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CHAPTER 7 THE BIOCHEMISTRY OF TERRESTRIAL SOILS A. D. MCLAREN INTRODUCTION In testing for life or for evidence for life processes in soil one can use either nutrient media in which organisms can grow or else choose reagents that will react specifically with products characteristically produced by living entities. The former presupposes either a universal medium or a medium in which some specific, universally-dispersed soil organism will proliferate. This type of test may fail in Martian soils if an unfamiliar biochemistry prevails. With the latter approach one must distinguish between compounds similar to those found in Miller-Urey experiments and those more charac- teristic of life such as enzymes and nucleic acids. These are usually mono- disperse with regard to molecular weight and possess non-random struc- tures. All of these exist in nearly undetectable amounts in Earth soils, since they are nutrients for at least some of the organisms present. On the other hand, a few of the soil organic constituents, the enzymes, may be revealed by their ability to "turn over" appreciable amounts of substrate. Here we make the assumption that although the nutritional requirements of organisms in strange soils may be unknown, the enzyme constituents released to the environment by decomposed organisms are universal in general properties and characteristic of life wherever it is found. Obviously, life forms on Mars and elsewhere may differ too much from 147

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148 RECOGNITION OF LIFE AND SOME TERRESTRIAL PRECEDENTS those here to be detected within this framework. However, the following discussion may suggest something concrete and accessible in the way of "life testers." GENERAL DESCRIPTION OF SOIL BIOCHEMISTRY Earth soils are aggregates of minerals, water, humus and microorganisms. As a kind of graveyard of microorganisms, a soil may be expected to contain almost any naturally occurring organic compound [Waksman, 1938]. The relative abundance of these extracellular compounds is far from that found in living tissues, however. Additional substances, perhaps characteristic of soil per se, are also present in abundance. From one point of view, the microbiologist is confronted with soil as a unique medium in which organisms live, not in pure culture but in highly complex populations comprising innumerable species; these exert a variety of associative and antagonistic effects [Waksman, 1945]. There seem to be countless numbers of different "species" of bacteria, yeasts, protozoa, fungi, as well as microfauna in soil. In assaying for unique biochemical activities, certain groups of these have been found to be geared to elemental cycles such as ammonitication, nitrification and nitrogen fixation. From a somewhat different point of view, Quastel [1946] adopted another con- ceptual scheme, namely that the soil as a whole be considered as an organ, comparable in some respects to a liver or a gland, to which may be added various nutrients, pure or complex, such as degraded plant materials, to- gether with rain and air, and in which enzymatic reactions can occur. The products of these reactions are important as steps in elemental cycles in the percolation (movement) of iron and aluminum, as humates, and in the formation of soil crumb structure. The notion here is that the soil bio- chemist is more concerned with the activity of microbes in soil than with precisely what they are with respect to size, shape or other characteristics that make up taxonomic schemes. In the form of enrichment cultures these two approaches come together. If glycine is perfused repeatedly through a sample of native soil, together with the products of microbial activity, those organisms capable of metabo- lizing glycine, ammonia and nitrite will increase greatly in numbers, and isolates of these, for example Nitrosomonas sp. can be more easily prepared. In this example, certain cells of the soil-organ are encouraged to multiply and to "cooperate" in the conversion of glycine to nitrate, carbon dioxide and water as principal products. Together with these reactions, however, and in the presence of carbohydrate, other soil organisms can synthesize

