Tải bản đầy đủ - 0 (trang)
III. Studies on Soil Polysaccharides

III. Studies on Soil Polysaccharides

Tải bản đầy đủ - 0trang

200



G. D. SWINCER, J. M. OADES, A N D D. J . GREENLAND



When preceded by complete hydrolysis into monosaccharides and removal of interfering compounds from the hydrolyzate, colorimetric

methods can give useful estimates of the carbohydrate content of the soil,

either before or after extraction, and of crude extracts. The colorimetric

methods are normally applicable only to single classes of monosaccharides, such as the hexoses, pentoses, uronic acids, or hexosamines, and

even within a particular class none of the methods gives the same color intensity for equimolar concentrations of the different individual monosaccharides. Moreover, optimum hydrolysis conditions vary at least from

one class of sugars to another, and probably also from one individual

sugar to another. Clearly, therefore, the most precise determinations of

the proportion of the total carbohydrates extracted would require the

measurement of the amount of each individual sugar in both the soil and

the extract.

Individual sugars, or the different classes of sugars, have been measured in soils and purified soil extracts (Graveland and Lynch, 1961;

Thomas and Lynch, 196 1; Ivarson and Sowden, 1962; Sowden and Ivarson, 1962; Gupta et al., 1963; Gupta and Sowden, 1965; Cheshire and

Mundie, 1966). Only Parsons and Tinsley (1961), Lynch et al. (1957,

1958), and Swincer et al. (1 968a,b) have attempted to relate the amounts

extracted with the amounts originally present in the soil or left in the

soil residue after extraction.

The results of Parsons and Tinsley are probably not accurate, particularly with respect to uronic acids, as the authors themselves admit. Although Lynch et al. (1 957, 1958) claimed to have measured the recovery

of the original carbohydrates in various extracts by separating and estimating seven different sugars, the yields reported were undoubtedly too

high as a result of the low values for “total soil carbohydrate” that must

have accompanied the very mild hydrolysis conditions used.

b. Polymer Degradation. The detection and evaluation of damage to

the polysaccharide molecules during the extraction process is by no

means easy. Positive and conclusive evidence against changes in the

carbohydrate polymers during extraction is virtually unobtainable because it is not yet possible to know the properties of these molecules before isolating them from the soil. Any information that can be obtained

must be either indirect or of a negative kind.

Whitehead and Tinsley ( 1964) made a useful indirect assessment of the

likely degradative effect of their extraction procedure on soil polysaccharides by subjecting several other natural polymers (starch, alginic

acid, chitin, cellulose, gluten) to the same treatment.

Bernier (1 958a) compared the viscosities of polysaccharides extracted



SOIL POLYSACCHARIDES



20 1



from soils by different reagents, and considered that the preparations with

higher viscosity were those that had suffered least degradation during

extraction. This is a worthwhile approach provided samples are free from

salt and ash.

Swincer et a f . (1968a) preferred to use the results from analysis of

extracts on Sephadex (3-25 as an indication of degradative treatments.

When reagents caused extensive fragmentation of polysaccharides by

hydrolysis or other means, a large proportion of the extracted materials

were “low molecular weight” according to gel filtration on Sephadex

(3-25. Thus, it was found that all methods involving high temperatures,

even methods based on water, led to extensive polysaccharide degradation, and 0.5 N NaOH at 20°C. was the most efficient, single, nondegradative extractant. Again it should be emphasized that such experiments

cannot provide conclusive evidence against degradation (e.g., by a relatively large average polymer size or the absence of specific degradation

products), even though they can give indications of whether damage has

occurred and thus help to rule out unsuitable extraction treatments.

3 . Extractants of Soil Polysaccharides

Extractants that have been used for obtaining polysaccharides from

soils include water, aqueous buffers and complexing reagents, dilute

mineral acids, organic reagents, and alkalis.

