Carbohydrates in aquatic plants and associated sediments from two Minnesota lakes

Geochimice

et ~osmorhimica

Acta 1065. Vol. 29, pp. 153 to 200.

Pergamon

Press Ltd.

I’rinted

in Xorihern

Irvl;~nd

Carbohydrates in aquatic plants and associated sediments from two Minnesota lakes* M. A. ROGERS Department

of Geology and Geophysics, University Minneapolis, Minnesota

(ReceiveJ.10 April

1964 and in revised form

of Minnesota

1’7 August

1964)

Abstract-Carbohydrate materials were studied in aquatic plants, lake waters, and luke sodiments of two eutrophic-alkalit,rophic lakes of central Minnesota. Both free sedimentary sugars and sugars liberated on treatment with mineral acid were recovered. They were identified by Glucose, galactose, xylose and arabinose were specific reactions and by paper chromatography. the dominant sugars in order of decreasing abundance in aquatic plants of the two lakes. Maxima and minima in these sugars, as well as the content of cellulose and hemicelluloso (determined by a calorimetric technique), show little rolat,ion to time of collection and appear to be a function of individual plant, species. Evidence indicates that aquatic plants and algae are not the only importimt source of carbohydrates in lake sediments. Acid hydrolysis of near-surface lake bottom sediments gave rise to eight sugars, arabinose, xylose, galactose, glucllronic acid, glucose, rhamnose, mannose and ribose, in concentrations ranging from 19.1 to 0.1 mg/g of dry weight sediment. The variety and amount of theso carbohydrate srtbstanccs demonstrate the importance of microorganisms in altering tho carbohydrat,e fraction prior to stabilization and preservation within t,he sediment. Acid hydrolysis of lake sediments from a deep core from Blue Lake, Minnesota, furnished in order of decreasing abundance the eight sugars, xylose. glucose, arabinose, galactose, mannose, rhnmnose, ribose and glucuronic acid. Sucrose. ~lWO.SC~,rn;mnost‘ anal sylose were recovered from Blue Lake waters as fret: sugars. Mannosc and xylose have not previously beon reported. Evidence was obtained for the presence of the fire (scdimrnt,ary) s~~g:nrx, maltose, sucrose, lactose, glucose, galactose, xylose and mannoso in aqueous cstrncts of stxtlirncnt. Mnnnoso and lactose arc previollsly unrecorded; however, this rrport of lactose, needs to bc confirmed by characterization. A natural stability series for carbohydrates in the lacustrino environment is: fairly stable: xylose, glucose, rhamnose, arabinose;, moderately stable: riboar. I:1:mnosc; fairly rmstable: galactose; very unstable: glucuronic acid. INTKODUCTION

AS part of a continuing project to evaluate the role of organic materials during diagenesis, carbohydrates were studied in aquatic plants. lake \\‘;Ltcrand sediments of two eutrophic-alkalitrophic lakes of central Minnesota, Blue Lake and Clear Lake. In order to supplement the knowledge of organic mutcri;rls in Incllstrinc clnvironments, the general seasonal distribution of carbohydrates in aquatic plants. sctliments and water was determined, their stability under thc,sc nat’llr;ll cotltlitions IV;~S cstimated and suggestions were sought as to correlations between nqllnt,ic plant source and accumulating sediment composition. (Because of the relative smallness of the watershed, the predominance of fine-gmincd organic ant1 marl!, sc~~lin~nts. ;Ln(l the absence of recognizable terrestrial detritus in the cores. thr author f(lclls tllat, :illochthonous terrestrial detritus can represent only a small portiotl 01 t 11~’r)l.y;ltlic cI(LI)t~is.) The limnology of Blue and Clear Lakes is summarized by Sw.\rx ( L!H~I: p. ;;??I and discussed in greater detail in a more recent article (SW~\I?YT. \‘~srr~nrs alld ‘I~IYG, * Contrihutlon of Minnesota.

no. 19, Llmnologtcal

7

R.esearch Cclntcr, School of 132:~r.~ 11S(.it%llc,css. L’llivvrsity 183

M. A.

184

ROGERS

1964). The relative structure and order of stability of amino acids in the same aquatic plants and associated freshwater sediments is discussed in the recent paper. Chlorinoid and flavinoid pigments from the kacustrine samples are to be detailed for these and other sediments in another report (SWAIN, PAULSEN and TIN+, 1964). Carbohydrate geochemistry is reviewed by VALLENTYNE in the volume edited by BREWER (1963). Table Plant

1. Blue Lake

Plant

Survey

(identification

Common

species

by R. B. KAUL,

name

Abundance

Comxrontf3 No roots, grows in all depths Prefer shallow water with no currents Floating, but submorscd plant Specimens of 3 meters common

1. CeratophyUum demereum L.

Coontail

very

2. Elodea cutaadetke Michx.

Waterweed

locally

3. Lemna triaulca L.

Duckweed

uncommon

4. My&phyUum

Water

milfoil

very

Yellow

water

exalbeacena Fem.

5. h+&UrSpp.

6. Nymphucu

odomta Ait.

