Occurrence and fate of carbohydrates in recent and ancient sediments from different environments of deposition

OCCURRENCE AND FATE OF CARBOHYDRATES

IN RECENT AND ANCIENT SEDIMENTS FROM

DIFFERENT ENVIRONMENTS OF DEPOSITION

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OCCURRENCE AND FATE OF CARBOHYDRATES

IN RECENT AND ANCIENT SEDIMENTS FROM

DIFFERENT ENVIRONMENTS OF DEPOSITION

OCCURRENCE AND FATE OF CARBOHYDRATES

IN RECENT AND ANCIENT SEDIMENTS FROM

DIFFERENT ENVIRONMENTS OF DEPOSITION

PROEFSCHRIFT

ter verkrijging van de graad van doctor aan de Technische Universiteit Delft

op gezag van de Rector Magnificus, Prof. drs. P.A. Schenck in het openbaar te verdedigen ten overstaan van

een commissie door het College van Dekanen daartoe aangewezen op donderdag 2 maart 1989 te 16.00 uur door

Maria Elisabeth Catharina Moers

geboren te Zoetermeer

doctorandus in de chemie en in de geologie

f

TR diss

1702

Dit proefschrift is goedgekeurd door de promotor Prof. drs. P.A. Schenck

Toegevoegd promotor Dr. J.W. de Leeuw

The author gratefully acknowledges the Koninklijke/Shell Exploratie en Produktie Laboratorium (Shell Research BV), Rijswijk, The Netherlands, for financial support of the investigations described in this thesis (agreement no 59643/Mkp/4).

voor Ron voor Debru

CONTENTS

page

SUMMARY 13 SAMENVATTING 17

1. INTRODUCTION

1.1 Natural occurrence of carbohydrates 21 Occurrence of saccharide containing polymers

Monosaccharides and their occurrence in polymers Organisms contributing to the organic matter in various depositional environments

1.2 Diagenesis of organic matter and of 25 carbohydrates in particular

1.3 Objective and framework of the thesis 27 Objective

Methods Samples

2. OCCURRENCE AND ORIGIN OF CARBOHYDRATES IN PEAT SAMPLES FROM A MANGROVE ENVIRONMENT AS REFLECTED BY ABUNDANCES OF NEUTRAL SACCHARIDES

2.1 Abstract 31 2.2 Introduction 32 2.3 Experimental 34 2.4 Results 36 2.5 Discussion 43 2.6 Conclusions 47

SAMPLES FROM A SUBTROPICAL OPEN MARSH ENVIRONMENT

3.1 Abstract 49 3.2 Introduction 50 3.3 Experimental 51

Analysis of carbohydrates as alditol acetates Analysis by Curie point pyrolysis-mass spectrometry

3.4 Results and Discussion 54 Analysis of alditol acetates

Py-MS mapping General discussion

3.5 Conclusions 67

4. CHARACTERIZATION OF TOTAL ORGANIC MATTER AND

CARBOHYDRATES IN PEAT SAMPLES FROM A CYPRESS SWAMP BY PYROLYSIS-MASS SPECTROMETRY AND

WET-CHEMICAL METHODS

4.1 Abstract 69 4.2 Introduction 70 4.3 Experimental 71

Analysis of alditol acetates

Analysis by pyrolysis-mass spectrometry Multivariate techniques

4.4 Results and Discussion 73 Analysis of carbohydrates as alditol acetates

Comparison of sugar data with literature data Py-MS mapping

Additional observations

FEN PEAT AND BOG PEAT SAMPLES FROM THE ASSENDELVER POLDERS (THE NETHERLANDS)

5.1 Abstract 89 5.2 Introduction 90 5.3 Experimental 91 5.4 Results and Discussion 94

Total sugar yields

Sugar yields in relation to peat type Factor analysis of minor sugars Special cases

5.5 Conclusions 105

6. ANALYSIS OF NEUTRAL SACCHARIDES IN

MARINE SEDIMENTS FROM THE EQUATORIAL EASTERN ATLANTIC (KANE GAP)

6.1 Abstract 107 6.2 Introduction 108 6.3 Experimental 108 6.4 Results and Discussion 111

SAMPLES FROM THE EASTERN MEDITERRANEAN

7.1 Abstract 119 7.2 Introduction 120 7.3 Experimental 121 7.4 Results and Discussion 122

Sugar yields

Sugar-sugar correlations Sugar-lipid correlations

Sugar-stable carbon isotope correlations Sugar-pollen correlations

Comparison with other sediments

7.5 Conclusions 132

8. ORIGIN AND DIAGENESIS OF CARBOHYDRATES IN ANCIENT SEDIMENTS

8.1 Abstract 135 8.2 Introduction 136 8.3 Samples 137 8.4 Experimental 141 8.5 Results and Discussion 142

Mahakam Delta Brandon Lignite Messel Shale DSDP-samples

Ancient marine (oil) shales

HYDROGEN SULPHIDE AND POLYSULPHIDES 9.1 Abstract 9.2 Introduction 9.3 Methods Simulation experiments Analysis of Standard Control experiments 9.4 Results

9.5 Discussion and Conclusions

APPENDICES REFERENCES DANKWOORD CURRICULUM VITAE 159 160 161 162 164

13 SUMMARY

The objective of the present study was to determine the contents of saccha­ rides in sediments from various depositional environments, to investigate to which diagenetical stage more or less intact saccharides can be encountered and to which extent they contribute to the organic carbon content. To this end neutral saccharides were determined in sediments from different depositional environments, geological ages and maturity.

The carbohydrates were hydrolyzed by sulphuric acid. The neutral mono-saccharides formed were converted to alditol acetates by reduction of the aldoses to alditols foliowed by acetylation of the alditols to alditol acetates. Gas chromatography and gas chromatography-mass spectrometry were used as analytical techniques to identify the alditol acetates and to quantify their yields.

The occurrence of neutral saccharides in peat samples is described in chapters 2, 3, 4 and 5. In chapters 3 and 4 the results of the characterization of the total organic matter by Curie point pyrolysis-mass spectrometry are given as well. The occurrence of saccharides in recent marine sediments is described in chapters 6 and 7. Chapter 8 deals with the occurrence of neutral saccharides in ancient sediments from various depositional environments and with different diagenetic histories. Results of an investigation on the inter-action of glucose and cellulose with sulphur compounds are comprised in chapter 9.

In the recent sediment samples with ages up to ca. 850,000 y. B.P. (chapters 2 through 7) about 40 to 50 different neutral saccharides could be identified. The monosaccharides concerned are hexoses (glucose, galactose, mannose, allose, altrose, idose), pentoses (xylose, arabinose, ribose), 6-deoxyhexoses (rhamnose, fucose), tetroses (erythrose, threose), glycerol, several heptoses and amino sugars, and partially methylated derivatives of the heptoses, hexoses, pentoses and 6-deoxy-hexoses mentioned. The fraction of the total organic carbon that can be ascribed to carbohydrate carbon ranges from ca. 1 to 15 wt% in the recent sediments investigated. This is a very conservative estimate of the amounts of carbohydrate present.

The distribution patterns of monosaccharides in recent sediments can be used to differentiate between different kinds of input of organic material. A major input of vascular plants can be deduced from relatively high glucose contributions, i.e. higher than ca. 40 wt% of the total sugar yield and from the total yields of saccharides. A major input of microorganisms to the organic matter is assumed when the relative contributions of partially methylated saccharides, glycerol, tetroses, allose, altrose, heptoses and amino sugars -collectively called 'minor sugars'- are together higher than 5 to 10 wt% of the total sugar yield.

Variations in the contributions of different types of 'higher' plants to peat sediments are in general reflected by variations in the relative contributions of glucose, xylose, arabinose and sometimes rhamnose. This can be explained by variations in the composition of hemicelluloses and pectins. The compositions of these polymers are often characteristic for certain groups of plants. Variations in the contributions of different types of microorganisms on the other hand are better reflected by variations in the contributions of the partially methylated saccharides, heptoses, amino sugars, tetroses, allose and sometimes altrose and glycerol. This is probably due to the specificity of compositions of cell walls of the various bacteria, algae, protozoa and fungi.

