20 Microbial Synthesis of Food Downloaded via UNIV OF CALIFORNIA SANTA BARBARA on July 12, 2018 at 06:23:43 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
From Coal-Derived Material MELVIN P. SILVERMAN,
1
JOAN N. GORDON and
IRVING
WENDER
Pittsburgh Coal Research Center, Bureau of Mines, U. S. Department of the Interior, Pittsburgh, Pa.
Cell yields of four strains of the yeast Candida grown on -hexadecane ranged from 433 to 719 mg. dry weight per gram of hydrocarbon added; growth on 1-octadecene decreased about one half. A normal paraffin fraction from petroleum gave yields of 244 to 430 mg.; three Fischer-Tropsch fractions and one low-temperature coal tar fraction gave yields within this range. Bacteria, but not yeasts, grew on three coal-acid mixtures and a synthetic mixture of polynuclear aromatic hydrocarbons with yields below 100 mg. In general, high growth yields of yeasts were favored by substrates that were high boiling, contained a high ratio of normal paraffins to olefins, and were low in phenols. The crude protein content of Candida grown on a FischerTropsch fraction was 41.1%. u
I ^he problem of the world's population explosion is compounded by an over-all global food shortage. New or unusual sources of food, especially high-quality protein, are needed as dietary supplements for both humans and animals. The Permanent Section on Food Microbiology and Hygiene, International Association of Microbiological Societies, called for an increased contribution of microbiology to world food supplies i n a unanimous resolution outlining several research areas, including hydro carbon microbiology, which might lead to increased world food production (UY Some recent reports have dealt with the microbial conversion of petroleum hydrocarbons to protein, vitamins, or amino acids. H i g h yields of yeast cells rich i n protein and vitamins have been obtained at the expense of the n-alkanes (preferably C or higher) i n crude petroleum v
1 0
Present address: Ames Research Center, Exobiology Division, National Aeronautics & Space Administration, Moffett Field, Calif. 1
269 Altschul; World Protein Resources Advances in Chemistry; American Chemical Society: Washington, DC, 1966.
270
WORLD PROTEIN RESOURCES
fractions (£-6), feedstocks (18, 16), or the pure hydrocarbons themselves (1, 4, 9,12) .Microbial synthesis of amino acids from petroleum products has also been reported (16, 19). E a r l y papers discussed the use of kogasin, a synthetic fuel derived from coal, for microbial substrates (8,10, 11). The rate of microbial synthesis of protein far exceeds the rate at which animals synthesize protein. A cow weighing 500 kg. when fed by grazing can synthesize 0.5 kg. of protein per day (18), whereas 500 kg. of micro organisms growing on paraffinie hydrocarbons could synthesize 1250 kg. of protein per day (8). According to one estimate, 3 million tons of protein per year (equal to the world's present protein deficit) could be produced by microorganisms at the expense of only 1% of the world's annual production of 700 million tons of crude paraffinie petroleum (8). Since coal is one of the world's cheapest and most abundant sources of fixed carbon, we investigated the question whether coal, like petroleum, could be converted by microorganisms into high-protein food. This report gives the results of a comparative evaluation of coal-derived materials, pure hydrocarbons, and petroleum-derived material as raw materials for microbial production of food. The use of coal-derived liquid fractions was emphasized because of the intriguing possibility of extending the utility of fuel and chemical products obtained from coal conversion processes.