White

I. Polygonum natana Eaton 8. Pohmog&on illinoenaie Marong

lily

pond lily

common

common

common

locally

common

locally

common

Smartweed Illinois pondweed

rare very common

Sago pondweed Flat&em pondweed

common common

latifoliu forma gracilis 11. Sagittati Wittcl. 12. Sciqmu~ validus Vahl. 13. Typhu l&folk L.

Arrowhead, potatoe Bulrush Cattail

rare

14. VaGmeriu

Wild Tape

9. Potumogebn pectitaatua L. 10. Potamggekm zoetetifornati Rydb.

15.

(Benn.)

americana Michx.

Also algae (Anabaenu and

Duck

rare common

celery, gram

PO&CySti-8) and abundant

common

diatoms

1960)

and desmids

Grow from huge underwater stems Grow from tubers rather than undorground stems Difficult to identify because of polymorphism, dopending upon environment Easily distinguished by linear leaves and flattoned stem Grows around Blue Lake in hays Fairly oommon in deeper parts of Bluo Laka

Carbohydrates

in aquatic plnnts and associated sediments from two Minnosota lakes PLANT

186

SURVEY

Tables 1 and 2 summarize the results of a plant aurvey made by R. B. KAIJL, graduate botanist, University of Minnesota, in September 1960. Both lakes have areas of mutted vegetation. In addition to the aquatic plants, a great number of diatoms and dosmids exist in both lakes. Species composition could be expected to change drastically from time to time throughout the growing season. Tablo 2. Clear Lake Plant Survey (identification by R. B. KAUL, Common name

Plant species 1. Ceratophyllum

demersum

Coontail

L.

2. Chma sp.

1960)

Abundance

Commonte

very common

No roots, grows at all dopths Most abundant, all depths, huge masses Specimens 3 meters long common -

very common

3. Myriophyllum

exalbmcena Fern.

4. Naja8 guatidupemia 6. Potamogeton illinoenaie

Marong

6. Potamogeton

nutana L.

7. Potamogeton 8. Potamogeton

pectinatue L. richardeonii (Berm.)

9. Potamogeton

zostetifwmie

Water milfoil

common

Illinois pondweed

very common common

Floating leaf or brownleaf pondweed Sago pondweed Clasping leaf of redhead pondweed Flatstem pondweed

Rydb.

(Benn) .

10. Scirpua valtiua Vahl.

Bulrush

11. Typha

Cattail

latifolia

L.

12. Also algae (Anubcrenu and Polycyetie) COLLECTION

locally common common common common

common

Difficult to identify because of polymorphism, depending upon environment Leaves float on surface Easily distinguishod by linear leaves and flattened stem Rather large mats along shore and in shallow parts Great mat toward center of lake

and abundant diatoms and desmide

AND

STORAGE

OF SAMPLES

Plant samples were obtained in shallow water by means of a long hook, either from a boat or through the ice. Homogeneous samples of all plant species represented at that season were collected and placed in polyethylene bags. The samples were then frozen and retained in that condition until required for analysis. All sediment samples were obtained from cores. Nearsurface bottom sediment cores were obtained with a free-fall Phleger type sampler weighing about 16 kg. Cores were collected in plastic, ‘Plexiglas’, tubes 1.2 m long and 4.5 cm in diameter. Blue Lake deep core sediments were obtained by means of a Davis peat sampler. All cores were 3

M. A. ROCSERS

186

extruded on to sluminum foil and frozen until ready for study. Blue Lake water analyzed for free sugars was collected at a 1.8 m depth in 19 liter glass containers and stored in the dark. No preservatives were added. Analytical procedures were begun immediately. ANALYTICAL

METHODS

Total carbohydrate analyses were determined by a calorimetric phenol-sulfuric acid test. (DWBOIS et al., 1960). Cellulose determinations, after hydrolyeis with 50 % H,SO,, and hemicellulose determinations, after extraction with 4 % NaOH, were run on these extracts employing the same calorimetric test. Procedures utilized in preparation for chromatography and chromatography follow those of PALACAS (1959). Sampies anaiyzed for acid-hydrolyzable sugars were boiled under reflux with 0% N H,SO, for 8-10 hr. Samples analyzed for free sugars were boiled under reflux with the appropriate solvents for standard times (VALLENTI-KE, 1957). Water samples for free sugars were treated to a preliminary reduction. These mixtures were neutralized with barium carbonate and desalted by ethanolic precipitation and use of electric-desalting equipment. Each hydrolysate was concentrated to a standard volume and examined by paper ~hromato~aphy (general solvent: la-butanol-acetic acid-water (4: 1: 5), general spray: aniline-phthalic acid-water saturated butanol (0.9 g-16-100 ml)). Identifications were made in comparison with known sugars and on the basis of R,- and &-values. Quantitative determinations were made using a recording densitometer in comparison with standard strips for each chromatogram. RESULTS Seasonal