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The Biochemistry of Terrestrial Soils 149 anionic, high polymeric substances, some of which are adsorbed on the clays. A kind of a steady state exists at any interval in time, depending on the rates of addition and loss of metabolites to and from the soil. In the following is described some of what is known about biochemical reactions in soil and the nature of soil organic matter. It is not clear how well, or poorly, this information will be useful in anticipating the biological chemistry of Martian soils, if indeed, the surface of Mars has any areas comparable to Earth soils. It may, however, lead to some guesses as to what one should look for on Mars if life as we know it has some counterpart there. ECOLOGICAL CONDITIONS IN SOIL First of all, let us consider the environment in which the soil micro- organisms dwell. It differs rather drastically from that of a culture flask or an agar slant. There is a point to point variation in concentration of all solutes, interspersed in a matrix of clays, sand and humus which charac- terizes a spectrum of microenvironments. Further, at the surfaces of these particles as well as at plant roots there is a variation in molecular environ- ment characterized by gradation in pH and redox potential [Thimann, 1955; Kubiena, 1938]. Morphologists have dissected soil into three principal "horizons": A) the upper or alluvial layer that receives litter and from which material is, or has been, leached; B) the underlying illuvial layer that is, or has been, enriched; and C) the mineral, parent material underneath A and B horizons. These horizons may be well delineated in profile or the B horizon may arbitrarily grade into the other two. Pore space occupies 30 to 45 per cent of the soil and varies in water and air content. From A to C the total numbers of microbes per gram of soil decline and the ratio of aerobes to anaerobes declines. Soils with greater humus or clay content tend to hold more water, because of hydration and the presence of capillary pore space associated with small particle size, than do soils with the larger, silt particles. In "waterlogged" soils there are practically no air spaces, and in "air dry" soils, the pore liquid water has almost disappeared, leaving only water films about the particles. Microorganisms cannot grow in the absence of water. The lower limit of relative humidity at which growth is possible depends upon the species. Their activities may depend on the thickness of the water film; for example, Rahn [1913] found the optimum film thickness to be 20 to 40 p. for Bac. mycoides, an aerobe. However, some bacteria can be stored under dry

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150 RECOGNITION OF LIFE AND SOME TERRESTRIAL PRECEDENTS nitrogen, without growth. Winogradsky [1924] determined depths to which an aerobe, Azotobacter, and an anaerobe, Clostridium could grow in wet soils: at a moisture content of 23 percent the former were limited to the extreme surface and the latter were found throughout the soil. Recently, from a study of widely different soils, it has been found that "changeover" from aerobic to anaerobic metabolism of organic materials takes place in widely different soils at an oxygen concentration less than about 3 X 10~(i M, a very low concentration indeed [Greenwood, 1961]. Decomposition below this concentration in soil leads to the accumulation of fatty acids. One implication is that water-saturated crumbs of a soil that are more than about 3 mm in radius have no oxygen at their centers, and since crumbs of this size are present in most soils it means that pockets with anaerobic conditions are ubiquitous, and this provides an explanation for the universality of strict anaerobes. Oxidative processes abound in rela- tively warm, dry soils. On the other hand, in relatively wet soils organic matter content increases with increasing rainfall [Jenny et al., 1948]. Waterlogged soils, high in organic matter, tend to become acidic due to fermentation, whereas well-aerated garden soils are near neutrality, i.e., the pH is of the order of 6 to 7.5 as measured with wet pastes and a glass electrode system. At higher pH, as in calcareous soils, growing plants may show Fe or Mn deficiencies because of the insolubility of the corresponding phosphates and carbonates. The addition of sulfur results in the lowering of pH and the liberation of these cations, as will be discussed below. Upon examining the effective pH at the surface of soil colloids, however, a new concept emerges. Wherever charged surfaces exist in contact with water, the effective pH of the surface (pH,) will be lower than the pH in bulk (pHb). This difference has been expressed by Hartley and Roe as pH. = pHb + 8 /60 at 25 ° C where 8 is the electrokinetic potential of the particle [McLaren and Babcock, 1959]. Thus, chymotrypsin acting on a protein adsorbed on kaolin has a different pH optimum for enzyme action than is found in solution with the same substrate. The optimum pH of succinate oxidation with cells of E. coli adsorbed on an anion exchange resin differs from that of free cells suspended in solution, and a study of the initial velocities of oxidation of this substrate with free and adsorbed cells at pHb = 7 revealed that the velocity reached a maximum at a ten times higher concentration with adsorbed cells than with free cells [Hattori and Furusako, 1959]. These observations show that the heterogeneity of soil as an environment for microorganisms extends from the gross particle to the molecular level. It is recognized that in a "living" soil the elements are continuously sub- jected to biochemical transformation and to biological, mechanical and hydrological translocations.