The yields of soil polysaccharides that have been obtained by various

workers using a number of different procedures are nearly all based on

gravimetric determinations and, therefore, provide only an approximate

guide to the efficiency of each extractant.

a. Water. Water has been a popular extractant for soil polysaccharides because of the simplicity of extraction, the relatively low

simultaneous extraction of humic materials, and the ease of subsequent

purfication. Most of the procedures involve extraction at temperatures of

at least 70”C., either with continuous shaking or by use of Soxhlet’s

apparatus (Mortensen, 1961; Keefer and Mortensen, 1963; Mortensen

and Schwendinger, 1963; Thomas et al., 1967). Forsyth ( I 950) found that

autohydrolysis of an extracted polysaccharide occurred in distilled water

at 1 OO’C., 90 percent of the arabinose and considerable amounts of ribose

being released in 24 hours. Furthermore, Swincer et al. ( 1968a) considered hot water extraction to be degradative on the basis of analysis of the

extract on Sephadex (3-25.

What quantitative information there is shows that hot water extracts

up to about 2 percent of the soil organic matter, which could represent

up to one-quarter of the total carbohydrates.



202



G. D. SWINCER, J. M. OADES, A N D D. J. GREENLAND



b. Aqueous Buffers and Complexing Reagents. Buffer solutions

(Bernier, 1958a) and complexing agents, such as sodium phyrophosphate

(Bernier, 1958a; Lynch et al., 1958; Mortensen and Schwendinger,

1963) and disodium EDTA (Barker et af., 1965, 1967), have been used

in attempts to obtain polysaccharides with a minimum of alteration from

their natural state. In general, yields were low (less than 5 percent of the

soil carbohydrate), and it is impossible to know whether the material

extracted was representative of the total. For this reason such mild reagents are probably of limited value for investigating the whole soil

polysaccharide fraction, in spite of their ideality in terms of the risk of

hydrolysis. A possible exception is the use of metal complex reagents

such as Schweitzer's reagent (cuprammoniurn hydroxide) for extracting

cellulose.

Schweitzer's reagent was first applied to soils by Daji (1932), and its

usefulness has been confirmed by Gupta and Sowden ( 1964). However,

it might well be replaced to advantage by one of the more recently developed metal complex reagents with high cellulose dissolving power

(Jayme and Lange, 1963).

c . Dilute Mineral Acids. Although inorganic acids are relatively inefficient reagents for extracting humic materials from soil (Mortensen and

Himes, 1964), they are more effective for the polysaccharide fraction.

Barker et al. ( 1 965, 1967) chose 0.6 N H2SO4as the extractant for routine

isolation of larger quantities of polysaccharide materials from an English

muck soil. Other reagents were superior in terms of yield, but the acid

extracts were more easily purified. Furthermore, treatment with acid

apparently hydrolyzed bonds between polysaccharides and humic materials, thus simplifying the separation of polysaccharides from these

materials. Acid hydrolysis of the polysaccharides themselves was prevented by carrying out the extraction at 3°C.

Black et af.( 1955) also used dilute mineral acid (0.09 N HCl) to extract

a polysaccharide complex from peat. The yield was low compared with

those obtained by other workers with different reagents: furthermore, it

is quite likely that some degradation occurred under the conditions used

( 1 hour at 60" to 70°C.).

H F at 20°C. was the most efficient acid extractant used by Swincer et

af.( 1 968a), but the extracts are not easily analyzed; thus, HCI was preferred and released 10 to 20 percent of the carbohydrate from a range of

great soil groups, being most efficient in soils rich in calcium carbonate

or free iron and aluminum oxides.