variation

AND DISCUSSION

of plant sugars

summarizes the results of this section of the article. The data are also in tabular form available from the author on written request. The total carbohydrate test was designed as a rapid quantitative check on concentrations of carbohydrates. The phenol~ulf~c acid eolorimetric test employed here expresses units in terms of the hexose, glucose, normally the predominant sugar, but errors will exist to the extent that other sugars constitute a portion of the sample. Preliminary analyses revealed that glucose, galactose, arabinose and xylose were the dominant sugars in the aquatic plants of interest. Other carbohydrate substances (hereafter grouped as sugars or monosaccharides) were chromatographically identified (Table 3). In general the other monosaccharides are present more in the summer than in the winter samples. Glucuronic acid is the most abundant and persistent of the minor carbohydrates, which is not surprising in view of its presence in hemicelluloses. Total sugar content expressed as per cent of total carbohydrate content indicates that in only a few instances do sugars other than galactose, glucose, arabinose and xylose make up a considerable portion of carbohydrate material. It is a biochemical fact that plant carbohydrates vary in type and quantity, depending upon their localization in leaves, stems or roots. Every attempt was made to utilize representative samples of portions in the natural state. Only ten plant samples, five species from each lake, are represented by more than five collections. The ten plant species provide the basis for the following discussion. Myriophyllum exalbescena, Typha latifolia, Chura sp., Najas guadalupensis and Potamogeton illinoensis are the species of interest from Clear Lake. The time of greatest carbohydrate content varies with the respective plant. In general, the concentrations of cellulose, hemicellulose and simple sugars vary directly with total carbohydrate content. Glucose is the predominant monosaccharide. Figure

I(a-d)

Carbohydrates

in aquatic plants and associated sediments from two Minnesota lakes

quodo&wnsis

Nojos

Clear Lake

lorifolio

Typho

Clear Lake

/’ ‘1,

600.

--..--

/

5oc-

‘,

400.

v-i Y z

d

-

\ %. ‘,\

-

hem~-c4llul0S4

-

..._._._

.‘\

,,*-.,

CCllUlDs4

________

\ /’

lololcorbohydrolc

---...---

‘\

-.--

“1

qowose

p,“COse carobmore xylem

\

‘\

.‘\

; N

i2

Fig. l(a)* Seasonal variation

in carbohydrate materials plant samples.

in predominant

aquatic

131

188

M. A. ROGERS

Myriophyllum exolbescens Clear Lake

500.

.

.,A ‘.\

4GQ~ .,

300. /-

/’

/’

‘\

/’

/

./

6M).

-..

/’

\ .‘.\ .-\

__.. --.

/’

200

\

___-

.A

-

_.

\

L.

‘.

‘.._

_/ __* ”

“.

500.

‘-5

Potomogeton illinoensis Clear Lake .. “‘..

‘.. ‘\ ‘\

\

400.

‘.\

\ “..__,._-..

1’

\

Cerotophyllum demersum Blue Lake

i i

Fig.

l(b).

Seasonal variation

in carbohydrate plant samples.

materials

in predominant

aquatic

Carbohydrates in aquatic plants and associated sediments from two Minnesota lakes Myriophythm &rOlh~~

Blue Lake

Blue

600.

soo.

‘1 \

\

\

Lake --..-l WO, c.yWI+* ---.. _- c*,lUfOY -----___ hm,-cll,“lW

Fig. l(c). Seasonal variation in carbohydrate materiate in predominant aquatic plant samples.

189

190

31.

A.

ROGERS

Algae Blue Lake - 600. x6 --. ---.

----

g500~ L 3

-- ------ .,.... -? 400 -.--

tow corb.mydrate ceII”IOIL hnru-cel,“lo,c q0kxt.m QlucO,S orobrme ry,orc

Fig. l(d). Seasonal variation in carbohydrate materials in predominant aquatic plant samples.

Table 3. Sugars other than glucose, galactose, xylose and arabinose chromatographically identified in aquatic planta Plant species Ceratophyllum Chara ap. Chara sp. Myriophyllum Najaa

demersum

exalbexen.9

guauklupeti

Potamogeton Potamogeton Potamogeton

illinoen.& illin0en-k pectinatua

Lake

Time of collection

Clear Clear Clear Clear

27 June 22 April 27 June 7 June

1961 1981 1961 1901

Clear

27 June 1961

Clear Clear Clear

27 June 1961 28 July 1901 28 July 1961

Algae

Blue

28 July 1961

canadenaia

Blue

28 July 1961

Myriophyllum eaxdbeacena Xymphaeu odora& Potamogeton illinoenati

Blue Blue Blue

27 June 1961 28 July 1901 28 July 1961

Potamogeton Potamogetfm

Blue Blue

28 July 1961 28 July 1961

Elodea

pectinalua zoater+rmia

Sugar glucuronic acid glucuronic acid galacturonic acid glucuronic acid galacturonic acid glucuronic acid fructoee glucuronic acid glucuronic acid ribose glucuronic acid galacturonic acid glucuronic acid ribose glucuronic acid glucuronic acid ribose rhamnoee ribose galacturonic acid