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151 SOME METABOLIC PROCESSES IN SOIL The oxidation of ammonia or sulfur in soil has been studied with a biochemical technique of broad application. A nutrient solution is con- tinuously perfused through a column containing about 30 grams of soil in a water saturated, aerated condition [Lees and Quastel, 1946]. The tech- nique is splendid for characterizing nitrification in soil nitrifying organisms until a "saturation" of numbers exists and that the conversion NH4+ —* NO2~~ took place largely on paniculate surfaces. In another study, sulfur was added to a sample of Fresno fine sandy loam (a black alkali soil initially of pH 10.2 and a salt concentration of less than 0.2%) in the proportion of 1 g to 30 g of soil [McLaren, 19636]. The mixture was perfused with 200 ml water and the perfusate was analyzed for hydrogen ion and sulfate ions as a function of time. After a few days of perfusion, sulphate was found in the perfusate and the pH fell from the overall reaction 1 !/2 O2 + S -j- HZO —* H2SO4. The addition of Thiobacillus thiooxidans to the soil did not greatly modify the rate of the reaction, showing that related or- ganisms existed in the field soil initially. After 20 days of perfusion the soil was washed free of sulfate and perfused again; oxidation took place at the maximum rate. Clearly, at this stage the soil was enriched with respect to sulfur oxidizers. Sulfide and ammonia are reduction products of decompositions of some common organic compounds; both may be oxidized by certain soil microbes and a few autotrophs can use these oxidations as sole sources of energy [Freney, 1958]. In a soil perfused under aerobic conditions, and contain- ing an indigenous population, practically all sulfur in cystine becomes sulfate and the corresponding nitrogen becomes nitrate. The acidity of the soil increases and other organisms may be inhibited. A single sulfur compound, however, may yield different products on dissimilation by dif- ferent microbes. From cystine, Achromobacter cystinovorum produces only elemental sulfur. The following pathway must be considered as a logical overall process for the soil as an organ but is not meant to pertain to any particular organism: cysteine ~* cystine: —* cystine disulfoxide ~* cysteine sulfinic acid —* cysteic acid —* sulfate [Freney, 19616]. It must be remembered that the amount of free cysteine normally in soil is minute and one of the great difficulties of interpreting such experiments is to decide if the reactions observed are typical of the microbial population under field conditions or only when the system is jammed with substrate. An abundance of cysteine changes the redox potential of soil and may lead in other ways to an enrichment of only a small proportion of the qualitative makeup of microbes present. Nevertheless, in some areas of the world, for

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152 RECOGNITION OF LIFE AND SOME TERRESTRIAL PRECEDENTS example New England and Australia, most of the soil sulfur is locked up in some form of organic matter, and pasture lands in this region are, at present, mainly dependent for nutrition on a release of this sulfur by mineralization. From solubility studies the organic sulfur appears to be in the form of high molecular weight fulvic acid sulfates (see Humus, below). Here, an element has been taken out of circulation by the forma- tion of macromolecules. A similar story pertains to nitrogen. Perhaps it is needless to say that in the organ approach to soil we have no way of deciding which reactions may be attributed to a given species. Historically, isolates of single species have been studied, but in doing so, the possible interactions, mutual inhibitions and stimulations between spe- cies are missed. This difficulty is not unique to the concept of soil as a tissue, however. A good illustration involves manganese metabolism. Bromfield and Sherman found that manganese sulfate could be oxidized with the formation of a brown deposit of manganese dioxide on agar plates if par- ticular pairs of species were growing as colonies near each other. Two such pairs had Corynebacterium in common; the other organism could be either a Flavobacterium or a Chromobacterium. Obviously, the existence of associative action between microbes makes it virtually impossible to count the number of Mn++ oxidizing organisms in soil [Bromfield and Sher- man, 1950]. An ascomycete, Cladosporium, could oxidize this ion without association. If manganese sulfate is perfused through a neutral or slightly alkaline soil it is found that after a few days all the Mn* * has disappeared from the perfusate. Above 0.02 M the rate of formation of manganese dioxide falls rapidly, indicating manganese toxicity. Metabolic poisons also, including azide and iodoacetate, bring about a marked inhibition of the oxidation. Detailed observations have been summarized as follows [Quastel et al., 1948]: Biological Oxidation Mn*' Dismutation and biological reduction Biological reduction with organics and sulfur Dismutation (belowpH8)