Another acid extractant that has been tried is a suspension in water of

the H+ form of a cation exchange resin (Amberlite 1 R-120) (Barker et al.,



SOIL POLYSACCHARIDES



203



1965, 1967). This treatment extracted slightly less carbohydrate material

than 0.6 N H&O, under the same conditions, and the mineral acid was

preferred for this and other reasons.

d. Organic Reagents. Parsons and Tinsley ( 1961) extracted polysaccharides from a variety of soils by double reflux with 0.2 N lithium bromide in anhydrous formic acid. The proportions of soil organic matter

(3.5 to 17.5 percent) extracted as polysaccharides are some of the highest

on record. However, after extraction, a considerable amount of carbohydrate still remained with each soil residue, and the authors themselves

admitted that the yields quoted were probably overestimates because

some of the C 0 2 evolved in the decarboxylation method for determining

uronic acids almost certainly arose from nonuronide materials. Deuel

et al. (1960) have established that ready decarboxylation is a general

property of the colored humic substances. I t is possible that the reducing

sugar content was also overestimated due to the presence of other reducing substances in hydrolyzates of the isolated polysaccharide materials.

Whitehead and Tinsley ( 1964) extracted high proportions of the organic

matter from several soils by refluxing with a mixed reagent consisting of

0.4 M boric acid, 0.4 M oxalic acid, and 0.2 M lithium chloride in dimethyl formamide (DMF). The polysaccharide contents of the preparations were not determined and attempts to separate the dissolved polysaccharides from the nonpolysaccharide components were not successful.

Although exposure to high temperatures was not prolonged, hydrolysis

of soil polymers almost certainly occurred as appreciable degradation

was observed when the procedure was applied to such natural polymeric

material as starch, alginic acid, chitin, and gluten. Swincer et al. ( I968a)

also considered this procedure to be degradative, based on analysis of

the extract by Sephadex (3-25.

DMF (without dissolved acids or salts) was also among the extractants

examined by Barker et al. (1965, 1967). Extraction was for 24 hours at

room temperature, and the carbohydrate yield was very low. However,

the yield was increased almost 30-fold if the soil was hydrogen ion saturated before extraction with DMF. Of the other organic extractants examined, N-methyl-2-pyrrolidone was only slightly more efficient than

DMF (without the pretreatment), but 8 M urea was quite superior. Not

enough data were given to allow adequate assessment of the potential

of the different reagents as extractants for soil polysaccharides.

The only soil examined was a muck, and relative yields could be quite

different in mineral soils where many of the polysaccharides are likely

to be strongly associated with inorganic colloids.

e. Alkalis. I n spite of some objections and criticisms, dilute NaOH,



204



G . D. SWINCER, J. M . OADES, A N D D. J. GREENLAND



the classical extractant for soil organic matter, has been used frequently

for isolating soil polysaccharides (Forsyth, 1947, 1950; Rennie et al.,

1954; Dubach et af., 1955; Chesters et al., 1957; Muller et af., 1960;

Graveland and Lynch, 196 1 ; Acton et al., 1963a; Dormaar, 1967; Oades

and Swincer, 1968; Swincer et al., 1968a,b). This extractant is more

efficient than most others, an observation which has been made repeatedly in studies of the extraction of other soil organic materials (e.g., Bremner

and Lees, 1949; Choudri and Stevenson, 1957; Evans, 1959). In contrast

to most other workers, Whistler and Kirby ( I 956) reported that hot water

and cold 0.5 N NaOH extracted comparable amounts of “purified” polysaccharide from an American soil. However, the amount of polysaccharide in their initial crude 0.5 N NaOH extract may have been much

higher than they reported, as considerable losses could have occurred

during the purification treatment.

Large amounts of humic materials are always extracted with the polysaccharides. The separation of these materials from the polysaccharides

has proved to be particularly difficult, and is a significant objection to

the use of this extractant.

Another objection to the use of NaOH has been the possibility of

damage either by hydrolysis of the polymers or by autoxidation (Bremner,

1954; Tinsley and Salam, 1961 ; Dubach and Mehta, 1963). There is good

evidence that some organic materials are, indeed, altered under alkaline

conditions (Bremner, 1950; Choudri and Stevenson, 1957; Evans, 1959)

although Kononova (196 1 ) discusses other evidence which, she says,

shows that extraction with alkaline solutions does not change the nature

of humic substances essentially. As these experiments were concerned

only with overall effects, the conclusions reached do not necessarily apply

directly to the polysaccharide materials, which constitute only a rather

small proportion of the extracted organic matter. Bremner (1 950) found

that the oxygen taken up during alkaline extraction of soils went into the

acid-insoluble (humic acid) fraction rather than into the fulvic acid fraction which contains most of the carbohydrates (Acton et af., 1963a;

Dormaar, 1967; Swincer et al., 1968a).