Carbohydrates

in aquatic plants and associrtted sediments from two Minnesota lakes

191

Myrophyllum exalbescens, Elodea canadensis, Ceratophyllum demersum, Nympha-ea odorata and “algae” are the species of interest from Blue Lake. Here, too, the time

of greatest carbohydrate content varies with the respective plant. Generally, cellulose, hemicellulose and monosaccharide concentrations vary with total carbohydrate content, but there are more erratic fluctuations than in Clear Lake. Glucose is also the dominant sugar in the Blue Lake plants. Present analytical data for carbohydrate content on these aquatic plants do not demonstrate any seasonal or lacustrine environmental trends. Instead, the observed variations appear to be a function of the particular taxonomy unit. Independent checks on cellulose, hemicellulose and total carbohydrate content, within the limitations of technique, prove that all fractions have been recorded accurately (overall 03 per cent of total carbohydrate content). Contrary to expectation, both cellulose and hemicellulose (a lower molecular weight, more soluble, and hence perhaps more unstable intermediate) frequently vary with respect to total carbohydrate level and exhibit no relationship independent of each other. The hemiceIlulose appears to be the more stable, a fact which may reflect its role as a reserve carbohydrate. Exceptions to the abundance sequence of glucose, galactose, xylose and arabinose most often involve the temporary dominance of galactose. Pentoses are of limited importance in the present group of plants. However the absence of other sugars does not preclude that they were originslly present. Overall individual averages for the carbohydra~s from Clear and Blue Lake plants as well as the average for both are listed in Table 4. Table 4.

Aquatic plant carbohydrate

averwee

Carbohydrates (mg/g dry weight aah free) Total cwbohydrste content

Ceil. and bemi.

Per cent of T.C.T.*

Cell.

Hami.

Gal.

Glu.

Arab.

XyI.

Total memo.

23

33

221

Per cent of T.C.T.

Blue Lake

326

299

92

216

64

70

SS

Clear Lake

415

369

94

256

133

64

113

31

49

247

69

AVC9rag0!3

371

344

93

236

108

62

104

27

41

234

63

*

T.C.T. = Total Carbohydrate

68

Test, p_ 4.

Sugars derived from acid hydrolysis of lake sediments

Recorded qualitative and quantitative data for free sugars showing few sugars and low concentrations prompted the author to investigate sugars released by acid hydrolysis of lake sediments. Eight bottom sediment samples were chosen in order to incorporate a variety of sediment types and seasonal collection times. Table 6 records the analytical data for these analyses. No correlation between sediment type and total carbohydrate content exists, other than the fact that the one pure marl sample was extremely low in quantity of sugars, while all other sediment samples were quite uniform. The close correspondence between the sum of cellulose and hemicellulose and the total carbohydrate test indicates that these constituents were recovered nearly quantitatively. On the other hand, the low yield of total simple sugars expressed as per cent of total carbohydrate content.indicates either that a considerable portion of carbohydrate was present in the form of an unidentified sugar or that losses during experimental determination were large.

Blue Lake (sapropel) Blue Lake (sapropel) Clear Lake (marl) Clear Lake (copropel & marl) Clear Lake (copropel & sand) Clear Lake (copropel & marl) Clear Lake (copropel) Clear Lake (copropel)

type)

Lake (sediment

32.4

49.4

5.3

26.2

21.7

34.1

44.0

18.2

215.4 26.9

28 June 1961

28 June 1961

21 Oct. 1960

22 April 1961

28 June 1961

22 April 1961

Total Average 214.1 26.8

12.0

23.3

26.2

4,7

29.6

35.8

21 Oct. 1960

36.5

30.1

.-

e 2

28 July 1961

Date

99.4 99.4

65.9

112.3

95.0

107.4

100.0

88,7

82.7

121.3

x

k ti 6

86.3 10.8

3.5

24.2

11.0

9.9

8.7

2.3

7.2

19.5

127.8 16.0

8.5

25.2

21.4

13.4

17.5

2.4

22.4

17.0

0.8

1.1

-

-

-

-

1.3

-

-

0.2

-

3.5 0.4

0.1

0.5

4.3 0.5

-

2.4

4.6 0.6

5.3 0.7

-

10.5 1.3

0.4

0.6

2.9

-

3.2

1.1

0.3

-

5.3

1.5

-

0.3

0.6

0.5

0.1

0.5 1.4

1.0

0.3

-

!!I 2

‘B

g-u 2

.2

1.8

-z j

B

P

sediments

2” rg -

.”