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The Biochemistry of Terrestrial Soils 153 Reduction of manganese oxides in soils can be brought about with added glucose, thiols and polyphenols. These laboratory observations provide a useful explanation for the fact that applications of manganese sulfate to deficient field soils are often ineffective as MnO2 represents an unavailable form of the element. An increase in concentration of available manganese can be brought about by the addition of reducing forms of organic matter, reduction of pH to shift the "cycle" toward Mn**, inhibition of oxidation by specific poisons and stimulation of manganese reducing organisms. Here one can "overlap" the sulfur and manganese cycles to advantage: the addition of sulfur favors the reduced form of manganese by formation of sulfuric acid, with a reduction in pH, and by slow thiosulfate produc- tion. The addition of sulfur to a "manganese deficient" soil may exert a beneficial effect on the growth of plants [McLaren, 1962]. Generally, if appreciable amounts of an organic nutrient, such as glucose, are added to soil the results are profound and ramified. We mention phosphate accessibility as another example. Sperber has found that certain fungi, actinomycetes and bacteria can solubilize apatite (Ca3(PO4)2CaF2) through production of lactic, glycollic, citric and succinic acids. Reports that soil microorganisms increase the availability of phosphates to plants are numerous [Sperber, 1958]. SOIL ORGANIC MATTER AND SOIL STRUCTURE The addition of glucose to soil can have important physical consequences as well. Certain of the bacteria synthesize large amounts of polysaccharides as by-products or storage materials. Some of the added glucose goes into protein formation, thus removing soluble nitrogen from the soil solution (making nitrogen temporarily unavailable for plants). The polysaccharides, particularly those with carboxyl groups, become adsorbed to the soil colloids. Organic matter in soil plays an essential part in securing the structure that is required for high fertility. From synthesis at the expense of sub- stances of plant origin, and from autolysis of microorganisms, macromole- cules are produced that have the capacity to bind to clay particles. This binding is presumably through bonds such as R—CO2—Ca—clay, and may be represented 65, where the lines indicate macromolecular chains and the circles depict negatively charged clay particles. Bacterial gums, alginic acid, pectic acid and a large class of synthetic compounds also have this capacity. The net effects of adding such substances to soils high in clay and low in organic matter are improved aeration, tilth, water percolation rate, and

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154 RECOGNITION OF LIFE AND SOME TERRESTRIAL PRECEDENTS sometimes crop yield, all resulting from the formation of a more stable crumb structure and an increase in resistance to compaction [Quastel, 1953]. ENZYME ACTION IN SOIL We have reviewed some implications of soil biochemical activities in the fields of soil chemistry, physics and fertility. In these cases, the microbe has been an active contributor. Some investigators, however, have asked how much of the enzyme action in soil is due to this activity and how much is attributable to an accumulated "background noise" of enzyme activity in soil itself, that is, to enzymes adsorbed on clays or humus. One approach is to add toluene to soil to suppress microbial activity. Although toluene and even gasoline are substrates for some organisms, in short-duration experiments life activities may be considered suppressed. Similar results are obtained if soil is first sterilized with an electron beam [McLaren et al., 1962]. With a Dublin clay loam, over half of the glycerolphosphatase activity was found after complete sterilization with a dose of 5 Mrep. It will be of significance to know if this residual enzyme activity is outside non-viable cells, and to what extent phosphatase in dead cells is capable of acting on substrate added to the sterile soil [Skujins et al., 1962]. Haig [1955] has fractionated soils and found that most soil esterase activity is in the clay fraction. How the enzyme is held by the clay, how it is accumulated and how it can be isolated for study are intriguing questions. An interesting and important problem of soil enzyme action requires a knowledge of the kinetics of enzyme action at solid-liquid interfaces. The phenomenon just described involves the action of an adsorbed enzyme on a soluble substrate. Conversely, microbes secrete soluble enzymes which act on insoluble substrates such as cellulosic debris, soil organic matter, chitin, etc. The kinetics of these reactions are not represented solely by equations of the Michaelis-Menten type for classical reactions in solution. Instead, the limiting rates of reaction involve diffusion of substrates to surfaces, adsorption of enzymes on particles and the local pHE discussed above. Equations for these situations are now available for evaluation [McLaren, 1962]. The role of water and the minimum activities of water at which enzyme action can take place have yet to be evaluated. SOIL DEVELOPMENT Studies of the microbiology of soil development have only begun. Sand- dune communities were chosen by Webley et al. [1952] because the devel-

