Extraction of amino acids from soils and sediments with superheated water

Oeoctdmicr. et Cosmochlmtca

Acta, 1974, Vol. 88, pp. 1843 to 1848. Pergamon Pnue.

Printed in Northern Ireland

NOTE Extraction of amino acids from soils and sediments with superheated water * C-N.

CHENG~

and CYR~, PONNAMPERUMA

Laboratory of Chemical Evolution, Department of Chemistry, University of Maryland, College Park, MD 20742, U.S.A. (Received 17 May 1973; accepted in rev&d

form 27 May 1974)

Ah&&-A method of extraction for ammo acids from soils and sediments involving superheated water has been investigated. About 76-97 per cent of the amino acids contained in four soils of a soil profile from Illinois were extracted by this method. Deep penetration of water into soil aggregates and partial hydrolysis of peptide bonds during this extraction by water at high temperature are likely mechanisms responsible for the release of amino acids from samples. This extraction method does not require subsequent desalting treatments when analyses are carried out with an ion-exchange amino acid analyzer. I??TRODlJCTION

THE EXTEACTIOPU’ of amino acids from soils and sediments is generally accomplished by using 6 N HCl to release chemically bound molecules from high molecular weight organic matter and the inorganic matrix. In so doing, large amounts of inorganic salts (e.g. salts of Fe, Al, Ca, Mg, etc.) are invariably present in the extract. Removal of these salts without the loss of amino acids is difficult. It is, therefore, important to develop a simple, effective extraction procedure in which desalting could be eliminated. Under ordinary conditions, water is generally considered a poor solvent for extraction of amino acids from soils, sediments and fossils. The efficiency of water extraction at room temperature or refluxing at 100°C at atmospheric pressure is low. The reports of work at low temperatures are somewhat conflicting. Although PAUL and Tn (1965) could not obtain satisfactory recovery, IV-ON and SOWDEN (1966) reported a marked release of water-extractable amino acids from soil by freezing-thawing pretreatment. SELLERS(1966) appears to be the first to have carried out a systematic treatment of a recent marine sediment and a Miocene mudstone with superheated water. In his study the samples were heated at temperatures ranging from 150’ to 47O”C, up to 50 hr, at a pressure of 500 atm in the presence of water. After this treatment, the whole sample was hydrolyzed with 6 N HCl. The results indicated that the per cent yield of the amino acids recovered at 235-240°C decreased as heating time increased. However, a slight relative increase in yields of amino acids occurred at

* A preliminary report was presented at the 8th ACS Middle Atlantic Regional Meeting in Washington, D.C., Jan1614, 1973. 7 Present address: Chemical Evolution Branch, NASA Ames Research Center, Moffett Field, California 94036, U.S.A. 1843

the heating times between 200 and 400 min. This increased yield could indicate that an additional amount of bound amino acids was released to replenish ihe original ones, and treatment with superheated water could help release ammo acids which were not extracted easily. When an ampoufe containing water is sealed and heated to 19O”C, the vapor pressure inside in the presence of liquid water may increase to more than 12 atm (WE&ST, 1967). It is the purpose of this study to investigate the recovery of amino acids from soils and sediments, utilizing hot water under pressure for t,he extractron

of amino acids. EXPERIMENTAL Four soils from a Cisne soil profile, numbered 20986, 20988, 20991 and 20993 were suppired by Dr. F. J. Stevenson. Sediment samples at depths of 1 m, 80 m and 108 m of a core from Cariaco Trench were supplied by Dr. lp. E. Hare. ‘rhe distilled wster used ~~~roughou~the study was prepared with an all-glass apparatus. Dilute HCl solutions were prepared ffom the reagent grad8 DuPont product. Analyses were performed on an automated Durrum D-300 amino aoid analyzer. A 250 mg sample of soil or sediment was introduaed into a 10 ml drying ampoule. D&%tXed water (2.5 ml) was added and a stream of Xz wes bubbled into the suspension for 10 mm %U remove the dissolved 0,. The ampode WM then seal8d and heated at 170°C ( &3”C) for i hr. After the heating, the S&I@8 was centrifuged at 3000 revjmin for IO min, and the supernatant recovered. The residue was washed and centrifug8d twice using 1 ml of divtilled water each time. The sup8rnatant and the two washings wer8 combined to give the tirst extract.. Tile second and third extra&a were obtcained by repeating the above steps on th8 residue wing the identical ampoule from. the first extraction. The 8xtR%%sW8l.Qhydrolyzed with 6 23 KC:1

for 16 hr. The hydrolyzatee w8re freeze-dried and dissolved in 400 ,ul of 0.01 N RCl solut.iotl. The solution was centrifuged st 15,000 rev/min for 10-15 min to remove suspended particlt~s and the supernatant tvaa analyzed for amino acids. RESTJZTS AND