P 0

P

(expressed as mg sugar/g of sediment, dry weight)

Table 5. Sugars released by acid hydrolysis of lake bottom

5.0 0.6

-

1.3

-

-

-

-

2.1

1.6

8

P 8

-

_-

P 2

28.6 3.6

0.8

19.1

2.2

-

1.8

0.8

1.9

2.0

11.2 1.4

0.3

2.3

1.2

2.5

2.1

1.0

0.2

1.6

3 -x”

P

B ._ e

73.0 9.1

1.6

28.6

4.7

5.1

13.5

3.9

7.3

8.3

33.9 33.9

8.8

65.0

13.8

23.5

51.5

73.6

20.4

27.6

E

B

? +&

g

Cnrhohytlrat~s

in nq~~at ic phmts and associatt>d s~~~iim~~tltsfrom

two llinncsota inkos 193

Comparison of similar sediment types (Tables 5, 6) indicates that almost all the xylose and mannose released by acid hydrolysis of lake bottom sediments are also obtainable as free sugars by alcoholic extraction of analogous sediments. These two monosaccharides also exist as free sugars in the water of these lakes. Variutions in sediment sugar from Blue Lake long core Analyses of sugars exhibit a range from 0.1 to 4.8 mg/g dry weight of totnl sediment (Fig. 2). No correction for ash content of sediments was made. In order to compare these values with those for plant sugars (‘ash corrected), one may assume 50 per cent ash content in the sediments. h’o distribution distinction between hexoses and pcntoses is apparent. The sporadic occurrence of glucuronic acid (only in the 135-145 cm and 1000-1010 cm intervals) may indicate the incorporation or preservation at these levels of some exotic or foreign hemicellulose compounds not represented elsewhere throughout the core. There is no gradual decrease in concentration toward the bottom of the core. Instead, lowest concentration occurs at either one of two depth intervals: 230-240 or 445-455 cm. These may represent periods of drought and low lake level. During such times partial emergence of the lake bottom and oxidation of sediments may have occurred. Parallel fluctuations in vertical distribution indicate to the author a relationship between depositional environment and extractable sugars. This relationship may include not only the source of the sediment, but Eh and pH conditions as well. The magnitude of experimental values and the observed irregularities confirm that errors of analysis cannot account for the cited fluctuations. A smoothly descending concentration curve might, on the other hand, be explained as due to the simple microbiological metabolism during diagenesis of small but uniform initial quantities of sugars. Thus the present analyses suggest that either the rate of contribution, the kind of organic matter or the conditions of pres:rvation have not always been uniform. In an analogous study of marine sediments, PRASHNOWSKY et al. (1961, p. 408) stated the same conclusions, which they confirmed by noting variations in the organic carbon and nitrogen simultaneously with those of the monosaccharide sugars. Swarm diasotved in water from Blue Lake The following free sugars were chromatographically identified in Blue Lake bottom water: glucose, mannose, suc~oso, xylose and “near-raffinose”. “Near-raffinose”, mannose and xylose have not previously been reported in the literature on lake waters. Interpretation of free sugar data should be made with reference to the solubility in water for sugars of organic geochemieal interest. In such a list, mannoso (the most soluble in water would be followed by sucrose, xylose, maltose, glucose, ribosc, fructose, arabinose, lactose, galactose, and finally raffinose (the least soluble in water). On this basis the presence of most of the free sugars detected here as well as those previously cited in the literature may signify (because of their wide distribution in nature) only their relatively greater solubility, and repre. sent little of geochemical interest. The particular absence of galactose, an otherwise abundant sugar, substantiates this conclusion.

Free avgw in extract8 of take sediments Seven sugars were identified and characterized extracts. These are presented in Table 6. Lactose

by paper chromatography in lake sediment is an extremely rare sugar in nature, with the

(marl & copropel) Clear Lake (marl & copropol) Blue Lako (sapropol) Blue Lako (sapropol)

Clear Lake (copropel) Clear Lake

Blue Lake (-propel) Blue Lake (-propel) Clear Lake (copropel)

21 Oct. 1960

21 Oct. 1960

21 Oct. 1960

6

7

8

21 Oct. 1960

5

22 April 1961

3

22 April 1961

28 July 1961

2

4

28 July 1961

Date

1

Lake (sediment type)

trace

trace

trace

70% EtOH 70 % EtOH & ultrssonica none

none

non0

nono

trace

none

none

water & ultrasonics

70% EtOH water

none none

none

70% EtOH water

none

P e s (13

none

trace

water

6 5 z

none

trace

none

none

none

none

none

none

tFaco

trace

trace

trace

none

nono

trace

none

none

none

0.9

none

non0

none

none

nono

none

1.1

non0

0.1

none non0

none

none

none

none

0.8

1.2

1.9

0.2

2.1

0.1

on bottom sediment samples from Blue Lake and Clear Lake, Minnesota (mg sugar/g dry weight)

Method

Tablo 6. Free sugar determinations

non0

1.9

0.9

1.8

none

none

0.8

none

? z 8 E (D

P

Carbohydrates in aquatic plants and associated sediments from two Minnesota lakes

195

exception of its occurrence in milk. This chromato~aphic identification needs to be confirmed by isolation and characterization. An unknown sugar commonly occupied a position near rafimose. As its identity is unknown, it is herein rofcrred to as “nearraffinose”. \f%wr’IXEER and VULEIFTE’NE (1957, p. 102) report a similar occurrence. In this investigation, development of the brilliant lemon-yellow color with the P-anisidine hydrochloride in n-butanol spray suggests FLUCTUATIONS

