H. M. L. Dieteren and A. P. H. Schovteten Deportment of Chemical Education Technical College Heerlen' Heerlen, Netherlands
Preparation of the Proteins by
I
Micro-Organisms A laboratory experiment
It is widely known that in developing nations there exists a considerable shortage of proteins in the human and animal diet. The various possibilities of eliminating this shortage a t least partially are under extensive study. In recent years scientific and technical laboratories paid much attention to the production of proteins by means of micro-organisms starting with hydrocarbons. Although Sijhngen proved as early as 1905 that hydrocarbons can be used as a source of carbon in the microbiological preparation of proteins, it was not until 1963 that Champagnat developed an economically practicable process (1). Oil companies such as Standard Oil of New Jersey, Gulf Oil, Sun Oil, Humble Oil, and British Petroleum are highly interested in this microbiological synthesis. Gulf Research and Development Company has put into use a facility a t Wasco, California where hydrocarbons are converted into proteins a t a semitechnical scale. British Petroleum has erected two plants, one a t Grangemouth, Scotland and and the other at Lavbra, France. The annual output of these factories will be 4000 and 16,000 metric tons, respectively, of protein concentrate, containing 65% of protein. The protein produced will be put on the market as a supplement to animal fodder. The major advantage is that in this synthesis the procedure is entirely independent of environmental conditions, such as climate, which makes it possible to prepare the protein in any possible location on earth. Because i t is expected that in the near future protein produced in this way will be used for human consumption a more close look a t the problem is generally considered necessary. 'Address: Dr. Jaegersstrartt 40.
An important problem yet to be solved is the acceptance of the protein-concentrate as a human food. An appropriate taste and shape of the product will undoubtedly be very important for future sales. Theory In the micro-biological preparation of proteins, microorganisms-preferably a yeast species-are allowed to multiply in a substrate containing hydrocarbons (normal alkanes). These alkanes are used in two ways, as a source of carbon and as a source of energy. For their development the micro-organisms do not only need carbon, but also a number of various other elements such as nitrogen, phosphorus, magnesium, oxygen; yeast in general needs an extra supply of growth factors (3). The composition of the culture medium and the reaction circumstances are directly related to the nature of the organism in use. When growing yeast it is recommended that the test is performed at or near pH = 4 to prevent bacterial infection and reproduction as much as possible. The micro-organisms grow, while consuming the hydrocarbons and the in-
Figure 1. Six stages in the gmwth curve of rnicro.organbrnr. 11 Lag phase; 21 transtion phore; 3) exponential growth phare; 41 transition phare; 51 rtationary phase; 6) endogenis phase.
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organic salt supplies. It produces proteins, lipids, 'and the other cell components, as well as water and carbon dioxide. The latter, being a waste product, is expelled from the cell. As is shown in Figure 1 the growth curve of microorganisms can be divided into six stages. During the lag phase the micro-organisms grow but their number does not increase. I n the exponential growth phase the increase in the number of organisms is directly proportional to the number present: the number N of the cells present after the elapse of time t is related to the number No of the cells present at the time t = 0 as N
=
No.@
where c represents a proportionality constant depending on the nature of the organisms in use and the conditions of the test. During the stationary phase the number of micro-organisms no longer increases, while in the eudogenic phase the organisms stay alive by consuming t.heir own cell components. This auto-oxidation appears in the exponential growth phase also, but in the exponential phase the degradation can be neglected as compared to the synthesis of its own components by the organism. Figure 2 shows the concentration of the n-alkane versus time when the production of protein is conducted in a hatch reactor.
Figure 2. Change of concentration of n-alkane with time for the pmduction of protein in 0 bokh reoctor.