Some alkaline degradation of the polysaccharides might be expected.

Neuberger and Marshall ( 1966b) and Horton and Wolfrom (1 963) state

that glycosidic linkages between monosaccharides are ordinarily stable

to alkali, but that in certain circumstances a slow stepwise degradation

may take place from the reducing end of a polysaccharide molecule.

Saccharinic acids are formed and eliminated and new reducing groups are

exposed until this “peeling” reaction is intercepted by a “stopping” reaction in which release of the saccharinic acid is inhibited by an unfavor-



SOIL POLYSACCHARIDES



205



able glycosidic linkage. The rate of the “peeling” reaction, which can

occur in the absence of oxygen, is influenced by the position at which the

penultimate sugar residue is attached (Kenner and Richards, 1957;

Whistler and BeMiller, 1958). If this linkage is (1 +. 3) the terminal

residue is readily detached, but ( I +. 4) and ( 1 + 6) links are much more

stable, and ( 1 -+2 ) links are resistant to the action of alkali even under

very vigorous conditions.

Thus under mild alkaline conditions, some shortening of polysaccharide chains is possible even though there is no random fragmentation

such as occurs in hot mineral acids. The presence of oxygen in an alkaline solution of a polysaccharide can lead to additional damage. I n this

case the individual sugars may be oxidized without changes in polymer

size (by hydrolysis of glycosidic bonds). In order to reduce this risk,

Choudri and Stevenson (1957) advocated the addition of stannous

chloride to the soil sample before extraction with NaOH; other workers

(e.g., Bremner, 1950) have carried out alkaline extraction of soil organic

matter under an atmosphere of nitrogen.

Tinsley and Salam ( 196 1) mentioned two other undesirable features of

NaOH as an extractant of soil organic matter. First, silica is dissolved

from the mineral matter in the soil and contaminates the organic fractions

separated. This can be overcome by concentration of a solution of the

partly purified polysaccharide; this results in polymerization and precipitation of the silica (Swincer et al., 1968a).

Secondly, NaOH is known to dissolve some protoplasmic and structural components of fresh organic tissues. The problem is eliminated if,

before extraction, the free organic residues are first removed from the

soil by flotation sieving (Roulet et al., 1963a) or densimetric fractionation

with heavy liquids (Ford et al., 1969).

$ Sequential Extraction Treatments. No single reagent has given

complete extraction of soil polysaccharides, and for maximum extraction multiple treatments are necessary. This is so with other organic

materials, e.g., humic materials (Choudri and Stevenson, 1957; Bremner

and Harada, 1959) and organophosphates (J. K. Martin, 1964). Studies

on ten great soil groups showed that a double treatment, using I N HCI

followed by 0.5 N NaOH, removed more than SO percent of the soil carbohydrate in all except two cases (Swincer er d.,1968a). A third treatment using acetic anhydride containing 2.5 percent concentrated H & 0 4

at 60°C. for 2 hours brought the total yield up to 80 percent or more except in an ando soil and a red earth. Extraction with NaOH can be increased by ultrasonic dispersion. Thus the sequential procedure should

give almost complete extraction in many cases and although certain soils



206



G. D. SWINCER, J . M . OADES, AND D. J . GREENLAND



may require special approaches this procedure would appear to have

general applicability (Fig. 1). These results were obtained on soils after

the removal of partly decomposed plant remains by a densimetric procedure (Ford et d . , 1969).

SOIL

Ultrasonic dispersion in a



LIGHT FRACTION - partly

decomposed plant and

animal remains



SOIL RESIDUE



1



I N HCI 20"



1 N HCI EXTRACT - add EDTA,

neutralize with NaHCO,.