OF CARBOHYDRATE

MATERIALS

WITH

DEPTH

IN DEEP

BLUELAKE

CORE

Fig. 2. Fluctuations of carbohydrate materisis with depth in deop Blue Lake core. that the unknown may be a ketohexose (LEDEHER and LEDERER, 1954, p. 166). The occurrence of free maltose has been investigated by WEITTAUR and VALAENTYNE (1957, p. 102). Chromatography and chemical tests convinced them that sediment extracts do indeed cont.ain free maltose. Sediment extracts of Blue and Clear Lakes contain no fructose, ribose, or arabinose, but mannose and what appears to be lactose, previously unrecorded as free sugars in lake sediment extracts, occur. Chromatographic evidence alone is insufficient to characterize mannose and lactose. These occurrences must also be confirmed by isolation and characterization. Otherwise the present data agree with previous work. The relatively high quantities of xylose and mannose may be related to the presence of these sugars in lake water. Minnesota lake sediments produce greater quantities of free sugars

196

-3%.A. ROGERS

than either the Connecticut (VA~ENT~E and BIDWELL, 1956) or Ontsrio (WRIT~AKER and This may be due to the greater eutrophication VALLENTYXE, 1957) lake sediment samples. state of the former. However, a variety of sugsrs ie not so evident in the Minnesota 8amplee. In summary, it appears best to 8gree with these authors and state only thst work on lake sediments ha9 ehown free sugars to vary both quantitatively and quelitativoly with sediment type, state of trophication and sampling locality.

DISCUSSION AND INTERPRETATION Source of carbohydrates Distinction between free sugars and those determined after acid hydrolysis is critical in a discussion of carbohydrate sources. Free sugars liberated by water or alcohol extraction presumably exist in aquatic plants, lake waters or lake sediments in the same form. Water extraction removes only a fraction of such sugars (PALACAS, 1959, p. 41). Ethanolic extraction quantitatively recovers sedimentary sugars (WHXTTAKER-VALLENTYNE, 1957). Sugars determined after acid hydrolysis of samples presumably represent degradation products of the more complex carbohydrate materials in the original sample. All such material should be recoverable under adequate conditions of acid hydrolysis. A direct origin for free sedimentary sugars would be the free sugars present in plankton. NORRIS,NORRISand CALVIN(1955) reported small amounts of maltose in Haematococcus and Spirogyra, and larger amounts in J’ontinulis. However, VALLENTYNE and BIDWELL(1956) rejected the possibility of a planktonic source for al1 free sedimental sugars except sucrose, glucose and fructose, for two reasons: the concentration of free sugars in sediments are too high, and free sugars commonly found in seston and those found in sediments are different. No free sugars were extracted from aquatic plants or other plankton during this investigation. There is no reason to doubt the preliminary conclusions of WH~AKER and VALLENTYNE (1957) that aquatic plants may contribute some portion of the free sedimentary sugars, but for reasons to be detailed later, this author feels that free plant sugars are an unlikely source for an important percentage of free sedimentary sugars. Acid hydrolysis may release carbohydrates from comminuted but microscopically visible aquatic plant fragments, which are often an important constituent of at least surface sediments. Other portions of the benthic biota have been considered by WHITTAK~Rand V~LENTYNE (1957, p. 106) as possible sources of free sedimentary sugars. They considered two groups of organisms : tendipedid larvae and bacteria. On the whole, they concluded “that some (perhaps all) of the sugars in samples with a very low sugar content may have been derived by the extraction of living benthic organisms, but such a source is quantitatively out of the question for mud samples rich in sugar” (WHITTAKERand VALLENTYNE,1957, p. 107). As carbohydrates are an integral constituent of animal tissues, acid hydrolysis of these tissues in sediments may be expected to produce monosaccharides. The kind and amount of monosaccharides should vary with the type and amount of living matter hydrolyzed.

A third sowce of free sedimentary sugar may be I~\-droiysis of the poiysacci~arides of se&on (pinnkton) both living and dead. On tile other hand, it is a fact that acid hydrolysis of sediments liberates monosaccharides from pol~sacci~nridcs. whatever the grosser structure of the poiyaacci~aritlc matrriai may be. It is possible that. ail or almost ail monosaccharides derived 1)~ acicl hydrolysis arc obtainrd from poiysncci~arides contained in stilt organized plant and animal mntcrini living and dead. However. it is also true that this material is much less important at moclfratc depth (1 to 2 m) in the sediments from take cores Kvidence that monosaccharide icvcis do not decrease regularly under discussion. with depth suggests t.o the author that some of the monosaccharides derived by acid iiydroiysis are contained in poiysacciiarides organized on a molecular or quasi-molecular basis and are held and stabilized within the sediment in some at present imperfectly known sorptive manner ( BADER, Hoon and SMITH, 1!)60). A gradation must, of course. exist between these two conditions. The phrase “organic matter of the sediment” includes both of these two conditions, but greater distinction is desirable. Two other tines of evidence confirm this suggestion of both organized and quasi-molecular organic matter sources. Relatively large amounts (up to 1.3 mgikg rock material) (PAIACAS, 1!359, p. 36) of hydrolyzable sugars in sedimentary rocks without dctrrminablc organized organic detritus as defined above, demonstrate an origin in an organic fraction, which is intimately related to the sediment particles. Stability of some sugars, demonstrated by occurrence in Ordovician Stonehenge limestone (PATACAS. 1!15!), p. 36) also substantiates this point. Although microorganisms produce a great variety of carbohydrates, glucose, (Aucuronic acid and amino sugars are the main products. With the addition of gaiacb tose and ribose these sugars constitute those of quantitative importance in the life cycles of animals. Lack of correspondence as to abundance among lists of sugars from take plants and sediments (Figs. l(a-d), 2) (Tables 5, 6) indicates no obvious and direct relationship between an aquatic plant source for a monosaccharide and its inclusion and preservation in the underlying sediments. The actual source probably involves synthesis by microorganisms. There is general correspondence in total sugars between the surface and deep sediments, although the relative increase in glucose and decrease of arabinose may be rt4ated to their relative stabiiit,y rather than to a source in the overlying aquatic plants. However, the abundance of hexoses in the plant samples examined here is in contra.st to the less clear-cut abundance of pentoses in t.he sediment samples. There is no reason here for supposing the sources of free sedimentary sugars to be different, although in both instances it is expected that the effects of stabilizing and preserving mechanisms would become dominant below a rather shallow depth.