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The Biochemistry of Terrestrial Soils 155 opment of a soil microflora could be traced from the simple conditions of early colonization of bare sand through a series of communities of increas- ing botanical complexity accompanied by the formation of a soil. Microbial counts were obtained from a transect across a dune at New- burgh (Aberdeenshire). In progressing from open sand past early fixed dunes to dune pastures and heath, the number of bacteria rise from a few thousand to millions per gram as soon as vegetation colonizes the sand. There is a fall in numbers of bacteria, but not of fungi, as heather, with an acid humus, enters as a dominant plant. In comparing rhizosphere soil with nearby soil there is a marked increase in microbes on passing from open sand to root surfaces of Agropyron and Ammophilia, with predominance of Cornebacteria, Mycobacteria and Nocardia among the "bacteria" through- out. Among the fungi, however, Penicillum sp. predominated in open sand and Cephalosporium near roots. Each plant species develops a unique rhizosphere flora which overlaps only in part those of other species. There can be little doubt that the activity of the microbes contributes to the development of the soil and the maturation of the habitat in a reciprocating way with higher plants growing from a sand and salt milieu. Elucidation of the development of a soil profile, involving the leaching and translocation of aluminum, iron, humic acids and other chelating agents, etc., together with co-precipitation, will require experiments of long duration. The evidence is presumptive and "ad hoc" as in evolutionary theory generally, but no one seems seriously to question the overall picture. SOME ORGANIC SUBSTANCES IN SOIL Soil Enzymes The bulk of organic substances in Earth soils undoubtedly arises from the decomposition of animal and vegetable residues by microorganisms. This does not imply liberation by autolysed cells, but rather cycling of the elements, first through the digestive and then the synthetic activities of soil flora and fauna. The microfauna are very important in the A horizon where leaf litter is degraded in the presence of abundant oxygen, particularly in acid, forest soils. Lower down in the soil, the flora tend to finish the catabolism begun by microfauna, and all recognizable vestiges of plant cytological structure vanish [Kononova, 1961]. These degradations release enzymes, the exoenzymes of digestion. On starvation, microflora and fauna may autolyse with release of intracellular enzymes. These details have not been worked out. In Table 1, Briggs and Spedding [1963] have listed some enzymes found in soil. We know nothing of the abundance of these en-

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156 RECOGNITION OF LIFE AND SOME TERRESTRIAL PRECEDENTS TABLE 1. Some Soil Enzymes (From Briggs and Spedding, 1963) Name of Enzyme Reaction Catalyzed 1. Urease 2. Amylases 3. Glycosidases 4. Asparaginase 5. Aspartate-alanine transaminase 6. Catalase 7. Invertase 8. Proteases 9. Dehydrogenases 10. Glutamate-alanine transaminase 11. Glycerophosphatase 12. Inulase 13. Leucine-alanine transaminase 14. Nuclease 15. Peroxidase 16. Phosphatases 17. Polyphenol oxidase 18. Tyrosinase Urea —» ammonia + carbon dioxide Starches —» sugars Glycosides —» sugars + aglycones Asparagine —» aspartate + ammonia Aspartate + pyruvate ^± alanine + oxalacetate 2H,O2 -» 2H,O + O2 Sucrose ~» glucose -)- fructose Proteins —» peptides Reduced substrate —> oxidized substrate Glutamate + pyruvate ^± alanine + a-ketoglu- tarate Glycerol phosphate —» glycerol + phosphate Inulin—» fructose and fructose oligosaccharides Leucine + pyruvate ;=; alanine + a-ketoisovalerate Purines, etc. —» ammonia + keto-purines (etc.) Substrate + H2O2 —» oxidised substrate + H3O Organic phosphates —» compound + orthophos- phate Polyphenols + O2 -~» quinones + H2O Tyrosine + O2 —» o-quinones + H2O zymes in soil and whether they may be found both in and outside soil microbes. Soil phosphatases are of several kinds, e.g., glycerol phosphatase and phytase. The latter is very important in the soil phosphorus cycle. About 60 per cent of soil phosphorus is typically organic but only a small fraction of this is of known structure, namely as in phytin. Of all the soil organic substances, individual enzymes are most easily tested for. Although only small amounts are present, the turnover of sub- strate, particularly in the cases where the products are volatile or fluorescent, is easily measured quantitatively. In fact, B. Rotman has developed a test for one or more enzymes which is sensitive at the one-enzyme-molecule level. Clearly, there cannot be much free enzyme-protein in soil as proteins are nutrients for organisms, but enzymes may be chemically cross-linked with humus. It is known that such bound enzymes may still function [McLaren, 1963a]. It is also of interest that soil sterilized by ionizing radiation can still respire, but at a reduced rate, indicating the presence of some residual organized enzyme systems [Peterson, 1962].