The absence of overall decreasing trends in total saccharide yields with increasing depth in the cases of the recent sediments studied indicates that no serious degradation of carbohydrates is taking place in recent immature sediments during the first million years or so after deposition. Most carbohydrate degradation appears to be limited to the upper cm's -if not mm's-of the sediment.

The data in chapters 2, 3 and 4 are derived from fractionated peat samples. The fine grained samples show rather low total saccharide yields and the saccharides are for the major part derived from microorganisms. The coarse grained samples show comparatively high total saccharide concentrations and the saccharides are for the major part derived from vascular plants. Multivariate analysis of the minor sugar data suggests that the remains of different microbial populations are present at different levels in the peats. Interpretation of these minor sugar data is seriously hampered by a general lack of knowledge of specific compositions in terms of partially methylated saccharides of the cell walls and membranes of the various microorganisms.

15

Characterization of fractionated peat samples by Curie point pyrolysis-mass spectrometry (Py-MS) (chapters 3 and 4) and factor-discriminant analysis of the Py-MS data reveal that the coarse grained samples show most of the markers for lignins and carbohydrates, which pointe to the presence of vascular plant material. A relative accumulation of lignified vascular plant material is observed with increasing depth. The fine grained samples show markers indicative for the presence of microorganisms and refractory matter. Chapter 5 shows the results of analysis of neutral saccharides in fen and bog peats. Eutrophic fen peats and oligotrophic bog peats can be distinguished on basis of total saccharide yields and the relative contributions (in wt% of the total saccharide yield) of glucose, xylose, arabinose and rhamnose. Factor analysis of the 'minor sugar' data shows that some minor sugars are associated with the presence of fungi living in symbiosis with heather rootlets. Another set of minor sugars is correlated with peats rich in cotton-grass or reed. In chapters 6 and 7 the occurrence of neutral saccharides in marine sediments is described. The carbohydrates are for the major part derived from marine microorganisms. Comparison of saccharide data with lipid data show that the results support each other in general. Increased primary production, presumably of cyanobacteria and/or diatoms, is correlated with increased contributions of specific mono-O-methyl-saccharides.

Chapter 8 shows that intact saccharides can be identified and quantified in ancient sediments, but increase in temperature has a pronounced effect on the quantity and quality of the saccharides present. In marine and lacustrine oil shales and in humic coals less than 0.1 wt% of the total organic carbon can be attributed to carbohydrate carbon. Glucose and mannose are selectively preserved with respect to the other monosaccharides, irrespective as to whether the original organic matter was predominantly 'marine' or 'terrestrial'.

Data on saccharides from Tertiary DSDP samples that have not been subjected to increased temperatures do not reveal any symptoms of diagenesis. Simulation experiments in which glucose and cellulose were reacted with hydrogen sulphide and polysulphides (chapter 9) reveal that organic sulphur containing compounds are formed that yield several thiophenes upon

pyrolysis/evaporation. It appears that interaction of carbohydrates with sulphur compounds is a possible way for carbohydrates to react in very recent sediments; so the resulting products become less susceptible to microbial attack.

17

SAMENVATTING

Het onderzoek waarvan de resultaten in dit proefschrift zijn beschreven, had tot doel om het voorkomen van sacchariden in sedimenten van verschillende herkomst te bepalen, om na te gaan tot welk diagenetisch stadium er nog min of meer intacte sacchariden in sedimenten kunnen worden aangetoond en om te bepalen in welke mate deze bijdragen tot het organisch koolstofgehalte. Ter realisatie van deze doelstelling werden sacchariden bepaald in een aantal sedimenten afkomstig uit verschillende afzettingsmilieus en van veschillende geologische ouderdom en rijpheid.

Om de sacchariden te analyseren werden zij allereerst met zwavelzuur gehydrolyseerd. De hierbij gevormde neutrale monosacchariden werden omgezet in alditol-acetaten door reductie van de aldoses tot alditolen, gevolgd door acetylering van de alditolen tot alditol-acetaten. Gaschromatografie en gaschromatografie-massaspectrometrie werden gebruikt als analysemethoden om de alditol-acetaten te identificeren en hun opbrengsten te kwantificeren.

Het voorkomen van neutrale sacchariden in veenmonsters wordt beschreven in de hoofdstukken 2, 3, 4 en 5. In de hoofdstukken 3 en 4 worden tevens de resultaten van de karakterisering van het totale organische materiaal met Curie punt pyrolyse-massaspectrometrie (Py-MS) gegeven. Het voorkomen van sacchariden in recente mariene sedimenten wordt uiteengezet in de hoofdstukken 6 en 7. Hoofdstuk 8 heeft als onderwerp het voorkomen van neutrale sacchariden in oude sedimenten afkomstig van verschillende afzettingsmilieus en met verschillende diagenese achtergronden. De resultaten van een onderzoek naar de interactie van glucose en cellulose met zwavelverbindingen zijn samengevat in hoofdstuk 9.

In monsters van recente sedimenten met leeftijden tot ca. 850.000 jaar (hoofdstukken 2 tot en met 7) konden ongeveer 40 tot 50 verschillende neutrale sacchariden worden geidentificeerd. De betreffende monosacchariden zijn hexoses (glucose, galactose, mannose, allose, altrose, idose), pentoses (xylose, arabinose, ribose), 6-deoxy-hexoses (rhamnose, fucose), tetroses (erythrose, threose), glycerol, verscheidene heptoses en aminosuikers, en partieel gemethyleerde derivaten van de bovengenoemde heptoses, hexoses, pentoses en 6-deoxy-hexoses. De fractie van de totale hoeveelheid organisch

koolstof die kan worden toeschreven aan koolhydraatkoolstof ligt tussen de 1 en 15 gewichtsprocenten in de onderzochte recente sedimenten. Dit zijn voorzichtige ramingen van de totale hoeveelheden koohydraten en koolhydraat­ koolstof die in sedimenten aanwezig zijn.

De verdelingspatronen van monosacchariden in recente sedimenten kunnen worden gebruikt om de bijdragen van verschillende soorten organisch materiaal te onderscheiden. Een belangrijke aanvoer van vaatplanten in het sediment kan worden afgeleid uit relatief hoge bijdragen van glucose, d.w.z. hoger dan ca. 40 gew%, en aan de opbrengst van de total sacchariden. Een hoofdbijdrage van microorganismen aan het organisch materiaal wordt verondersteld wanneer de gesommeerde relatieve bijdragen van partieel gemethyleerde sacchariden, glycerol, tetroses, allose, altrose, heptoses en aminosuikers -gezamenlijk 'minor sugars' genoemd- hoger zijn dan 5 è 10 gew% van de total saccharide opbrengst.

Variaties in de bijdragen van verschillende typen 'hogere' planten aan venen worden meestal weerspiegeld in variaties in de relatieve bijdragen van glucose, xylose, arabinose en soms rhamnose. Dit kan worden verklaard als een gevolg van variaties in de samenstelling van hemicelluloses en pectinen. De samenstelling van deze biopolymeren is namelijk vaak karakteristiek voor bepaalde groepen planten. Variaties in de bijdragen van verschillende typen microorganismen uiten zich echter in variaties in de bijdragen van partieel gemethyleerde sacchariden, heptoses, aminosuikers, tetroses, allose en soms altrose en glycerol. Dit is waarschijnlijk een gevolg van de specificiteit van de samenstelling van de celwanden en celmembranen van de verschillende bacteria, algen, protozoae en schimmels.

De afwezigheid van een globale dalende trend aan saccharide opbrengsten in hun totaliteit met toenemende diepte in de gevallen van de bestudeerde recente sedimenten, is een aanwijzing dat in recente, onrijpe sedimenten geen belangrijke afbraak van koolhydraten plaatsvindt gedurende de eerste millioen jaar na afzetting. De sterkste afbraak van koolhydraten lijkt te zijn beperkt tot de bovenste centimeters -misschien zelfs millimeters- van het sediment. De saccharide data zoals beschreven in de hoofdstukken 2, 3 en 4 zijn het resultaat van bepalingen aan gefractioneerde venen. De fijnkorrelige monsters vertonen tamelijk lage opbrengsten aan sacchariden en deze sacchariden zijn voor het grootste gedeelte afkomstig van microorganismen. De grofkorrelige monsters vertonen relatief hoge concentraties aan sacchariden die voor het

19

grootste gedeelte afkomstig zijn van vaatplanten. Multivariantie analyse van de 'minor sugar' resultaten laat zien dat overblijfselen van verschillende microbiele populaties aanwezig zijn op verschillende niveaus in de venen. Interpretatie van deze 'minor sugar" gegevens wordt ernstig bemoeilijkt door een algemeen gebrek aan kennis van de specifieke samenstellingen in termen van partieel gemethyleerde suikers, van celwanden en celmembranen van de verschillende microorganismen.