Because of the nearly complete insolubility of the hydrocarhons in water it is an absolute necessity to distribute the oil in the water in droplets as small as possible in order to allow the organisms to develop at a fast rate (3). I n industrial fermentation reactors it is hardly possible to achieve this by stirring, but the goal may be reached by the use of homogenization devices (4). Erdtsieck (6) proved that the growth of yeast with n-alkanes may be explained by assuming that the organisms gather near the oil-water boundary which enables a direct contact between the cell and the hydrocarbons. As a source of carbon it is possible to use the straightchain hydrocarbons containing 12-18 carbon atoms. Branched hydrocarbons, aromatic hydrocarbons, and naphthenes (cyclic nou-aromatic hydrocarhons) are generally not attacked at all or to a far less degree. The hydrocarbons that can be used are components of the gas oil fractions, and they can be isolated by means of molecular sieves. The yeast cells, however, do grow on crude gas oil, but the major disadvantage is that the yeast produced in this way contains a t least partially the nonassimilated hydrocarhons. These hydrocarhons can be removed by extraction; the extraction and the
subsidiary drying are to be performed more easily after a preliminary hydrolysis or plasmolysis of the yeast cells (6,7). Experimental Description of the Apparatus
A vessel A of 2000 ml volume is equipped with a double propeller stirrer D, an air inlet tube B, a thermometer F, and a reflux condenser E (see Fig. 3). A is charged with the culture medium; B is used to provide the cells with the oxygen (air) needed for their respiration, while F controls a thermostatic device necessary to keep the contents of A at a constant temperature. The superfluous air escapes from the flask through E which serves as a device to prevent or diminish the evaporation of the liquid in A . C is an extra mouth that is used to introduce liquids, etc. into the flask and to extract samples from the flask.
Figure 3.
Diagram of the react.&.
Prepare a culture solution by dissolving 7 g NHIC~, 5 g KHIPOn, 2.5 g MgSOs, 400 mg CaC12, 1 mg MnSOn, and 1 mg ZnSOn in 1000 ml of tap water. Introduce this solution in the flask and set the stirrer in motion, velocity 1000 rev/miu. Add 1 mg of yeast extract to the solution and set the pH a t 4 using diluted HC1 or NH40H. Maintain the temperature of the contenance of the flask continuously a t exactly 30°C. Allow air to pass through the inlet tube at a rate of 1000 ml/min. The tube should dip as deep in the solution as possible hut it should reach at least beneath the lower blades of the stirrer in order to ensure a good contact between the air and the solution. Introduce 40 ml of tetradecane, followed by a sample of Caudida Lypolitica Harrison (C.B.S. 59g3) and allow the organism to develop. The procedure involved is a modification of the method used by Erdtsieck (6). a Candida. Lypalitica Harrison (C.B.S. 599) was purchased from the Centraal Bureau voor Schimrnelcult res (C.B.S.), Delft, Netherlands.
At regular intervals check the pH of the solution and if necessary adjust the pH to 4 by means of 2 N NH,OH solution. At the end of the test, indicated by a decrease in the consumption rate of the NH3, pour the mixture into a beaker and add 100 ml of 5% sodium laurylsulfate solution. Stir and centrifuge the mixture at 3000 rev/min for 20 min. The yeast settles to the bottom of the tube. Wash the yeast two times with 5% sodium laurylsulfate solution, followed by three successive washings with distilled water. Dry the p o d & at 50°C preferably at diminished pressure. The protein contents of the product may be determined by the Kjeldahl-Gunning determination of nitrogen (8). Mercury was used as a catalyst throughout. The progress of the test may be checked in various ways 1) By determining the amount of dry material. This may be done as follows: Pipet an aliquot of the test solution from the Bask and separate the yeast by centrifuging as described above. Dry till constant weight a t diminished pressure. 2) B y measuring the amount of N&OH solution that is r e quired t o maintain the pH a t a. constant value. There is a direct relationship t o the amount of organisms formed.
'Antifaam A is manufactured by Midland, Michigan.
ow Corning Corporation,
Critical Remarks 1) n-Tetrsdecnne was used throughout the test because the compound is liquid a t room temperature. A hydrocarbon containing s, Longer carbon chain is solid a t room temperature which could cause crystallization a t the wall of the vessel due t o splashing of the solution. Shorter-chain hydrocarbons in general show rt smaller reaction rate. 2) To prevent flotation it is possible t o add antifoam agents. "Antifoam A"' was found useful. 3) It is not necessary t o sterilize the apparatus and the chemicals before starting a test but it is recommended t o do so.
Literature Cited
1964. (4) CBAMPAGNAT. A , , AND LAINE.B.. "Pedeotionnemont d la oullure de mioro-ormniames sur substrat hydroesrbures, lcr. 1.357.040-24-21964. (5) ERDTSIBCK, B.. Thesia, Teobnioal University, Eindhoven, Netherlanda.
1965.
(8) VoanL, A. I., "A Textbook of Quantitative Inorganic Analysis." (3rd Ed.) Longmans, London. p. 256-7.
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