DISCUSSION

Te~~~~tu~e and time of dwuticm

Samples of soil 20993 were treated at ~rn~rat~s between 100°C and 19O’C. In an experiment in which the sample was heated for 16 hr at a temperature that did not exceed 125°C only a emall amount-not more than 100 nmolesjg or less than 10 per cent of total-of amends acids could be extracted. However, more could be recovered when the temperature was increases1 to 146°C and above. The totai amounts released after 3-hr heating at 145’, 160”, 170”, i.s0 and 190% am shown in Fig. 1. A maximum was reached at 170°C. Figure I also presents amounts of each amino a&d recovered at various temperatures. The recovery of giucarme acid, glycine, and alanine increased with temperacure up to 170°C and decreased at 180°C and 190°C. The amount6 of serine and aspartic acid extracted tend to decrease with temperatur?. and the quantities of other amino acids extracted generally remain constant. VALLENTYNE f 1964) reported that by heating amino acids xith water, @yeme can be ~orrmri from dine, se&e, threonine and methionione; and alanine from serine and methionine. it VGUS, therefore, n8C8aSSryto see if glyeine and alanine recovered from extractions at 16O”C, 170% and 180°C for 3 hr oont&in products of this inter-transformation of amino acids. The thermal stability of a mixture of standard amino acids (100 nmoles of ench) in aqueous solution was treated at about 170°C for periods of 0.5, 1, 2 and 3 hr under Nz or air using the drying ampouk. The results are tabulated in Table 1. The recovery of glycine, &nine and glutamic acid increased signifkntly at 3 hr heating under N,. Apparently, inter-transformation of glyCii3e and alanine did take place in %hhe aqueous solution when heated long enough; but the rt?~tsorf. for the increase of gh_&amicacid is not cfear. Soil 20993 was then extracted at 16%172% under I-$ for 0.5, 1, 2 and 3 hr. In each case only the first extract and washing were analyzed. About 300 nmoles/g of amino acids were extraoted after the 0.5 hr heating while more than 5 times as much was recovered after i t,!.. There was a slight decrease after 2 hr of heating. The results indicate that the 1 hr heanng

Extraction

of amino acids from soils and sediments with superheated water

0

I 140

I 160

I 160 TEMPERATURE

I

1845

1

2%

lC

Fig. 1. The extraction of amino acids from soil 20993 with superheated water at 145°C and above with 3 hr heating under air. Each value represents the average of triplicate. Average deviations for the individual amino acids are f2- &-6 per cent. One extraction was made for eaoh sample. Table 1. Per cent recovery of amino acids at 170°C ( 13 “C) N2 Aepartie acid Threonine Serine Glutamic acid Glycine Alanine Valine Methionine Isoleucine Leucine Phenylalanine Hi&dine Lysine Arginine

l/2 hr Air

100 100 96.5 95.6 98.5 97.5 97.7 82.6 95.3 96.6 94.0 92.0 98.2 97.2

96.0 93.5 95.2 96.0 101 92.8 93.1 59.2 92.0 92.0 89.2 96.0 91.2 93.0

1 hr h’, 78.5 93.5 83.0 104 110 102 95 31.6 92.9 93.0 89.7 83.4 93.0 93.0

2 hr

3 hr

Air

N,

Air

h’,

Air

85.0 75.5 77.0 92.0 95.5 88.5 79.0 72.4 70.3 48.5 54.0 74.5 74.0

61.2 89.2 82.0 110 109 96.2 95.7 30.6 94.1 93.6 91.0 84.0 93.0 93.2

52.0 65.5 63.7 82.5 102 82.5 69 55.3 48.5 21.4 24.8 61.0 56.5

55.5 86.7 51.0 108 110 105 96.0 28.0 957 95.2 91.8 82.0 93.9 94.0

14.0 40.5 37.0 70.9 110 77.8 48.2 33.2 24.8 7.2 4.3 40.0 33.0

* Each value is the average of duplicate. under N, seems to be suitable for the soil sample. Because the amino acids appear rather unstable toward the higher temperature treatment when heated beyond 1 hr, the observation by SELLERS(1966) of the increasedglycine and alanine with prolonged heating may be an indication of an inter-transformation of amino acids, Table 2 summarizes the results of three serial