Karakterisering van gefractioneerde veenmonsters met Curie punt pyrolyse-massaspectrometrie (hoofdstukken 3 en 4) and factor-discriminant analyse van de Py-MS gegevens laten zien dat de grofkorrelige monsters de meeste markers voor ligninen en koolhydraten bevatten. Dit wijst op de aanwezigheid van materiaal afkomstig van vaatplanten. Met toenemende diepte wordt het organisch materiaal relatief rijker aan gelignificeerd materiaal afkomstig van

vaatplanten. De fijnkorrelige monsters bevatten 'markers' die kenmerkend zijn voor de aanwezigheid van microorganismen en van refractair materiaal. De resultaten van de analyse van neutrale sacchariden in laagvenen en hoogvenen worden behandeld in hoofdstuk 5. Eutrofe laagvenen en oligotrofe hoogvenen kunnen van elkaar worden onderscheiden op basis van de totale opbrengsten aan sacchariden en de relatieve bijdragen van glucose, xylose, arabinose en rhamnose daaraan. Factor analyse van de 'minor sugar' resultaten laat zien dat de aanwezigheid van sommige 'minor sugars' is geassocieerd met de aanwezigheid van schimmels die in symbiose leven met heidewortels. Een andere groep 'minor sugars' is gecorreleerd met venen die rijk zijn aan pijpestrootje of riet.

In de hoofdstukken 6 en 7 wordt het voorkomen van neutrale sacchariden in mariene sedimenten beschreven. De koolhydraten zijn voor het grootste gedeelte afkomstig van mariene microorganismen. Toegenomen primaire productie, naar verwachting van cyanobacteriën en/of diatomeën, is gecorreleerd met toegenomen bijdragen van enkele specifieke mono-O-methyl-sacchariden.

Ook in oude sedimenten kunnen intacte koolhydraten worden geïdentificeerd en gekwantificeerd (Hoofdstuk 8). Toename van de temperatuur waaraan de sedimenten zijn blootgesteld tijdens diagenese, heeft een uitgesproken effect op de kwantiteit en kwaliteit van de aanwezige sacchariden: Minder dan 0,1 gewichtsprocent van de totale hoeveelheid organisch koolstof kan worden

toegeschreven aan koolhydraatkoolstof in mariene en lacustriene olie schalies en in humuskolen. Glucose en mannose worden selectief gepreserveerd in vergelijking met de andere sacchariden. Dit is onafhankelijk van een oorspronkelijk mariene of terrestrische samenstelling van het organische materiaal.

Saccharide resultaten van Tertiaire DSDP monsters die niet aan verhoogde temperaturen blootgesteld zijn geweest, vertonen geen kenmerken van diagenese. Met simulatie-experimenten waarin glucose en cellulose werden behandeld met waterstofsulfide en polysulfides (hoofdstuk 9) is aangetoond dat organische zwavelbevattende verbindingen worden gevormd die tijdens pyrolyse

verschillende thiofenen opleveren. Het blijkt dat interactie van koolhydraten met zwavelverbindingen een mogelijke manier is waarop koolhydraten kunnen reageren in recent sedimenten; op deze wijze ontstaan verbindingen die minder gevoelig zijn voor microbiele afbraak.

21

1. INTRODUCTION

1.1 NATURAL OCCURRENCE OF CARBOHYDRATES

Occurrence of saccharide containing polvmers

Carbohydrates are important and omnipresent constituents of the living biomass. They are. present as cell constituents in all organisms from protists to vascular plants and mammals. In bacteria they comprise 20 to 40 percent of the dry weight and in vascular plants ca. 75 wt% of the biomass is attributable to carbohydrates (e.g. Kirk, 1973; Sjöström, 1981; Aspinall, 1983).

In living organisms carbohydrates perform various different functions; some are metabolically active and are used for energy storage (e.g. starch, inulin, glycogen, laminaran, mannitol). Other saccharides are associated with cell walls and membranes where they may provide protection, stability and strength. This concerns polymers like cellulose, hemicelluloses, pectins, chitin, agar, alginic acid, peptidoglycans, lipopolysaccharides, teichoic acids,

glyco-proteins and glycolipids. DNA and RNA constitute still another category of biopolymers which contain saccharide moieties.

The kind of polymers present is often specific for a certain group of organisms, for example: Cellulose, hemicelluloses, pectin and tannin are widespread in 'higher' plants. Peptidoglycans are important constituents of bacterial cell walls. Lipopolysaccharides are typical constituents of the cell membranes of Gram-negative bacteria and cyanobacteria. Teichoic acids are present in cell membranes of Gram-positive bacteria. Agar and alginic acid are structural polysaccharides in certain algae. Chitin is an important

constituent of many fungal cell walls and of the exoskeletons of insects and crustaceans. Acid mucopolysaccharides, glycoproteins and glycolipids are of normal occurrence in cell coats of animal cells.

Monosaccharides and their occurrence in polymers

The sacchande monomers contained in the polymers mentioned above vary widely both quantitatively and qualitatively. The monomers present may be specific for certain kinds of polymers and hence for certain groups of organisms or may be even specific for a certain species. Fig. 1.1 shows structures of monosaccharides that occur frequently in organisms.

Glucose is a very abundant, omnipresent monomer and occurs for instance in starch, glycogen, cellulose and hemicellulose. Other monomers frequently encountered in various polymers are galactose, mannose, xylose, arabinose, ribose, fucose, rhamnose and 2-deoxy-ribose.

Partially methylated derivatives of the monosaccharides mentioned above are reported to occur in cell membranes of various microorganisms, for instance in their lipopolysaccharides (e.g. Laskin and Lechevalier, 1982; Aspinall, 1983). Many of them are thought to be species specific (Weckesser et al., 1979).

Apart trom neutral sugars, a number of sugar alcohols (e.g. glycerol, ribitol, mannitol and inositol), sugar acids (uronic acids, aldonic acids, aldaric acid) and amino sugars (e.g. glucosamine and galactosamine) occur in nature.

The monomers may occur in homopolysaccharides like mannans, xylans, galactans etc, but they may also occur in heteropolysaccharides like hemicelluloses and acid mucopolysaccharides or they may form polymers with non-sugar constituents like proteins or lipids (e.g. steroids) and form for example glycolipids or glycoproteins.

For instance, the major hemicelluloses are polymers of xylose, mannose, galactose, glucose, arabinose and 4-O-methyl-glucuronic acid. Acid muco­ polysaccharides in the cell coats of 'higher' animals usually contain two types of alternating monosaccharide units, often a uronic acid and an amino sugar. Teichoic acids contain either glycerol or ribitol in their backbones. Partially methylated galacturonic acid forms the backbone of pectin and mannuronic acid is an important constituent of alginic acid. The backbone of the rigid peptidoglycan framework of bacterial cell walls consists of glucosamine and muramic acid. Chitin is also built up of N-acetyl-glucosamine units.

C no.

1

2

3

4

5

6

HCO

HCOH

HOCH

HCOH

HCOH

I

H

COH

HCO

HCOH

HOCH

HOCH

HCOH

I

H

COH

glucose galactose

HCO

HCOH

HCOH

HCOH

H

COH

ribose

HCO

HCOH

HOCH

HCOH

H

COH

xylose

HCO

HCOCH3

HOCH

HCOH

HCOH

H

COH

2 0 m e t h y l

-glucose

HCO

HCOH

HCOH

HCOH

HOCH

CH

fucose

H

C0H

HCOH

HèoH

HCOH

H,COH

ribitol

H

COH

OH

H,6

OH

glycerol

HCO HCO HCO

HCOH HCNH

HOOC HC-NH-C(0)-CH,

HOCH HOCH HC-O-CH

HCOH HOCH H

C HCOH

HCOH HCOH HCOH

COOH H,COH H

C0H

glucuronic galactosamine

acid

N—acetylmuramic acid

Organisms contributing to the organic matter in various depositional environments

It was mentioned above that the various saccharide containing polymers and their monomeric building blocks may be specific for various groups of organisms. For investigations of this kind it is necessary to know which organisms occur in which environments.