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157 Organic Nitrogen Compounds in Soil Among the recognizable organic nitrogen compounds in soil are ammo sugars, ami no acids and nucleotides, Table 2. Ammo sugars such as glucosamine are derived from chitin. It is clear from the table that spectroscopic examination of soil for peptide bonds or amino acids, without suitable extraction from soil humus, is out of the question. Some infrared spectra of soil organic matter have been reported; the evidence for soil protein is almost nil [Jenkinson and Tinsley, 1960]. Further, the amino acids derived from soil organic matter following hydroly- sis are not of protein origin. The liberated amino acids may have been in condensation products of phenols or quinones [Kononova, 1961]. Free amino acids may range from 2—4 mg per kg soil; in so.il the polybasic amino acids are adsorbed to the clays; tryptpphan is not [Putnam and Schmidt, 1959]. Organic Sulfur Compounds in Soil At present we can conclude only that there are some organic sulfur compounds in soil; in fact, in some Australian soils most of the soil sulfur may be organic [Freney, 196la]. Some of it is, of course, in amino acids (Table 2). Organic Phosphorus Compounds in Soil As already mentioned, most of the soil phosphorus is organic [Bower, 1949]. Very little nucleic acid exists in soil (1 ppra), perhaps no more than in the microbes themselves. In one such test the total organic phos- phorus ranged from 300-500 ppm [Adams et al., 1954]. Several forms of inositolhexaphosphate are found in soil [Cosgrove, 1962]; they occur from 100 to 300 ppm [Anderson, 1961]. Carbohydrates Sugars added to soil are quickly metabolized and free sugars are absent in natural soil. On the other hand, polysaccharides of microbial origin are present in soil [Whistler and Kirby, 1956], to the extent of 0.1% [Lynch et al., 1958]. These contain galactose, glucose, mannose, arabinose, etc. Humus The major portion of soil organic matter is called humus. It is insoluble in water, but about one third of this organic matter can be removed from

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158 RECOGNITION OF LIFE AND SOME TERRESTRIAL PRECEDENTS S 1 0 's \o ^ trt •§ o\ o\ .—. 8 3 ^ to .. U 0emne 1 c C I — i a -H § U k. i— i 09 X K 03 ' ' a.^1 Z ^ 8 .— . o e L^ ca O '1 '§5 a cs o ? «*1 9 c •o " C/l § c £s£ C5 O d 'c u a B o U TJ O ~l C 1 •„ 00 Z (§£ ti i .. tN ZIM 0 o H U < (X) H "g & .2 1 •a f U 1 c •a •a V '5 .0 '% '3 U •3 u ca q > a < < 3? Z Z i 2 . a «0 •o CO •o 'i U O 0 "*- u 'i c O o Q c rt "O C c C s M f. 1 X) t« D 0 00 H E | 2 a.

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The Biochemistry of Terrestrial Soils 159 soil by extraction with alkali. The extraction is improved by prior treat- ment of the soil with chelating agents, since humus seems to be held to clay by bonds involving di- and tri-valent metals. Alternately, treatment of soil with hydrofluoric acid removes sand and solubilizes clay minerals, thereby leaving the organic matter. Following neutralization of alkaline extracts, humic acid is precipitated and polymeric fulvic acid remains in solution. These composite acidic substances contain chemically bound nitrogen, phosphorus and sulfur. Of course, the solution phase also con- tains the nitrogen, sulfur and phosphorus micromolecular compounds described above, and polyuronic acids. Humus may be found in low amounts, about one per cent in desert soils and up to 80 per cent in so-called organic soils. About one-half of the nitrogen in fulvic acid occurs as compounds that are deaminated on hydrolysis; about one-fourth appears as amino acid precursors and one-tenth as amino sugars [Stevenson, I960]. Humic acid from one source has been found to contain about seven per cent methoxyl groups [Johnson, 1959], together with a number of other functional groups (Table 3). A summary of infrared absorbances of organic matter from A and B horizons is given in Table 4. [Wright and Schnitzen, 1959]. REMARKS CONCERNING TESTS FOR LIFE IN SOIL This problem can be divided into two parts, namely the test for living creatures and testing for products of metabolism. The latter products must be distinguished from chemicals formed under the action of other natural agents, such as radiation. (For a review see McLaren and Shugar [1954].) The former products, as we have tabulated, include enzymes, polysaccha- rides, and certain aromatic substances of unknown structure that occur in a percentage that is perhaps higher in humus than in products formed by radiation. Test methods for enzymes in soil are already under development, for example, phosphatase in the Multivator. We do not now know about the relative abundance of enzymes in soil and this should be actively explored. For example, urease is prevalent and urea labeled with radioactive carbon should provide a sensitive assay, comparable to that for phosphatase in the Multivator. A difficulty with Gulliver, and the Wolf Trap, is that we do not know what universal substrate system can be supplied to the Martian "soil" and a test for autotrophs may require growth under light. On the other hand, all microbes contain some enzymes in common and a battery of enzyme