Experimental Materials. Three fractions of Bureau of Mines Fischer-Tropsch syn thetic liquid fuel (iron catalyst) were used: fraction F T L (boiling range 0 to 204°C), fraction F T D (boiling range 204 to 316°), and fraction F T W (boiling point > 316°). The analysis of Fischer-Tropsch fraction S A S O L (from the South African Coal, Oil, and Gas Corp.) is given i n Table I. Two fractions of hexane-soluble material from Rockdale lignite lowtemperature tar were obtained from the Texas Power and Light C o . : hexane-soluble foreruns ( H S F ) and a hexane-soluble distillate ( H S D ) . The H S F fraction constituted 7%, and the H S D fraction 4 6 % of the primary tar. The compositions of fractions H S F and H S D are given i n Table I I . Phenolic compounds were removed by chromatographing fractions H S F and H S D on alumina with petroleum ether as the eluent to obtain phenolfree fractions H S F 0 and H S D $ . About 20 weight-% of starting material was removed by this procedure. A paraffin-rich fraction ( C T P ) and a linear paraffin-olefin fraction ( C T P O ) , both derived from the neutral oil of low-temperature tar, were supplied by the Bureau of Mines' Morgantown Coal Research Center. Their analyses of fractions C T P and C T P O are given i n Table I I I . The normal paraffin fraction ( P E T ) was derived from petroleum by an indus trial firm. Our mass spectrometric analysis of this fraction is given i n Table IV. Three mixtures of coal acids were tested asg rowth substrates: D O W , a 5 6 % aqueous solution from the Dow Chemical C o . ; H O W , a watersoluble mixture of aromatic acids from alkaline oxidation of coal by the Carnegie Institute of Technology; and A N T H , the acid-soluble residue
Altschul; World Protein Resources Advances in Chemistry; American Chemical Society: Washington, DC, 1966.
20.
SILVERMAN ET AL.
Table L
Microbial Synthesis
Analysis of Fischer-Tropsch (SASOL) Fraction
Hydrocarbon
FIÀ Analysis
Carbon No,
%
Compounds
%
C„ Ci2
2 11 17 16 15 13 11 9 4 2
Aromatics Olefins* Paraffins* Oxygenates
Nil 33 64 3
C13
c4 Ci6
c8 C19 α
271
Normal Hydrocarbons ca. 90%
from 1000-hour nitric acid oxidation of anthracite by the Bureau of M i n e s ' Anthracite Research Center (7). Substrate C T A , a synthetic mixture of polynuclear aromatic hydro carbons found in relatively large amounts i n high-temperature coal tar, consisted of 1 -methylnaphthalene, 59.2; 2-methylnaphthalene, 16.3; naphthalene, 11.9; and phenanthrene, 12.5 w t . - % . Microorganisms and Culture Media. Cultures were obtained from soil by standard enrichment culture techniques or from the culture collections of the University of Pittsburgh, Syracuse University, and the University of Iowa. Basal medium Ν was prepared by adding N H N 0 (5.0 grams), K2HPO4 (2.5 grams), and M g S 0 - 7 H 0 (1.0 gram) to 1 liter of tap water. The p H was adjusted to 7.0 and any insoluble salts were removed by filtration. The clear medium was sterilized by autoclaving. M e d i u m N X was prepared by adding sufficient filter-sterilized yeast extract to medium Ν to make a final concentration of 0.01%. 4
4
3
2
Measurements of Growth Yields. Yeast inocula were prepared from cultures grown overnight i n 50 m l . of M y c o p h i l broth (Baltimore Biological Laboratory, Baltimore, M d . ) . The resultant growth was collected by centrifugation, washed twice i n sterile medium N , and resuspended i n 20 m l . of the same solution. One milliliter of washed yeast-cell suspension was the standard inoculum for a l l experiments. F o r studies on growth yield of yeasts, these standard inocula were added to 50 m l . of medium N X i n 300-ml. Erlenmeyer flasks i n triplicate. A quantity of substrate equivalent to 0.3 m l . was weighed into each flask. Controls consisted of triplicate inoculated flasks without added substrate. Cultures were incubated at 30°C. on a rotary shaker (225 r.p.m.) for 6 days. The resultant growth was collected on 2-inch diameter Alpha-8 Metricel filters of 0.20 μ pore size (Alpha Metricel, a regenerated cellulose (rayon) membrane, is a product of the Gelman Instrument Co., A n n Arbor, M i c h . ) . The growths were washed with 10-ml. volumes of acetone and
Altschul; World Protein Resources Advances in Chemistry; American Chemical Society: Washington, DC, 1966.
272
WORLD PROTEIN RESOURCES
Table Π.