1846

C-N. CEENG and CYBIL PONNANFERU-NA Table

2. Successive extraction of amino acids from soil 20993 at 170°C (53°C) for 1 hr (in nmoles/g) .-Extraction* 1st 2nd 3rd Total

Aspartio acid Threonine Serine Glutamia acid Glycine Alanine Valin0 Methionine Isoleucine Leucine Phenylalanine Histidine Lysine Arginine Total

229 112 146 317 346 287 69.2 25.6 48.‘” 79.6 42.7 ? 31.6 -r 1634

-

53.0 17.7 27.0 36.7 74.5 43.6 11.6 8.2 IO.8 14.7 3.8 f T

301

17-O 5.3 11.0 14.2 31.8 17.2 3.3 5.2 2.6 6.4 ? -

,

114

299 135 184 267 452 348 84.1 39.0 61.6 101 46.5 31.6 2049

--

* Run triplicate, the average deviations for individual amino acids at the 1st extraction: *2- &6 ‘A, the 2nd: &2- kg.5 ‘A and the 3rd: &3--&B:.{; total _~2-&6%. extractions on the soil at about 170°C under Nz for 1 hr. The results indicated that at this temperature, the first extraction accounts for about 80 per cent of the total, the second For 14 per cent and the third for only about 5 per cent. Hydrolysis of the extracts In Table 3 are listed amounts of amino acids before and after hydrolysis of the water extract with 6 N HCl. These results show that the amounts of amino acids d&e&xl after hydrolysis approached twice that recovered before hydrolysis, when the extractions were made at 1;~ ‘. 125” and 145”C, and about Z-2.5 times as much was obtained in the 170°C extract. Each v:-tiua was the average of dupliaates. The results here indicate that hydrolysis is necessary. VALLENTYXE (1964) reported that the glutamic acid molecule becomes dehydrated q>or: heating to form pyroglutamia acid which is not ninhydrin sensitive, therefore, not deter+-cl by nn amino acid analyzer, and is easily converted back to glutamio acid by acid hydroiysls. In t.he present study, glutamic acid in the unhydrolyzed samples could hardly be detec,retl, while the amount detected after hydrolysis at 170” for 3 hr amounted to more rh:tn 350 nmoleslg. APPLICATION AND IMPLICATT~N

Samples of the soil profile and the Cariaco sediment core were extracted according to the described procedure. The total amounts of amino acids extracted from t,he soils and the sediments are in Table 4, together with the values obtained by other extraction procedures-CHENo et al. (1974), DEGENS and RETJTER(1964), arid HARE (1972)-for comparison. The extraction of amino acids from the soils by the current procedure, B, is seen to be quite effective. The recovery represents about 75-97 per cent of that obtained by CHENG et aE. (1974) A, and is better than that by DEGENSand REUTER(1964), C. In the case of the sediments, the current procedure is the poorest, accounting for only about 25 per cent of the recovery by A and about 33-75 per cent of the recovery by C.

Extraction

of amino acids from soils and sediments with superheated water

1847

Table 3. The amino acids in the water extract of soil 20993 determined before and after 6 N HCl hydrolysis

Treatment Ammo acid Aspartic acid Theonine Serine Glutamic acid Glycine Alanine Valine Methionine Isoleucine Leucine Phenylalanine Histidine Lysine Arginine Total

100°C @moles/g) 12 hr Un-H* H 6.67 2.0 5.2 10.0 11.1 5.7 40.6

11.2 5.5 8.8 -t 21.1 18.1 + ? ? 7.6 ? 72.3

125°C @moles/g) 6 hr Un-H H 6.3 6.2 7.2 + 19.0 14.0 P ? 2.5 6.6 ? ? 60.8

7.4 8.8 11.1 5.0 26.4 22.9 4.6 ? 5.6 8.9 100.7

145°C @moles/g) 3hr Un-H H 130 27.2 38.0 + 127 102 16.9 6.1 12.6 23.2 10.1 ? 492.1

170°C (nmoles/g) 3 hr Un-H H

152 605 74.1 199 223 181 29.6 20.7 23.2 38.1 21.4 1.0 14.0 1037.6

39.1 31.4 34.2 + 197 124 27.0 7.4 19.0 20.6 4 499.7

67.8 75.8 82.2 352 330 238 63.7 17.2 39.4 49.5 30.8 0.9 35.2 ? 1382.5

* Un-H unhydrolyzed. H hydrolyzed. 7 Each value represents the average of duplicate. Table 4. A comparison of amino acids extracted by different methods Cisne soils and Carisco Trench sediments*