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160 RECOGNITION OF LIFE AND SOME TERRESTRIAL PRECEDENTS .2Pin ^ ^ ,__, r— i ,__, r— i ,__, o ^ ". i ^ ON ON ON ON NO NO r-i c >o in NO in ON ON .~ i ON i— i ON ON ON •1 r— i ON Johnson [1959 and Schnitze o\ m W0ight and Schnitzen [1 Johnson [1959 W0ight and Schnitzen [1 W0ight and Schnitzen [1 ,__, r~ W0ight and Schnitzen [1 ON in W0ight and Schnitzen [1 ON NO Wald0on and Mo0tensen [ References ON ON in in i — i in 1 I 1 •* c ON C 1 P .SP •C O 3 M oa k. u E X § .S '1 M 'v i 8 ts c 1 -C $ m CO NO o Ou "rt ri NO 00 1^ ON B 0 cfl E Q. ,^ u 8 CO 0 i o 00 u u c o u &* cr u E tn eo Tf 00 00 00 LU < S. 1 O "3 ^a >, ^ 1 u 4> u Methoxyl Carbonyl Carboxyl Unknown Aromatic cx E -S Quinone Phenolic 'S. Unknown D- u 2 0 SO X 2 a PC S u 2 U i u o

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The Biochemistry of Terrestrial Soils 161 TABLE 4. Major Bands and Relative Absorbances in the Infrared of Extracted Organic Matter [Wright and Schnitzen, 1959] Fre- quency (cm.-i) Relative Absorbance A horizon B horizon Interpretation 3,380 2,910 2,840 2,600 1,720 1,620 1,450 1,400 1,250 1,200 1,030 strong strong medium medium shoulder shoulder strong strong O weak O medium shoulder O shoulder strong strong O medium O medium O Hydrogen bonded—OH and bonded— NH groups Aliphatic C—H stretching vibrations Aliphatic C—H stretching vibrations Carboxyl C—H stretching vibrations Carboxylic carbonyl Joint interaction of hydroxyl and carboxyl with carbonyl [McLaren, 19636], also possibry carboxylic groups associated with metals so as to give the carboxy- late structure [Hattori and Furusako, 1959] CH3 or CH2, or both, in plane deforma- tion vibrations Carboxylate Phenoxyl C—O Carboxyl Si—O of silica due to the presence of clay assays may be more likely to succeed as a presumptive test for evidence of life. When water is added to soil, after it has been air-dried, there is a burst of biological activity and a liberation of carbon dioxide [Birch, 1958]. This suggests another approach to the experimental recognition of life. One would expect that this metabolic activity would result in the liberation of heat and that the heat could be measured calorimetrically [Gloser, 1964]. (Cf., Chapter 20, Section 15.) A microbiological fractionation of isotopes might also be useful in life detection; for example, fractionation of sulfur by Desulfovibrio desulfuri- cans is rather dramatic [Kaplan and Rittenberg, 1964]. Finally, since living things generally contain free radicals, and humus contains trapped free radicals, probably of the semiquinone and quin- hydrone type, the finding of free radicals in Martian soil could be taken as presumptive evidence. Offhand, both the calorimetric and free radical measurements appear to be potentially very important; they are non-specific in the sense that

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