Concentrate for gel filtration on

Sephadex and Biogel



SOIL RFSIDUE

Ultrasonic dispersion in



SOIL RESIDUE

Acetic anhydride + 2.5%

coric. H2SO4 60"



SOIL RESIDUE



0.5 N NaOH extract - pass

through H+ Dowex 50 and

Polyclar AT columns.

Concentrate for gel filtration

on Sephadex and Biogel

REACTION MIXTURE -dilute

tenfold with water and extract

acetylated polysaccharides in

CHCI,. Concentrate the extract

for gel filtration on Sephadex

LH-20



Hydrolyze to

determine

residual

carbohydrate

content



FIG.1. Procedure for isolation of polysaccharides from other soil materials. The light

fraction usually contains from 10 to 50 percent of the total soil carbohydrate. Of the remainder, 10 to 20 percent occurs in the acid extract, 30 to 50 percent in the NaOH extract,

20 to 30 percent in the acetic anhydride extract, and 10 to 20 percent may be left in the final

residue. Details of this procedure are described by Swincer et af. (1968a,b).



C . PURIFICATION

OF SOILPOLYSACCHARIDES



I . Introduction

After removal of solids by centrifugation, the first step in the recovery

of polysaccharides from an extract depends on the particular extractant

used. The specific primary treatments that have been applied will be discussed very briefly before consideration is given to the more general

procedures used for subsequent purification.



SOIL POLY SACCHARIDES



207



2 . Primary Purijication Treatments

With water extracts, the initial purification treatment is simplified because the problem of removing solutes contributed by the extractant does

not arise. In fact some workers (Duff, 1952a; Whistler and Kirby, 1956)

have omitted the “primary treatment” and have applied the more general

procedures for purifying polysaccharides directly to the concentrated

extract. However, Mortensen and co-workers (Mortensen, 196 1 ; Keefer

and Mortensen, 1963; Thomas et al., 1967) favored an initial acidification step (adjustment to pH 2 with N HCI) to precipitate humic materials.

To neutralize acid extracts, Black et al. ( 1 955) used 40 percent NaOH

while Barker et al. (1 965, 1967) used NaHC03. The latter authors examined polysaccharides both in the resulting precipitate and in the

supernatant. The precipitated polysaccharides were brought into solution with 0.3 N HCI before the application of further purification treatments.

To avoid precipitation of polysaccharides during the neutralization of

acid extracts Swincer et al. (1968a) added EDTA before NaHCOa. The

EDTA prevented precipitation of di- and trivalent cations in the acid

extract and consequently hydroxides of metals did not occur as precipitates associated with polysaccharides.

Parsons and Tinsley (1 96 I ) used diisopropyl ether to precipitate

organic materials from anhydrous formic acid extracts of soil and Whitehead and Tinsley ( 1 964) treated DMF extracts with diethyl ether in the

presence of acetic acid. The precipitates were dissolved in water and

formic acid, respectively, in readiness for further purification treatments.

Where alkaline solutions have been used for polysaccharide extraction,

the first purification step has been acidification to pH 2 to 3 to precipitate

humic acids. The acidification treatment removes a large proportion of

the organic components of the extract, as well as some of the inorganic

materials, but the precipitate may also contain significant amounts of

carbohydrate material (Lynch et al., 1957; Acton ef al., 1963a; Dormaar,

1967). Dormaar ( 1967) reported that some humic acid precipitates contained up to 9 percent of the total soil carbohydrates, and Acton et al.

obtained values as high as 12 percent.

This association of carbohydrates with the humic acid fraction is

probably due to coprecipitation involving metal cations. Swincer et al.

(1968a) found that 8 percent of the soil carbohydrate was precipitated

with the humic acid fraction obtained by direct addition of acid to the

NaOH extract, but only 1 percent of the soil carbohydrate was present

in the humic acid fraction obtained by passage of the NaOH extract

through a column of coarse H+ Dowex 50. The humic acid fraction was



208



G . D. SWINCER, J. M. OADES, AND D. J. GREENLAND



trapped in the resin column, and the fulvic acid solution emerging from

the column was free of metal cations and was easily concentrated without the formation of precipitates.