Amounts of sugars Hydrolyzed sediment sugars may constitute approximately 10 per cent of the amount recovered from an equivalent mass of aquatic p1ant.s. The amount of free sedimentary sugars may equal the amount of hydrolyzed sediment sugars, but is often less. The amount of free sugars in take waters is very minor compared to any of the above sources. Surface sediments of Blue Lake contain on the average less than one per cent of the total carbohydrate derivable from the standing crop of

19S

;M. .k

~OCERR

aquatic plants. In summary, it will be necessary to relate monosaccharide sugars to their carbohydrate source more clearly before the quantitative data accumulated for carbohydrates may be utilized with precision and usefulness. Types of sugars Occurrence of mannose as an important free sugar in Blue Lskc water, as well as its occurrence along wit.h maltose among the f&e sedimentary sugars, confirms a non-plant source. Presence of glucuronic acid in sediments suggests that microorganisms may be important. Large amounts of galactose also imply a microzoological source, or at least the importance of galactans. Dominance of the pentoses arabinose and xylose in hydrolyzates of lake sediments are even more difficult to relate to a carbohydrate source. It is most likely that these pentoses are derived from hemicelluloses which, as the data from the Blue Lake deep core indicate, become more and more important a constituent with depth. As the decrease is gradual and persistent, even beyond the zone of extensive microbiological activity, it would appear that this is related to relative stabilities of cellulose and hemicellulose. In summary, the types of sugars indicate also that although Clear and Blue Lake sediments derive some of their carbohydrate content from aquatic plants, other sources, particularly microbiological, are important. Changes or jhctuntions

in sugars

Fluctuations in total amount of sugars with depth, such as those observed in sediments studied during the present investigation, may have been caused by variations in supply of organic matter, by variations in Eh at the time of deposition or during subsequent diagenesis, or by variations in amount of inorganic sediment entering the lake. It is probable that reducing conditions as they exist today have prevailed only intermittently in Blue Lake. PLUXKETT (1957) has pointed out that a change in redox potential may have a marked effect on preservat.ion or elimination of the carbohydrate fraction. PHASHXOWSKY et al. ( 1061, p. 4 10) investigated marine sediments from California and suggested that changes in total am0unt.s of sugars may be interpreted as largely due to fluctuations in Eh at their time of deposition. As this tendency is not shown by total organic matter content, two conclusions may be drawn : first, kerogenous materials are quite resistant to diagenetic attack; second, carbohydrates in sediments may not yet be an intimate portion of the kcrogen fraction. This author supports the latter possibility. Preservation

of sugars

It is known that microorganisms remove the last traces of free sugar when such free sugars arc added to their growth medium. Therefore, the discovery of free sugars preserved in lake sediments presents an interesting problem. Finding hydrolyzable sugars is also a problem because of the known abilities of microorganisms to hydrolyze polysaccharides. Several possible physical and chemical protective mechanisms that may serve for the preservation of carbohydrates have been proposed : the retardation of microbiological decomposition in an anaerobic environment; adsorption of carbohydrates

Carbohydrates

in nqunticplantsand wociated sedimentsfrom two MinnesotaIakes 199

by suspended clay particles in the upper water thereby protecting them from microbial decomposition until they settle to more favorable bottom environment; and formation of larger molecular or colloidal complexes with lignin, marine humus, kerogen, and chitin. VATJ,ENTYNE and BIDWELL(1056, p. 400) prefer to view the problem of the preservation of free sedimentary sugars in terms of a steady-state equilibrium. The balance is between recruitment of free sugar molecules (from sinking plankton and polysaccharide hydrolysis) and emigration (breakdown of sugars during bacterial metabolism). Demonstration of free sedimentary sugars suggests that formation of free sugar is faster than breakdown. The present author suggests that a similar steady-state analogy might be applied to the situation of hydrolyzable sugars. Balance here is between recruitment of carbohydrate material (sinking organic debris, incorporation with sediments or resistant organic material) and emigration (slow leakage from resistant preservative mecha.nism). Here the presence of appreciable quantities of hy~olyzable sugars indicates the dominance of accumulation and preservative stabiliiation. Thus the steady-state analogy is applicable only on gross terms, and then perhaps only to sediments and rocks for which kerogen or kerogen-like compounds constitute the dominant organic fraction.

