

Arginine g

Glutamic acid 8

Leucine 6

Proline 5

Isoleucine 3.5

Threonine 3.5

Valine 35

Phenylalanine 2.0

Methionine 1.0

Cystine 1,0

Potassium 20%

Phosphorus 1-5%

Magnesium 0.003-0.3%

Silver I 0.001-0.003% Chromium )

B Vitamins

Aneurine 41-49 /tg g-1

Calcium pantothenate 14.5-21.5 /igg-1

Nicotinamide 30-40 /ig g-1

Riboflavine 3.9-4.7 /ig g 1

Also niacin and pyridoxine for conversion to microbial protein (SCP) for economic animal and/or human consumption. Sharp (1989) gives a very good account of market considerations of changes in price and how this would affect the price of SCP. The SCP would have had to have been cheaper or as cheap as soya meal to be marketed as an animal feed supplement. It is evident that both ICI pic and Shell pic made very careful assessments of likely future prices of soya meal during process evaluation.

SCP processes were developed by BP pic (Toprina from yeast grown on n-alkanes), ICI pic (Pruteen from bacteria grown on methanol), Hoechst / UBHE (Probion from bacteria on methanol) and Shell pic (bacteria on methane). Only BP pic and ICI pic eventually developed SCP at a production scale as an animal feed supplement (Sharp, 1989). BP's product was produced by an Italian subsidiary company, but rapidly withdrawn from manufacture because of Italian government opposition and the price of feed stock quadrupling in 1973. At this time the crude oil exporting nations (OPEC) had collectively raised the price of crude oil sold on the world market. In spite of the significant increase in the cost of crude oil and its derivatives, as well as recognizing the importance of competition from soya bean meal, the ICI pic directorate gave approval to build a full scale plant in 1976. Pruteen was marketed in the 1980s but eventually withdrawn because it could not compete with soya bean meal prices as an animal feed supplement.

Drozd (1987) has made a detailed study of hydrocarbon feedstocks and concluded that the cost of hydrocarbons does not make them economically attractive bulk feedstocks for the production of established products or potential new products where feedstock costs are an appreciable fraction of manufacturing costs of low-value bulk products. In SCP production, raw materials account for three quarters of the operating or variable costs and about half of the total costs of manufacture (Sharp, 1989; see also Chapter 12). It was considered that hydrocarbons and their derivatives might have a potential role as feedstocks in the microbial production of higher value products such as intermediates, pharmaceuticals, fine chemicals and agricultural chemicals (Drozd, 1987).

NITROGEN SOURCES Examples of commonly used nitrogen sources

Most industrially used micro-organisms can utilize inorganic or organic sources of nitrogen. Inorganic nitrogen may be supplied as ammonia gas, ammonium salts or nitrates (Hunter, 1972). Ammonia has been used for pH control and as the major nitrogen source in a defined medium for the commercial production of human serum albumin by Saccharomyces cerivisiae (Collins, 1990). Ammonium salts such as ammonium sulphate will usually produce acid conditions as the ammonium ion is utilized and the free acid will be liberated. On the other hand nitrates will normally cause an alkaline drift as they are metabolized. Ammonium nitrate will first cause an acid drift as the ammonium ion is utilized, and nitrate assimilation is repressed. When the ammonium ion has been exhausted, there is an alkaline drift as the nitrate is used as an alternative nitrogen source (Morton and MacMil-

lan, 1954). One exception to this pattern is the metabolism of Gibberella fujikuroi (Borrow et al., 1961, 1964). In the presence of nitrate the assimilation of ammonia is inhibited at pH 2.8-3.0. Nitrate assimilation continues until the pH has increased enough to allow the ammonia assimilation mechanism to restart.

Organic nitrogen may be supplied as amino acid, protein or urea. In many instances growth will be faster with a supply of organic nitrogen, and a few microorganisms have an absolute requirement for amino acids. It might be thought that the main industrial need for pure amino acids would be in the deliberate addition to amino acid requiring mutants used in amino acid production. However, amino acids are more commonly added as complex organic nitrogen sources which are non-homogeneous, cheaper and readily available. In lysine production, methionine and threonine are obtained from soybean hydrolysate since it would be too expensive to use the pure amino acids (Nakayama, 1972a).