Cellulose extracted from soils with Schweitzer’s reagent was recovered by precipitation with 5 volumes of 80 percent ethanol (Gupta

and Sowden, 1964). The precipitate was washed successively with water,

N HCl, and water before being dried. This product was reported to be

reasonably pure without further treatment.



3 . Secondary Purification Treatments

The purification procedures commonly applied to soil polysaccharide

extracts after the appropriate initial treatment include: (a) dialysis to

remove salts, solvents, and other low molecular weight materials; (b)

charcoal filtration to remove colored compounds; (c) precipitation of the

polysaccharides from aqueous solution by the addition of acetone,

alcohol, or other reagents; and (d) various deproteinization procedures.

Dialysis in Visking tubing against distilled water has been the most

commonly used purification treatment, having been applied in all cases

of extraction with hot water (Duff, 1952a; Whistler and Kirby, 1956;

Clapp, 1957; Mortensen, I96 1 ; Keefer and Mortensen, 1963; Mortensen

and Schwendinger, 1963; Thomas et al., 1967) and acid (Black et al.,

1955; Barker et al., 1965, 1967) as well as to phosphate buffer extracts

(Bernier, 1958a), a sodium pyrophosphate extract (Mortensen and

Schwendinger, 1963), and an acidified NaOH extract (Muller et al.,

1960). Losses of carbohydrate during dialysis of various extracts against

distilled water are considerable (Clapp, 1957; Muller et al., 1960).

Swincer et al. (1968a) showed that much of this material could be recovered from the dialysis water as low molecular weight fragments, but

not as monosaccharides. However, significant amounts were sorbed on

dialysis tubing and could not be removed by washing with water.

Forsyth (1 947) introduced the use of acid-washed animal charcoal for

the sorption of organic materials from classical fulvic acid extracts.

Charcoal will remove most of the organic materials and all the color from

such extracts and has been used to purify polysaccharides not only from

the fulvic acid fraction of NaOH extracts (Dubach et al., 1955; Muller

et al., 1960; Swincer et al., 1968a), but also from a hot water extract

(Whistler and Kirby, 1956) and phosphate buffer extracts (Bernier,

1958a). However, recoveries of polysaccharides from charcoal are poor.

Whistler and Kirby (1 956) measured a recovery of 14 percent and

Swincer et al. (1968a) reported a 12 percent recovery. Thus while relatively pure polysaccharides can be obtained by this procedure, losses

are very high. Swincer et a f . (1968a) increased recoveries to 30 percent



SOIL POLYSACCHARIDES



209



by using 8.5 N acetic acid as eluant, and even up to 65 percent by pretreating the charcoal with stearic acid as described by Dalgleish (1955),

but results obtained were difficult to reproduce.

Polyclar AT (cross-linked polyvinyl pyrrolidone) which is a strong

adsorbent of polyphenolic compounds (Sanderson and Perera, 1966)

proved to be most useful for separating colored materials from polysaccharides in “fulvic acid” solutions. Recoveries of carbohydrates from

columns of Polyclar AT were greater than 95 percent, and almost complete removal of colored compounds occurred when the pH was lowered

to 2 (Swincer et al., 1968a).

Very often polysaccharide materials have been recovered from

aqueous solution by precipitation with excess ethanol or acetone (Forsyth,

1947, 1950; Duff, 1952a; Rennie et al., 1954; Black et al., 1955; Chesters

et al., 1957; Bernier, 1958a; Mortensen, 1961; Salomon, 1962; Acton

et al., 1963a; Dormaar, 1967). Polysaccharides have been purified by

precipitation from 0.5 N lithium chloride solutions at pH 7 by the addition of cetyl trimethylammonium bromide (Parsons and Tinsley, 196 I ) ,

from formic acid solution with diisopropyl ether containing 1 percent

acetyl chloride (Whitehead and Tinsley, 1964),and from alkaline solution

with copper sulfate (Forsyth, 1950). The purity of some preparations was

increased by redissolving the precipitate in water and repeating the step,

sometimes with an alternative precipitant such as ether (Black et al.,

1955) or a mixture of alcohol and acetic acid (Duff, 1952a).