Recognizing that some individual plant species show greater seasonal variation in carbohydrate content than others, and that sedimenta contain qualitatively and quantitatively different carbohydrates depending upon their sediment type, depth, and organic fraction, it is possible to draw conclusions regarding carbohydrate stability, If carbohydrates of these aquatic plants and lacustrine sediments are arranged in order of their natural preservation on the basis of their natural stability to seasonal and depth fluctuations in the plants and sediments studied, the following series is obtained. Fairly stable: xylose, glucose, rhamnose, arabinose moderately stable : ribose, mannose Fairly unstable : galactose Very unstable: glucuronic acid This may be interpreted as an order of carbohydrate stability under natural conditions in the lakes studied. The unexpectedly greater relative stability of hexoses is at present unexplained, but perhaps in view of the.complex factors operating in even this simple natural environment, strict correspondence to chemical stabilities should not be expected. The abundance and geologic occurrence of glucose, rather than its stability, explains its inclusion as a fairly stable carbohydrate. SuimmY

AND

CONCLGSIONS

The pathways of carbohydrate geochemistry will be elucidated only as other and varied environments are studied. However, two of the more pressing immediate needs are: (1) An identification of the “polysaccharide-mineral” precursors of the extractable monosaccharides and (2) further determination and confirmation of the

200

M. A. Roasts

stability of these carbohydrate species. microbial origin, and possible fixation of ~dimenta~ carbohy~a~s at the water-sediment interface will more easily be followed in terms of the larger structures. A knowledge of the relative stabilities will allow further elucidation of organic geochemical diagenesie. AoircnowZt&~~~&+--The writer is indebted to Dr. FREDERICKM. SWAIN, Department of Geology and Geophysics, Univeraity of Minnesota, for suggestions, guidance, and support during the investigation. Gratitude is also extended to Dr. FRED SMITHof the Department of Biochemist,ry, University of Minnesota, for permitting the writer to carry out research in his laboratory and for his guidance in biochemical portions of the work. To the National Science Foundation, Grant No. 9392, and to the American Chemical Society, Petroteum Research Fund, Grant No. 716-AZ, both to F. M. SWAIN, the writer is grateful for financial assistance. REVERENCES BADER R. B., HOOD D. W. and SMITH J. B. (1900) Recovery of dissolved organic matter in sea-water and organic sorption by particulate material. Geeoehim. et Coemochim. Acta 19, 236-243. BREUER IRVING A. (Ed.) (1963) Orgunic Qeochemdy, p. 658. Macmillan, New York. DUBOIS M., GILLES K. A., HAMILTONJ. K., REBERS, P. A. and SMITH F; (1956) Calorimetric method for determination of sugars and related substances. Ana&. Chem. a8, 350-356. LEDERER E, and LEDERER M. (1964) Chromatography, p. 460. Elsevier, New York. Namers L., NORRISR. E. and CALVINM. (1955) A survey of the rates and products of short-term photosynthesis in plants. J. Exp. Bat. 6, 64-74. PALACAS J. G. (1959) ~ochemistry of carbohydrates, p. 103. Unpublish~ Doctors Thesis, University of Minnesota. PLWNKETZM. A. (1957) The qualitative de~rmination of some organic compounds in marine sediments. Deep-Sea Res. 3, 23-45. PRASHNOWSKYA., DECZENSE. T., EMERY K. 0. and PXMENTAJ. (1961) Organic materials in recent and ancient sediments,I. Sugars in marine sediment of Santa Barbara Basin, California. N. Jb, Qeol. Pateontol. Mh. 8, 400-413. SWAIN F. M. (1961) Limnology and amino-acid content of some lake depoeita in Minnesota, Montana, Nevada, and Louisiana. Qeol. Sot. Amer. BtdZ. ?‘!& 519-646. SWAIN F. M., PAULSEN 0. W. and TINT F. (1964) Chlorinoid and flavinoid pigments from aquatic plants and associated lake and bay sediments. J. Sed. Petrol. 24, 561-598. SWAIN F. M., VEHTERIS G. and TINU F. (1964) Relative abundance and order of stability of amino acids in some aquatic plants and associated freehweter sediments. J. Se& Petrol. &&24--4&L VALLENTYKEJ. R. (1957) The molecular nature of organic matter in lakes and oceans, with lesser reference to sewage and terrestrial soils. Fisher&s Res. Board Canad. J. 14, 33-82. VALLENTYXE J. R. and BXDWELLR. G. S. (1956) The relation between free sugars and sedimentary chlorophyl1 in lake muds. EcoEogy 37, 495-500. WR~AKER J. R. and VALLENTYNEJ. R. (1957) On the occurrence of free sugars in lake sediment extracts. LimnoE. Oceanogr. 2,98-110.