Approximate Composition of Rockdale Lignite LowTemperature Tar Fractions (Volume-%)
Type of Constituent
HSF
HSD
Caustic-solubles Acid-solubles Neutral oil Paraffins Olefins Alpha-olefins Aromatics
6-8 2-4 88-92 13-15 40-55 17-20 30-47
10-15 1-3 80-90 15-20 40-50 17-20 35-45
n-hexane, dried overnight i n air, and then weighed. A l l reported data are corrected for growth of controls. Bacterial inocula consisted of 1 ml. of cultures grown for 3 days in 50 m l . of medium Ν or N X supplemented with 1 weight-% of coal acid ( D O W or H O W ) or 0.5 m l . of C T A . Procedures for determining bacterial growth yields were similar to those for yeast cultures, except that 1 weight-% of coal acid ( D O W or H O W ) or 0.5 m l . of C T A was weighed into 50 m l . of medium Ν or N X . Incubation times were 4 days for C T A cultures and 6 days for coal-acid cultures.
Results More than 200 cultures of bacteria, yeasts, and fungi were screened for viability on coal-derived materials. Only bacteria (no yeasts or fungi) grew on the coal acids and C T A . Conversely, yeasts and fungi produced the Table ΙΠ. Analyses of Paraffin-Rich (CTP) and ParaffinOlefin (CTPO) Fractions from Rockdale Lignite Low-Temperature Tar (Weight-%) CTP Carbon No.
n-Paraffin
c8 c9
0.2 3.2 11.7 18.2 22.4 20.7 10.8 0.8
Cio
Cn Cl2 Cl3
C,4 Ci6
Ci6 Ci7 C
— — —
1 8
88.0
CTPO n-Olefin — —
0.7 1.5 3.3 2.8 3.7 — — — —
12.0
n-Paraffin
n-Olefin
—
—
0.1 1.3 3.6 5.8 7.8 7.5 6.7 4.6 2.3 4.2
0.2 1.6 5.3 7.6 9.5 10.9 9.4 5.6 2.9 3.1
43.9
56.1
Altschul; World Protein Resources Advances in Chemistry; American Chemical Society: Washington, DC, 1966.
20.
SILVERMAN ET A L .
273
Microbial Synthesis
most vigorous growth on Fischer-Tropsch, low-temperature tar, and petroleum-derived fractions. Of these, the yeasts Candida lipolytica strains 409, 409A, 409B, and Candida tropicalis strain 410 were selected as the most promising cultures with respect to total cell yield and ability to grow in reasonably high concentrations of substrate. These studies also indicated that growth yields on Fischer-Tropsch fraction F T L , low-temperature tar fractions H S F and H S F 0 , and the coal acid A N T H were negligible. N o further studies were made with these materials. Table IV.
Analysis of η-Paraffin Fraction (PET) from Petroleum
Carbon No. C ClO 9
c„
Cl2
Ci3 C 1 4
Volume- % 0.5 7.2 37.9 29.1 23.4 1.9
Growth Yields of Yeasts on Pure Compounds and Coal-Derived
Ma
terial. Absolute growth yields, in milligrams of dry weight of yield per gram of added substrate, and relative growth yields are given i n Table V . The highest yields for all cultures (433 to 719 mg. dry weight) were obtained with n-hexadecane as substrate; C. lipolytica 409 and 409A gave the best yields with n-hexadecane. Growth yields of 300 mg. or higher were obtained when 1-octadecene (C. lipolytica 409 and C. tropicalis 410) and Fischer-Tropsch fraction F T W (C. lipolytica 409B) served as growth substrates. Other substrates yielding more than 200 mg. dry weight were paraffin-rich low-temperature coal tar fraction C T P (all cultures), and Fischer-Tropsch fractions F T D (C. lipolytica 409B) and F T W (C. lipolytica 409). A l l cultures yielded less than 100 mg. dry weight on low-temperature tar fraction H S D ^ . On all substrates tested, with the exception of lowtemperature tar fraction HSD