Although they have been widely used, these precipitation methods are

far from satisfactory, particularly when not preceded by a desalting step.

They are not specific for polysaccharides, as some noncarbohydrate

materials are also precipitated, and they leave polysaccharides in the

supernatant (Acton et al., 1963a; Dormaar, 1967). In fact, Acton et al.

( 1963a) found that the precipitate obtained by one purification procedure

(Rennie et al., 1954; Chesters et al., 1957) had an ash content of more

than 60 percent and a carbohydrate content of less than 20 percent, measured by an anthrone method (Brink et al., 1960). They also found that

there was much more carbohydrate in the supernatant than in the precipitate.

Much of the protein material that invariably remains with soil polysaccharide preparations, even after several different purification treatments, has been removed either by the emulsification procedure of Sevag

et al. (1938) (Bernier, 1958a; Mortensen, 1961; and Thomas et al.

1967) or by calcium sulfate precipitation and sorption on Fuller’s earth

in N acetic acid (Bernier, 1958a). However, amino acids were always

detected in hydrolyzates of the “deproteinized” polysaccharides.

Roulet et al. (1963b) used gel filtration and ion exchange chroma-



210



G . D . SWINCER, J . M . OADES, AND D . J. GREENLAND



tography with the primary purpose of purifying the humic substances in

acid extracts. The best separation of high molecular weight carbohydrates (measured as uronic acids) from “nitrogenous compounds” (as

well as from humic substances) occurred with Sephadex G-75, but no

pure fractions were recovered. The “nitrogenous substances” were

mainly of low molecular weight, but they occurred in all fractions. Oades

and Swincer (1968) and Swincer et al. (1968a,b) have confirmed the

partial separation of nitrogenous materials from carbohydrates by means

of gel filtration. This separation occurred because materials of low

molecular weight obtained by gel filtration invariably contained higher

proportions of amino acids than larger polymers. Thus materials separated

as the high molecular weight fraction on Biogel P- 100 contained the least

proportions of amino acids.

A more direct application of gel filtration to the purification of soil

polysaccharides has been made by Barker et af. (1 965, I967), who used

Sephadex G- 100 to remove humic materials from the high molecular

weight polysaccharides. Limitations of the technique were that good

separations could be achieved only with acidic extracts, and the problem

of separating humic materials from the smaller polysaccharides remained

unsolved.

Other purification procedures that have been used to advantage with

soil polysaccharides are desalting by ion exchange (Mortensen, 196I),

and removal of clay minerals by treatment with a mixture of 0.3 N with

respect to both H F and HCI for 10 minutes at 40°C. (Mortensen and

Schwendinger, 1963).

In summary, it can be said that in spite of numerous intensive attempts

to purify the polysaccharides extracted from soils, it is doubtful if any

of the resulting preparations have been completely free from noncarbohydrate materials. The possibility of carbohydrate-humic and carbohydrate-protein links will be considered later.



D. FRACTIONATION

OF “PURIFIED”

SOILPOLYSACCHARIDES

1 . Introduction

The large number of different sugars in the hydrolyzates of “purified”

polysaccharides and viscosity and ultracentrifuge studies (Bernier,

I958a; Ogston, 1958) which indicate a wide range of molecular size and

shape show that soil polysaccharides are complex mixtures. Their fractionation has thus proved to be even more difficult than fractionation of

other polysaccharide mixtures. Most of the well-established methods of

polysaccharide and polymer chemistry have been used, but in spite of a



Tài liệu bạn tìm kiếm đã sẵn sàng tải về

III. Studies on Soil Polysaccharides

Tải bản đầy đủ ngay(0 tr)

×