Depositional environments important in the present context are swamps, marshes, lakes, seas and oceans.

In the open marine environment planktonic organisms can be considered as the major primary producers of organic material and consequently of carbohydrates (e.g. Degens and Mopper, 1976). Planktonic organisms concerned are protozoa (e.g. foraminifera, silicoflagellates), algae (e.g. diatoms, coccolithophores, dinoflagellates, radiolaria) and cyanobacteria. During transport of the remains to the sea floor and incorporation into the sediment, carbohydrates are partly degraded due to bacterial and benthic activity. Hence, bacteria and benthic organisms can be regarded as an additional source of carbohydrates in the sediment. The contribution of terrestrially derived organic matter is in general rather small.

In lacustrine environments fresh water counterparts of the marine organisms mentioned above may cause an important contribution to the organic material. Depending on the specific circumstances, remains of vascular plants may also contribute to the organic material in the sediment.

In terrestrial environments like swamps and marshes, remains of land plants form the major source of carbohydrates. Additional contributions may be derived from algae or cyanobacteria, depending on the specific ecological conditions. Degradation of the primary organic material due to fungal and bacterial activity results in contributions of carbohydrates derived from these organisms to peats as well.

13, DIAGENESIS OF ORGANIC MATTER AND

OF CARBOHYDRATES IN PARTICULAR

After death of an organism (and in some cases during senescence), its molecular structures are for the greater part degraded, either by autolyse, or by grazing and predation, or by the activity of bacteria and fungi, or abiotically. Metabolic activity results in the formation of new biopolymers which in their turn are also prone to degradation. The result is that the more labile organic components participate in cycles of synthesis-degradation-resynfhesis as part of the foodchain in the biosphere. A small proportion, generally less than 1%, consisting of the more recalcitrant organic components accumulates in sediments and participates in cycles associated with the formation of kerogen, oil and coals in the geosphere (e.g. Tissot and Welte, 1984).

It appears that compounds with a protective function in a living organism are often rather recalcitrant after death of the organism as well. Compounds with a metabolic function, such as storage saccharides, on the other hand are often destroyed during senescence or very shortly after death of the organism and do not contribute to the organic matter in sediments (e.g. Ittekkot et al, 1982; Klok et al., 1984b).

Non-storage organic compounds may be preserved in the biosphere and geosphere long after the death of the organisms from which they originated. This recalcitrant behaviour of certain organic compounds is determined by several factors:

1) Some compounds -e.g. lignins and structural lipids- show recalcitrant behaviour and are selectively preserved in sediments as a result of their natural molecular composition, molecular configuration and degree of crystallinity in living organisms. In many cases recalcitrance appears to be a consequence of inaccessibility for enzymatic decomposition. In a living plant recalcitrance may be aimed at as a means of protection against invasions from outside or as protection against stress due to the environment. Examples are lignification, cutinization, suberinization of leave, stem and root.

2) Natural compounds that are labile by themselves may show recalcitrant behaviour due to covering by or incorporation into recalcitrant compounds in living organisms. For instance, carbohydrates become more recalcitrant when they are protected by lignin in lignocellulosic complexes (e.g. Boon et al., 1988), when they are incorporated into the highly aliphatic biopolymer that is

present in cuticulae (e.g. Nip et al., 1986a,b, 1987), when they are encapsulated in a mineral matrix (e.g. Liebezeit et al., 1983).

3) Microbially initiated (stereo)chemical transformations may also render a compound recalcitrant. For example the transformation of cholesterol into 50(H)-cholestanol (coprostanol) in the intestinal tract of mammals (Eyssen et al., 1973). This latter compound is less degradable than its precursor and it is used as a tracer for (polution by) sewage.

It should be kept in mind that recalcitrance is a relative concept. The preservation of organic material in general and of carbohydrates in particular will also depend on a number of environmental factors, such as primary production, sedimentation rate and anoxicity of the water column and sediment. Increased primary production, higher sedimentation rates and lack of oxygen in the water column and sediment all favour enhanced preservation of organic material and hence of carbohydrates as well.

Structural saccharides are often assumed to be relatively rapidly degraded upon burial in sediments, despite their protective function in the living organism. Hence their contribution to the organic matter in mature sediments and kerogen is often assumed to be negligible (e.g. Tissot and Welte, 1984).

Nevertheless, literature data show that hydrolyzable saccharides may account for 2 to 20 wt% of the total organic carbon in recent sediments (e.g. Degens and Mopper, 1975, 1976; Mopper, 1977; Mopper et al, 1978; Ittekkot et al., 1982; Cowie and Hedges, 1984; Klok et al., 1984a,b,c; Liebezeit, 1986; Steinberg et al., 1987; Hamilton and Hedges, 1988). Total' lipids (i.e. free plus hydrolyzable by acid and base) on the other hand constitute generally less than 10 wt% of the total organic carbon (e.g. Boon, 1978; Hunt, 1979, Klok et al., 1984c).

Acid extractable and identifiable saccharides have also been analysed in ancient sediments: Cretaceous black shales appeared to contain between 0.2 and 0.0003 g carbohydrate carbon per 100 g total organic carbon (Michaelis et al., 1986), and averages of ca. 1 and 3 ng sugar/g sediment could be extracted from Devonian shales and (Pre)cambrian rocks respectively (Swain and Rogers, 1966; Swain et al., 1970).

These literature data indicate that it may be somewhat premature to dispatch the presence of carbohydrates in sediments as insignificant and negligible.

27

1.3 OBJECTTVE AND FRAMEWORK OF THE THESIS

Objective

The objective of the present study was to determine the contents of sac-charides in sediments from varying depositional environments, to investigate to which diagenetical stage more or less intact sacchatides can be detected and to which extent they contribute to the organic carbon.

To this end a variety of natural sediment samples from different depositional environments, geological ages and maturities was studied.

Apart from this it was investigated whether carbohydrate carbon could be preserved in other ways. To this end simulation were carried out. Methods

The methods used comprise hydrolysis by sulphuric acid of the carbohydrates in the sediment samples to their monomeric building blocks, reduction of the neutral monosaccharides released to alditols and derivatization of the alditols to alditol acetates. Identification and quantification of the alditol acetates was achieved by gas chromatography and by gas chromatography-mass spectrometry (chapters 2 through 8).

Details on the procedures foliowed during the simulation experiments can be found in chapter 9.

During acid hydrolysis the various glycosidic bonds behave differently and the various monosaccharides are degraded by acid at different rates (e.g. Cheshire and Mundie, 1966; Dutton, 1973; Mopper, 1977; Mopper et al., 1978; Moers, unpublished results). It is important to ensure that hydrolysis of the carbohydrates is as complete as possible, although this might cause partial decomposition of small amounts of the less stable monosaccharides. The results of quantitative determinations of the released monosaccharides are also influenced by the reduction and acetylation procedures (e.g. Albersheim et al., 1967; Torello et al, 1980). Therefore it is important to ensure maximum reproducibility during treatment of the samples in order to obtain results that can be mutually compared. The hydrolysis and derivatization conditions for the analysis of the sediment samples have therefore been kept as much identical as possible. The only exception are the experiments with the samples

from the Mediterranean (chapter 7). Control experiments have shown, however, that this will not lead to misinterpretation of the results.

The sacchandes analysed by the method used in the present case comprise only the neutral sacchandes. This implicates that the carbohydrate yields reported constitute only a fraction of the total amount of carbohydrates and carbo­ hydrate carbon present in the sediments. Uronic acids for instance may be present in ca. equal quantities as the neutral monosaccharides (e.g. Steinberg et al., 1987). Moreover, in the present case sediment samples were treated with dilute hydrochloric acid and cold water for the removal of carbonates and other inorganic salts, prior to analysis for carbohydrates. This procedure inevitably leads to the removal of small amounts of soluble carbohydrates as well.

The methods used for determining concentrations of identifiable saccharides do not take into account the fraction of carbohydrate carbon that is not immediately recognizable as such due to alterations by which carbohydrates moved out of the analytical windows. This implies that the concentration of carbohydrate carbon in sediments may be much higher than would be deduced from the data of identifiable saccharides.