Other proteinaceous nitrogen compounds serving as sources of amino acids include corn-steep liquor, soya meal, peanut meal, cotton-seed meal (Pharmamedia, Table 4.8; and Proflo), Distillers' solubles, meal and yeast extract. Analysis of many of these products which include amino acids, vitamins and minerals are given by Miller and Churchill (1986) and Atkinson and Mavi-tuna (1991a). In storage these products may be affected by moisture, temperature changes and ageing.

Chemically defined amino acid media devoid of protein are necessary in the production of certain vaccines when they are intended for human use.

Factors influencing the choice of nitrogen source

Control mechanisms exist by which nitrate reductase, an enzyme involved in the conversion of nitrate to ammonium ion, is repressed in the presence of ammonia (Brown et al., 1974). For this reason ammonia or ammonium ion is the preferred nitrogen source. In fungi that have been investigated, ammonium ion represses uptake of amino acids by general and specific amino acid permeases (Whitaker, 1976). In Aspergillus nidulans, ammonia also regulates the production of alkaline and neutral proteases (Cohen, 1973). Therefore, in mixtures of nitrogen sources, individual nitrogen components may influence metabolic regulation so that there is preferential assimilation of one component until its concentration has diminished.

It has been shown that antibiotic production by many micro-organisms is influenced by the type and

Table 4.8. The composition of Pharmamedia (Traders Protein, Southern Cotton Oil Company, Division of Archer Dariels Midland Co.)

Component

Quantity

Total solids Carbohydrate Reducing sugars Non reducing sugars Protein

Amino nitrogen

Components of amino nitrogen

Lysine

Leucine

Isoleucine

Threonine

Valine

Phenylalanine

Tryptophan

Methionine

Cystine

Aspartic acid

Serine

Proline

Glycine

Alanine

Tyrosine

Histidine

Arginine

Mineral components Calcium Chloride Phosphorus Iron

Sulphate

Magnesium

Potassium

Vitamins Ascorbic acid Thiamine Riboflavin Niacin

Pantothenic acid

Choline

Pyidoxine

Biotin

Folic acid

Inositol

4.5% 6.1% 3.3% 3.3% 4.6% 5.9% 1.0% 1.5% 1.5% 9.7% 4.6% 3.9% 3.8% 3.9% 3.4% 3.0% 12.3%

2 530 ppm 685 ppm 13100 ppm 94 ppm 18000 ppm 7 360 ppm 17200 ppm

16.4 mg kg

1.6 mg kg 10800 mg kg"

In shake flask media experiments, salts of weak acids (e.g. ammonium succinate) may be used to serve as a nitrogen source and eradicate the source of a strong acid pH change due to chloride or sulphate ions which would be present if ammonium chloride or sulphate were used as the nitrogen source. This procedure makes it possible to use lower concentrations of phosphate to buffer the medium. High phosphate concentrations inhibit production of many secondary metabolites (see Minerals Section).

The use of complex nitrogen sources for antibiotic production has been common practice. They are thought to help create physiological conditions in the trophophase which favour antibiotic production in the idiophase (Martin and McDaniel, 1977). For example, in the production of polyene antibiotics, soybean meal has been considered a good nitrogen source because of the balance of nutrients, the low phosphorus content and slow hydrolysis. It has been suggested that this gradual breakdown prevents the accumulation of ammonium ions and repressive amino acids. These are probably some of the reasons for the selection of ideal nitrogen sources for some secondary metabolites (Table 4.9.).

In gibberellin production the nitrogen source has been shown to have an influence on directing the production of different gibberellins and the relative proportions of each type (Jeffetys, 1970).

Other pre-determined aspects of the process can also influence the choice of nitrogen source. Rhodes (1963) has shown that the optimum concentration of available nitrogen for griseofulvin production showed some variation depending on the form of inoculum and the type of fermenter being used. Obviously these factors must be borne in mind in the interpretation of results in media-development programmes.

Some of the complex nitrogenous material may not be utilized by a micro-organism and create problems in downstream processing and effluent treatment. This can be an important factor in the final choice of substrate.

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