Cariaco trench sediment

Cisne soils Methods A B C D

from

20986

20988

20991

20993

1m

80 m

108 m

13,900 11,400 11,300

4990 3990 2580

2590 2450 961

2580 2040 1020

3930 913 2710 1410

1670 439 899 461

1020 285 383 316

A: HF pretreatment, HCl hydrolysis and HF desalting (CHENG et al., 1974). C: The current extraction procedure. C: HCl hydrolysis and NH,OH desalting (DEGENS amd REUTER, 1964). D : Water and dilute NaOH extractable ammo acids in fine fractions by HARE ( 1972). * The values are in nmoles/g, the average deviations of triplicate are &2- &6 %.

These results seem to indicate that the efficiency of this water extraction depends very much on the tspe of samples which might concentrate a particular form of amino acids. The most obvious compositional difference between these soils and sediments is the carbonate contents. Hare (personal communication) pointed out that the Cariaco Trench sediment contains a very significant amount of carbonate materials which are mostly micro- and nanofossils. The organic materials of these fossils seems to be embedded in the calcareous matrix of the fossil shells. Therefore, water (the solvent of the present procedure) could hardly reach the amino acid contained in this organic residue unless the inorganic matrix is first broken down (ABELSOR, 1963). Under the higher temperature and pressure, e.g. in the current

1848

C-N. CHENO and CY&IL PONNM.ZPZR~~~~

study, about 8 atm at li0” C’. (WEAST, 1967), water may penetrate into aggregates of the sample and extract amino acids therein. The values by HARE (llf52), D. in Table 4, are the total protein amino acids (excluding proline and tyrosine) extracted from the fines {particle size CC1 pm) of t,he same sample as used in this study. The corresponding values of B and D are about equal. It appears that most of t,he amino acids of these sediments contained in fractions other than the coarse one can be recovered by the current treatment. The mechanism by which this extraction method gives recovery of amino acids from these soils is not exactly known; however, water will hydrolyze peptide bonds (GREENBERG and BURK, 1927). Furthermore, certain peptide bonds, e.g. aspartyl, glycgl, seryi, threonyl, are more easily hydrolyzed than others (H.~RIs et al., 1956). Since these soils contain very large amounts of these particular amino acids (52 per cent of total in soil 2099S1 Table _“), it is highly probable that the amino acids remain as the constituents of peptides in these soils. The high temperature-pressure conditions during this water extraction promote partial hydrolysis and hence extraction. This extraction method is simple, and does not require a desalting procedure. It should be of value for the rapid survey of soils for amino acids. dcknourledgement-We thank Dr. I?. J. STEVENSONof the University of Illinois for tile soil samples and his valuable suggestions, Mr. GLENN E. POLLOCK of NASA-Ames Research C+_nter for helpful discussions, and Dr. P. E. I%RE of Geophysical Laboratory, Carnegie Instltutc of Washington, for the Cariaco Trench sediment samples. The study is wpported through grants NGR 21-002-317 Chemical Studies on the Origin of Life, and NCR 21-002-329 Proposal for the Analysis of Carbon Compounds in the Apollo 15-17 Samples.

REFERENCES P. H. (1963) Geochemistry of amino acids. In International Series oj Xovwgrophs ix Eurth Sciences Xo. 16. (editor I. A. Breger), pp. 431-455 Pergamon Press. CRENG C-N., SHL~ZDT R. C. and STE’(‘ENSONF. J., (1974) An improved desalting u\rac!locl for amino acid analysis of soils and sediments. Soil Biol. Biochem. DE~ENS E. T. and REUTER J. H. ( 1964) Analytical techniques in the field organic geooborn~~rr~ In ddvances i?~ Organic #eochemistr?J, Proo. Int. Meeting in Milan, 1962, (editors U. Colw:bo and G. D. Cobson), pp. 377-402. (GREENBERG D. 31. and U~CR~N. F. (1927) The rate of hydrolysis of solutions of protenis in acids ss measured by the formation of amino nitrogen. J. Amer. Chem. Sot. 49, 275-286. H;LRE P. E. (1972) Amino acid geochemistry of a sediment core from the Car&o trewh. Carnegie Inst. Wash. Yea&. 71, 592-596. HAERIS J. I., COLE R. D. and Pow N. G. (1956) The kinetics of acid hydrolysis of dipeptr