Samples

Chapters 2, 3, 4 and 5 deal with the occurrence of neutral saccharides in various swamp and marsh systems. Chapters 2, 3 and 4 describe the occurrence of neutral saccharides in fractionated peat samples from cores procured from sub-tropical environments in the Florida Everglades and the Okefenokee Swamp, both in the southeastern USA. It was investigated how differences in input of vascular plant species and differences in input of microorganisms were reflected in the contributions of the individual monosaccharides, their partially methylated derivatives and the total sugar yields. The total organic matter in the peats was characterized by Curie point pyrolysis-mass spectrometry (Py-MS) and multivariate analysis of the pyrolysis data. Both saccharide and Py-MS data were examined for trends with increasing depth, c.q. age, of the peat layers.

Chapter 5 covers the results of analysis of neutral saccharides in a peat sequence from a temperate climate. The samples represent a terrestrialization sequence during which the environment changed from eutrophic to oligotrophic, foliowed by deposition of oligotrophic raised bogs. It was studied how these

29

changes in environment and associated changes in the flora were reflected by the saccharide data.

In chapters 6 and 7 the results of analysis of neutral saccharides in sediment samples from marine environments are presented. Chapter 6 is concerned with the results of saccharides from sediment samples ranging in age from ca. 9,000 to 845,000 y. B.P. from the equatorial eastern Atlantic Ocean and chapter 7 reports on the saccharide results from sediment samples ranging in age from ca. 6,500 to 200,000 y. B.P. from the eastern Mediterranean. The saccharide data were used to study relative inputs of marine versus terrestrial organic matter, to investigate variations in the marine microbial assemblages and to explore changes in saccharide abundances attributable to prolonged times of burial c.q. diagenesis.

Chapter 8 deals more specifically with the effects of abiotic diagenesis on the contributions of saccharides to sediments. To this end neutral saccharides were determined in sediment samples ranging in age from Tertiary to Jurassic, from different depositional environments and with different diagenetic histories, namely Tertiary marine samples from DSDP-cores, a Miocene oil shale from the Monterey Formation (USA), Tertiary humic coals from the Mahakam Delta (Indonesia), an Oligocene lignitic wood sample from the Brandon Lignite (USA), Eocene oil shales from Messel (FRG), Cretaceous oil shales from Julia Creek (Australia) and from Jurf ed Darawish (Jordan) and Jurassic samples from the Paris Basin (France) and from the Posidonia shale (FRG). It was investigated how saccharide contents were affected by increasing maturity and age of the sediment and whether original sources of input of organic material could still be recognized.

Chapter 9 deals with simulation experiments investigating the interaction of glucose and cellulose with hydrogen sulphide and polysulphides. In this way the feasibility was explored of carbohydrates escaping biomineralization by formation of organic sulphur containing compounds that are more resistant to microbial attack and hence have a greater potential for survival during diagenesis than their carbohydrate precursors.

2. OCCURRENCE AND ORIGIN OF CARBOHYDRATES

IN PEAT SAMPLES FROM A MANGROVE

ENVIRONMENT AS REFLECTED BY ABUNDANCES

OF NEUTRAL SACCHARIDES

M.E.C. Moers, M. Baas, J.W de Leeuw, J J . Boon and P.A. Schenck Submitted

2.1 ABSTRACT

Acid hydrolysates of fractionated peat samples from Jewfish Key in the Florida Everglades were analysed for neutral saccharides. Two major sources of carbo­ hydrates could be determined: 1) vascular plant carbohydrates derived from

Rhizophora mangle and 2) microbially derived carbohydrates. Significant

correlations exist between the relative contributions of most sugars and the total carbohydrate concentration: Low total carbohydrate yields with high microbial contributions in the fine grained samples and high total carbo­ hydrate yields with high vascular plant contributions in the coarse grained samples. It is estimated that the greater part of the sugars analysed in the fine grained samples originates from microorganisms. The absence of a trend in total carbohydrate concentrations with depth suggests that microbial degradation is limited to the upper levels of the peat and that the microbial sugars determined at lower peat levels are derived from nonviable or dormant microorganisms. Results from factor analysis indicate differences in microbial populations in the various peat samples.

12INTRODUCTION

Jewfish Key is located in the Florida Everglades at the mouth of the Shark River. The present vegetation on this small island consists of a forest of

Rhizophora mangle (red mangrove) with occasional trees of Avicennia nitida

(black mangrove) and Laguncularia racemosa (white mangrove). The area is under the influence of fresh water currents due to outflow firom the Shark River and of strong tidal action because of the proximity of the Gulf of Mexico.

The peat at Jewfish Key is ca. 3.4 m thick and is underlain by a Pleistocene limestone (Miami oolite). The basal peat layer is dated at ca. 4000 y. B.P. (Spackman et al., 1966). The peats in the Florida Everglades have been depo-sited under the influence of a transgressing sea, so at several locations peat cores consist of upper layers deposited under saline water conditions while the deeper layers were deposited under progressively less saline (from brackish to fresh) water conditions (Spackman et al., 1966). Changes in water conditions and consequently in vegetation were also observed in the peat core under investigation; the upper four samples consist of material derived from

Rhizophora mangle (red mangrove) and the peat layers at these depths have

been deposited under saline water conditions. The lower two samples have been deposited under brackish to saline water conditions with Mariscus

jamai-censis (sawgrass) as constituent in the sample from 245-254 cm. The deepest

sample shows contributions of fresh Rhizophora rootlets. These are attri-buted to the presently living Rhizophora vegetation at the site (Spackman et al., 1981; Given et al., 1983).

The core under investigation has been studied before by Rhoads (1985) and by Ryan (1985). They took samples from different depths in the core and frac-tionated these samples into coarse and fine grained fractions. The coarse grained fractions ( + 80 and +20 mesh) consisted of plant tissues and their fragments and the fine grained fraction (-80 mesh) consisted of amorphous matter (Ryan, 1985).

Microscopic observations of the coarse grained fractions by Ryan (1985) showed that the samples derived from tissues and tissue fragments of Rhizophora were in different stages of degradation. The uppermost level (0-10 cm) con­ sisted of unaltered and slightly altered roots and rootlets. The sample from 64-74 cm consisted of remnants of roots and rootlets with most tissues largely

degraded. Many cell inclusions were present. The samples from 122-132 and 183-188 cm showed small amounts of highly disrupted rootlets, no roots were observed. In the sample from 244-254 cm, derived from Mariscus, virtually no identifiable tissues or organs were noted. The peat at this level consisted predominantly of cell inclusions. A large amount of slightly disrupted rootlet tissue from Rhizophora embedded in a mineral rich matrix was observed in the sample from 305-310 cm. For explanation of the terminology used in the microscopic descriptions see Cohen and Spackman (1977,1980).

Rhoads (1985) and Ryan (1985) analysed the peat samples by Fourier-transformed infrared spectroscopy (FT-IR) and by Curie point pyrolysis-mass spectrometry and Curie point pyrolysis-gas chromatography-mass spectrometry (MS and Py-GC-MS), respectively. These methods provide a general view of the occurrence of cellulose, hemicellulose and lignin in the peat samples. They noticed a strong decrease in the abundance of carbohydrates with increasing depth when compared to the abundance of lignin. This is in agreement with the generally held opinion that carbohydrates are labile compounds that are degraded rapidly during and after incorporation into sediments (e.g. Hatcher et al., 1981; Benner et al., 1984a,b; Given et al, 1984; Hedges et al., 1985).

In contrast to former studies, the present study focusses in a quantitative fashion and in much more detail on the abundance of individual sugars in peats. This should give more insight into the molecular aspects and occurrence of carbohydrates than is possible with the more general wet chemical and spectroscopie methods used previously for the analysis of carbohydrates in peats.

Jewfish Key is a very interesting site for studying the occurrence of carbo­ hydrates in peats, because there seems to be only one major source of vascular plant organic material to the peat at nearly all levels, namely Rhizophora

mangle roots. This precludes the occurrence of differences in carbohydrate

abundances due to differences in vascular plant input in the coarse grained fractions. The mangrove root system is so devised that fine grained detritus is easily trapped between the roots. This implies that the fine grained material in these peats may also contain contributions from trapped alloch-thonous material in addition to those from decayed mangrove tissues and the degrading organisms themselves.

2.3 EXPERIMENTAL

Twelve samples, six coarse grained and six fine grained, taken from different depths in the peat (see Table 2.1) were analysed for neutral saccharides. Four samples were analysed in duplicate to obtain an indication of the reproduci-bility of the carbohydrate determinations.

The samples had been fractionated by Rhoads (1985). For information on the fractionation procedures see Given et al. (1984) or Rhoads (1985). The results from analysis by Py-MS, Py-GC-MS and FT-IR showed that the two coarse grained fractions (+20 and +80 mesh) behaved very similarly (Rhoads, 1985; Ryan, 1985). Therefore in the present investigation only the +20 mesh and -80 mesh fractions were studied. The organic carbon content of the peat fractions on a dry weight basis is ca. 60 wt%, with a carboh/nitrogen ratio of ca. 22 (Rhoads, 1985).

The samples of the fine grained fraction had been washed with 0.1 M HC1 for removal of carbonates and all samples had been Soxhlet extracted by benzene/ ethanol 2/1 v/v for 48 h at 100° C for removal of soluble lipids, phenols and pigments, also as part of the investigation by Rhoads (1985). About 6-7 % of the material on a dry weight basis appeared to be extractable by benzene/ ethanol (Rhoads, 1985).

For the present study 100 mg of Soxhlet extracted, dry and pulverized peat was mixed with 5 mi's of 12 M H2S04 at room temperature for 2 h. The acid was

diluted to 1 M and the polysaccharides were hydrolyzed for 4.5 h at 100°C. Myo-inositol was added as an internal Standard. The monosaccharides released by hydrolysis were reduced to alditols by NaBH4 (16 h, room temperature) and

acetylated to alditol acetates by acetic anhydride/pyridine (3h, 100°C). The alditol acetates were analysed by gas chromatography (GC) on a Carlo Erba Fractovap 4160 gas chromatograph and by gas chromatography - mass spectrometry (GC-MS) on a Hewlett-Packard 5890 gas chromatograph coupled to a VG 70-250SE mass spectrometer. Gas chromatographic separations were performed on a CPsil88 fused silica capillary column (1=25 m, i.d.=0.32 mm, df=0.12 /im; Chrompack, Middelburg, The Netherlands). Samples were injected at 50°C (GC-MS) or 70°C (GC). The temperature was rapidly raised to 150°C and from thereon further programmed at 3°C/min to 230°C and held at this tempera­ ture for 40 min. The mass spectrometer was operated in the electron impact mode at 70 eV, with a source temperature of 250°C.

The alditol acetates were identified on the basis of known relative retention times established by the analysis of Standard mixtures of partially methylated alditol acetates (Klok et al., 1982) and on the basis of mass spectra (Stoffel and Hanfland, 1973; Schwarzman and Jeanloz, 1974; Jansson et al., 1976; Radziejewsky-Lebrecht et al, 1979; Wong et al., 1980; Klok et al., 1982). In the case of two heptoses and two amino sugars no retention times were available. Therefore identification was based on mass spectra only. Small amounts of mono-O-methyl-heptoses were present in nearly all samples as were partially formylated alditol acetates.

Quantification of the alditol acetates identified was achieved by peak area integration using a Maxima Chromatography Workstation (Dynamic Solutions Company, Ventura, CA, USA) coupled to the gas chromatograph. The responses of all alditol acetates were presumed to be equal on a weight basis, so that absolute quantifications became possible on the basis of the amount of internal Standard (myo-inositol) added. In some cases when peaks in the gas chromatogram overlapped, quantification was performed on GC-MS results by peak area integration of the responses of selected characteristic mass fragments with the aid of the software available with the mass spectrometer.

The duplicate analyses indicate that the errors in the determinations of the individual major monosaccharides are 10% or less. The errors in the deter­ minations of the minor components are larger: up to 50% for components present in quantities smaller than 0.02 mg/g.

Multivariate analysis was applied to normalized yields of 33 minor components -listed in Table 2.2- and to a set consisting of normalized yields of all sugars analysed plus the total carbohydrate yield. The data were subjected to factor analysis using a modified ARTHUR computer package (Infometrix, Seattle, WA, USA). The principles and the application of this procedure are described by Windig et al. (1982).

36 2.4 RESULTS

Results of the analyses of neutral saccharides are listed in Table 2.1. From this table it can be seen that the samples in the coarse grained fraction yield systematically more carbohydrates upon hydrolysis than those from the fine grained fraction, with exception of the coarse grained sample from 244-254 cm. It seems that the samples from neither coarse nor fine grained fraction show a significant decrease in total carbohydrate yields with increasing depth. The coarse grained sample from 0-10 cm yields, however, a markedly greater amount of neutral sugars than the other samples. The sugars collectively grouped as "minor components" in Table 2.1 are listed in full in Table 2.2. The yields of these minor sugars are listed in Appendix 1. Glucose,

Table 2.1 Sugar y i e l d s In mg per gram dry peat f r a c t i o n . -80 mesh Depth 0 64 122 183 244 305 (cm) -10 -74 -132 -188 -254 -310 Rhamnose 3.3 2.6 2.2 2.5 1.8 1.3 Fucose 0.8 0.6 0.8 1.6 0.9 0.7 Rlbose 0.5 0.3 0.4 0.7 0.3 0.7 Arabinose 8.0 2.0 2.1 7.3 0.9 1.5 Xylose 4.0 2.5 6.1 10.2 3.5 2.4 Mannose 3.6 3.6 5.2 4.6 4.0 2.6 Galactose 4.8 4.1 5.3 5.4 3.9 2.8 Glucose 12.3 9.8 17.2 23.3 11.7 5.7 Mln.comp. 2.9 2.9 4.3 3.6 3.4 2.9 SUM COHC/TOC* 40.2 28.4 2.7 1.9 43.6 2.9 59.2 3.9 30.4 2.0 20.9 1.4 +20 mesh Depth (cm) 0 64 -10 -74 122 -132 183 -188 244 -254 305 -310 Rhamnose Fucose Rlbose Arabinose Xylose Mannose Galactose Glucose Mln.comD. 5.4 1.1 0.5 23.8 24.4 6.4 9.5 50.5 1.7 5.4 1.0 0.4 20.3 16.9 4.9 6.9 38.2 2.1 3.0 1.0 0.6 13.2 18.0 6.0 7.4 30.3 3,3 4.2 1.3 0.6 18.4 15.0 6.0 7.5 42.8 2.9 2.6 1.1 0.6 5.2 6.5 4.9 6.3 12.5 2.7 2.8 1.0 0.4 15.4 14.6 4.8 6.0 42.6 1,9 SUM 123.3 96.1 82.8 98.7 42.4 89.5 COHC/TOC* 8.2 6.4 5.5 6.6 2.8 6.0 *: grams carbohydrate carbon per 100 g total

galactose, mannose, xylose, arabinose, ribose, fucose and rhamnose are regarded as "major" sugars.

The relative yields of major and minor sugars as percentages of the total carbohydrate yield are depicted in Fig. 2.1. They are plotted versus the total carbohydrate yield (in mg/g dry peat fraction). This way of presentation il-lustrates that in most cases definite relationships can be discerned between the relative contributions of sugar monomers, the total carbohydrate concen-tration and the grain size fraction. Galactose, mannose, fucose, rhamnose,

Table 2.2 Minor sugars identified and their loadings on the first flve factors obtained from factor analvsls.

variance preserved 26% 19% 13% 11% 9% Fl F2 F3 F4 F5 1) a heptose +0.68 -0.03 -0.20 +0.09 +0.01 2) glucoheptose +0.83 -0.15 -0.18 +0.44 -0.02 3) an amino sugar +0.81 +0.11 +0.22 +0.29 +0.00 4) 6-O-methyl-mannose +0.70 -0.32 +0.37 -0.12 +0.29 5) 6-O-methyl-galactose +0.51 -0.46 +0.19 -0.28 +0.39 6) 2-O-methyl-arabinose +0.09 -0.79 -0.16 +0.23 +0.34 7) 4-0-methyl-arabinose +0.18 -0.73 -0.13 +0.14 +0.47 8) 2/5-O-methyl-mannose- +0.22 -0.87 -0.07 -0.06 +0.24 9) 2/5-O-methyl-galactose- +0.01 -0.66 -0.26 -0.35 -0.43 10) 2/4-0-methyl-ribose~ -0.37 -0.52 -0.22 +0.08 +0.57 11) 2/4-0-methyl-xylose~ -0.56 -0.74 -0.02 +0.23 +0.25 12) 3-O-methyl-arabinose -0.54 -0.46 +0.57 -0.15 -0.09 13) 3-O-methyl-xylose -0.59 -0.39 +0.31 -0.57 +0.00 14) 4-0-inethyl-rhamnose -0.70 -0.15 +0.35 -0.07 -0.28 15) erythrose -0.77 +0.06 -0.18 -0.14 -0.21 16) threose -0.85 -0.05 -0.12 +0.20 -0.20 17) 6-0-methyl-glucose -0.77 +0.02 +0.04 +0.22 +0.32 18) a heptose -0.77 -0.04 +0.02 +0.20 +0.14 19) 3-O-methyl-fucose -0.69 +0.15 +0.29 +0.36 +0.09 20) glycerol -0.82 +0.29 -0.09 +0.25 +0.02 21) 3-O-methyl-rhamnose +0.22 +0.71 +0.27 +0.19 -0.06 22) glucosamine +0.42 +0.56 -0.11 +0.56 -0.02 23) 3/4-O-methyl-mannose- -0.03 +0.37 +0.36 -0.29 +0.31 24) 3/4-0-methyl-galactose~ -0.16 +0.42 +0.75 -0.18 +0.18 25) 3-O-methyl-glucose -0.04 +0.38 +0.56 -0.55 +0.30 26) 2-O-methyl-rhamnose -0.23 +0.42 -0.69 +0.04 +0.20 27) 2-O-methyl-fucose -0.39 +0.13 -0.64 -0.30 -0.08 28) allose +0.21 -0.04 -0.62 -0.66 -0.01 29) altrose +0.05 +0.06 -0.56 -0.46 -0.09 30) 4-O-methyl-glucose +0.51 +0.30 +0.23 -0.75 -0.03 31) 2-O-methyl-glucose -0.19 +0.29 -0.62 -0.42 +0.18 32) 4-0-methyl-fucose +0.12 -0.55 +0.54 -0.01 -0.47 33) an amino sugar -0.01 -0.47 +0.12 -0.24 -0.58 -: Enantinttieric alditol acetates are not separated on CPsil88.

ribose, and the minor components (allose, altrose, the heptoses, amino sugars, tetroses, glycerol and partially methylated aldoses) show an increase in their relative contributions with decreasing total saccharide concentration irres-pective of sample depth. In Fig. 2.1 only the relative contribution of the sum of the minor components is shown, because the individual minor components all show the same negative correlation between their relative contributions and total sugar yield. Arabinose and xylose tend to show increased contributions with increasing total carbohydrate concentration. A trend in contributions of glucose with increasing total carbohydrate concentration is not clear. The results of the coarse grained sample from 244-254 cm are remarkable: This sample has a low overall carbohydrate yield and shows at the same time large contributions of the minor components and of mannose, galactose, ribose,

<: MINOR COMPONENTS • - 5 D * cn Z5 (f) • + 3 •+4 + « • «+2 • - 2 - 1 '-« ■ ~ 5 »+S • -3 • - 4 R H A M N O S E • +2 •+4 •+ •+3 , «+". , , FUCOSE —2 . - 1 ■-3 *tr. "75T (D . > t 5Ml _5 '+2 160 ARABINOSE SOSSL •+2 •+4 ' 1ÓO -p.±i •*: ,-£~2 -+5 • -5 • -3 • -1 ■ ■ 5b ■ ■ GALACTOSE •+3 ■ 4-S ' ' 100 ' ' • -5 -> •-4 •-2 -6 *-1 ' ' 5b ' ' "+« •+4 ■+ •+2 •+3 ' ' 100 ' '

Yield total sugars in m g / g

Fig. 2.1 Relative yields of the individual "major" sugars and of the collective "minor components" as wt% of total determined sugars versus the yield of the total of determined sugars in mg/g. The numbers next to the dots within the figures indicate the depth of the peat sample: 1= 0-10 cm, 2= 64-74 cm, 3= 122-132 cm, 4= 183-188 cm, 5= 244-254 cm, 6= 305-310 cm. "+" and "-" indicate coarse grained (+20 mesh) and fine grained (-80 mesh) samples respectively.

Table 2.3 Logarithmic regression of rel, vields on total sugar yields.

_r slope s.d. Rhamnose Fucose Ribose Arabinose Xylose Mannose Galactose Glucose 0.75 0.88 0.74 0.75 0.73 0.95 0.87 0.52 -6.43 -3.31 -2.67 18.85 10.74 -14.64 -13.53 10.99 1.75 0.55 0.77 5.32 3.17 1.56 1.63 5.69 Minor comp. 0.97 -15.88 1.35 r -correlation coëfficiënt s.d.-standard deviation in slope

fucose and rhamnose, so that it plots with the fine grained samples.

The correlation coefficients between the relative contributions of various monomers and total carbohydrate concentration determined by logarithmic regression are listed in Table 2.3. Application of the student's t-test (10 degrees of freedom) on the regression results reveals that the correlation between galactose, mannose, fucose, and minor components and the total carbohydrate concentration highly significant (> 0.1% probability level). The correlation between the relative contributions of rhamnose, ribose, arabinose, and xylose and total sugar yield are significant (> 1.0% probability level). In all cases logarithmic regression gives higher correlation coefficients than linear regression.

Fig.'s 2.2 and 2.3 show the distribution patterns of the major and minor components, respectively. The contributions of the individual sugars are calculated as wt% of the total concentration of "major" plus "minor" sugars (in mg/g dry peat fraction). The differences in sugar distribution patterns for coarse and fine grained samples are clearly seen in Fig. 2.2. The enhanced contribution of minor components in the fine grained fraction and in the coarse grained sample from 244-254 cm when compared to the other coarse grained samples is evident from Fig. 2.3.

Factor analysis applied on all individual sugar data plus the total sugar concentration, yielded a major first factor that describes 63% of the total variance. Arabinose, xylose, glucose and "total sugar" load on the positive side of this function and all other sugars load on the negative side (results not shown).

50-3 - 1 0 cm. + 2 0 mesh 3 0 10

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mam fuc rib ora xyl man gal gluc mam fuc rib ara xyl man gal gluc mam fuc rib ara xyl man gal gluc

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rham fuc rib ara xyl man gal gluc rham fuc rib aro xyt man gal gluc rham fuc rib aro xyl man gal gluc

Fig. 2.2 Distribution patterns of the "major" sugars (wt%) in the peat samples. The total yield of all "major" sugars is taken as 100%.

Rham= rhamnose, fuc= fucose, rib= ribose, ara= arabinose, xyl= xylose, man= mannose, gal= galactose, gluc= glucose.

• 8 1011 14161620 22 242628» 32 2 4 6 6 1012 141616» 22 242629» 32 2 4 6 6 1012 1< - r - , 1 3 5 7 9 1113 151719 21 2325Z72B 3133 1 3 5 7 9 1113 151719 21 2325Z729 3133 1 3 5 7 9 1113 151719 21 23252729 3133 \J 2 4 9 B 1012 14191820 22 24292B30 32 2 4 6 0 1012 14161820 22 24202830 32 2 4 6 6 1012 14161820 22 242628» 29 <U > -•-3 « <ü 1 0-10 cm, +20 rnaah 64-74 cm, +20 mMh t I . . T 7 T ' . . 7 T T . T ? , . 7 . T F 7 I I 7 T . . T J 0 « ' ■ ■ n m i W n W i i^WiOhwfti , M 0 T M , , ,,,90,998, . ^ . J . W . B l f a l w J r . 122-132 cm. +20 r 1 3 9 7 9 1113 181719 2123252729 3139 1 3 5 7 9 1113 181719 2123252729 3133 1 3 5 7 9 1113 191719 2123252729 3133 I I 9 • 1012 141916» 22 «3»2630 32 2 4 6 8 1012 14161620 22 24282830 32 2 4 6 6 1012 14101820 22 24262830 32 « s B 7 « n i s lft*Ti4 91 U M 7 7 H 3133 1 3 6 7 6 1113 181716 21 23262729 3133 1 3 8 7 9 1113 151719 21 23282729 3133

\\',\mi'iï£,n"a Jmnma » ♦ • . 1011 i4i6i620 22 242928» 32 1 4 9 6 1012 14161520 22 24202a» 32

Fig. 2.3 Distribution pattems of the "minor" components (wt%) in the peat samples. The total yield of all "major" and "minor" sugars is taken as 100%. The numbers correspond to the numbers and components listed in Table 2.2.

several factors, the first (Fl) describing 26%, the second (F2) describing 19% and the third (F3) describing 13% of the total variance. The scores of the peat samples on the first two factors are shown in Fig. 2.4. The arrows indicate the directions in which the contributions of the individual sugars increase, starting from the "average sugar distribution of the 12 peat samples" that is represented by the intersection of the Fl and F2 axes. Table 2.2 lists which individual minor sugars load on the first three factors.

Figure 2.4 shows that the fine grained samples all plot on the positive side of the first factor and they seem to show little variation in their scores. This is in contrast to the coarse grained samples, which show great variations in their scores on the first factor. It can also be seen that the fine grained samples tend to plot more on the positive side of the first factor than the coarse grained samples from corresponding depth. It seems that the peat sam­ ples can be divided into three groups: One group (+3 +4 -1 -3 -5 in Fig. 2.4) scores on the positive side of both Fl and F2. Another group (+1 +2 +6) scores

F 1

-"©

6gluc, hept ^ gluchept

-F2

Fig. 2.4 Score plot of the peat samples in the multivariate space described by the first two factors.

The numbers next to the dots within the figures indicate the depth of the peat sample: 1= 0-10 cm, 2= 64-74 cm, 3= 122-132 cm, 4= 183-188 cm, 5= 244-254 cm, 6= 305-310 cm. "+" and "-" indicate coarse grained (+20 mesh) and fine grained (-80 mesh) samples respectively.

The names of the sugars are abbreviated, the Ml names are listed in Table 2.2.

(highly) negative on the first factor. A third group (-2 -4 -6) scores high on the negative side of the second factor. The coarse grained sample from 244-254 cm (+5 in Fig. 2.4) seems to be an outlier.

2.5 DISCUSSION

The lower yields for the fine grained samples compared to those for the coarse grained samples and the relationships between relative sugar contributions and total sugar concentration can be explained as the result of microbial degra-dation. Most saccharides analysed are known to occur in vascular plants, bacteria, algae and fungi, though not in the same quantities. Vascular plant tissues contain a high proportion of carbohydrates and within this fraction glucose, xylose and sometimes arabinose are the main constituents (Kirk, 1973; Lowe 1978; Cheshire 1979; Sjöström, 1981; Aspinall, 1983). Microorganisms on the other hand have a lower carbohydrate content than vascular plants. They show a more uniform distribution of the "major" neutral saccharides and contain higher proportions of partially methylated sugars, heptoses and amino sugars than vascular plants do (Percival and McDowell, 1967; Lowe 1978; Cheshire, 1979; Stewart, 1974; Laskin and Lechevalier, 1982; Aspinall, 1983; Klok et al., 1984a,b). On the basis of literature data and of the present results which indicate covariance of minor components with rhamnose, fucose, ribose, mannose and galactose (see Fig. 2.1), it is concluded that high contributions of rhamnose, fucose, ribose, mannose, galactose, mono-O-methyl-aldoses, heptoses, allose, altrose, tetroses, glycerol and amino sugars in the hydrolysates of our samples point to the presence of microorganisms, and that relatively high contributions of arabinose and xylose point to vascular plant sources. This implies that the fine grained samples contain relatively more microbially derived saccharides than the coarse grained samples. Dual input to the peat samples is confirmed by factor analysis where glucose, xylose, arabinose and the total sugar yield load on the positive side of the first factor and all other sugars load collectively on the negative side of the first factor.

Analyses of neutral saccharides in cyanobacterial mats (Klok et al., 1984b) and in recent marine sediments derived from algae (Klok et al., 1984a) have shown that in those cases the minor components comprise 5 to 15 wt% of the

total yield and that the relative contributions (in wt%) of rhamnose: fucose: ribose: arabinose: xylose: mannose: galactose: glucose are approximately in the proportion of 10: 9: 5: 7: 15: 14: 19: 21 respectively. On the basis of these results it can be estimated that the greater part of all carbohydrates analysed in the fine grained peat samples are derived from microorganisms and not from vascular plants.

The lower carbohydrate abundance in the fine grained samples is probably due not only to the lower carbohydrate content of microorganisms but more to degradation of vascular plant polysaccharides by microorganisms resulting in catabolic products of which only a small fraction is re-used for the bio-synthesis of microbial polysaccharides and other saccharide containing polymers.

The significant correlations between the contributions of various sugar monomers and the total carbohydrate yield make clear that difference in grain size is indirectly a result of biodegradation of material derived from identical vascular plant precursors. It should be kept in mind, however, that in this particular environment part of the fine grained material may be allochtonous, transported to the mangrove environment by tidal movements and retained in place due to the mangrove root system.

The direct cause for formation of different grain sizes appears to be the fractionation procedure itself. Decaying vascular plant tissue apparently loses its mechanical strength due to microbial action and the influence of abiological factors (e.g. tidal action, acidic micro-environments). The disrupted tissue is more accessible to microorganisms and may consequently house a larger microbial population. A larger microbial population results in more intensive decay of the vascular plant polysaccharides with a conco-mitantly lower total carbohydrate concentration and it results at the same time in higher relative contributions of microbial and lower relative con­ tributions of vascular plant polysaccharides.

The absolute and relative carbohydrate yields from the fine grained samples show no trends with increasing depth. This indicates that degradation as outlined above occurs during the initial stages of peat formation in the uppermost peat layers and that carbohydrates derived from vascular plants that are possibly remaining in the fine grained fraction are resistant to microbial attack. This is in agreement with the analytical data of the coarse grained samples, in which the most extensive decay as apparent from the total sugar yields takes place at depths less than 65 cm from the surface (the sample from

244-254 cm is regarded as an outlier as will be discussed below). It is also in agreement with the fact that fractionation of the peat into coarse and fine grained fractions did not show an increase in the proportion of the fine grained fraction with increasing depth (Ryan, 1985). So, it seems that the conditions for microorganisms to be active in the peat become unfavourable even at shallow depths. Deactivation of microorganisms appears to be the main cause determining the quantities of carbohydrates remaining in the coarse grained samples of the peat. Mangrove roots are highly lignified which greatly reduces the digestibility of these tissues for microorganisms after the more accessible carbohydrates have been digested.

The line of reasoning foliowed above is supported by investigations into the activity of microorganisms in peat samples from the Florida Everglades and the Okefenokee Swamp in Georgia. Measurements of dehydrogenase activity, plate counts of numbers of bacteria, streptomycetes and fungi, and colonization studies of peat material with hyphal organisms (streptomycetes and fungi) show that in most cases this activity is restricted to the upper peat levels (Given, 1972; Dickinson et al., 1974; Given and Dickinson, 1975; Given et al., 1983). Observations by light microscopy (Dickinson et al., 1974) show no strong fluctuations in numbers of bacterial cells and of pieces of hyphae. The middle and lower parts of the cores usually contained the highest numbers. It should be noted, however, that it is difficult to distinguish nonviable from viable cells with microscopic techniques, or to determine whether the cells are actively growing or dormant (Spackman et al., 1981). Combination of our results with the results of determinations of microbial populations, indicates that the "microbial" sugars analysed in all samples in this inves-tigation except the ones from 0-10 cm, will be mainly derived from nonviable or dormant microorganisms.

It has been suggested that the rather high levels of tannins in Rhizophora tissues could act as preservatives by inhibition of fungal attack (Given 1972; Spackman et al., 1981). Extra preservative action of tannins on carbohydrates, however, cannot be deduced from the total sugar concentrations, because yields of neutral saccharides in peat samples from an open marsh environment in the Florida Everglades (chapter 3) and in peats derived from cypress, waterlily and saw grass in the Okefenokee (chapter 4) are similar to those in peat samples derived from mangroves in the present case. The yields from the present site are not markedly high at all.