A study of the formation of high molecular weight compounds during the decomposition of a field diatom population

Estumine, Coastal and Shelf Science (1983) 17, 189-196

A Study of the Formation of High Molecular Weight Compounds during the Decomposition of a Field Diatom Population

E.-L. Poutanen’and Institute of Oceanographic

R. J. Morrisb Sciences, Wormley, Godalming, Surrey, U.K.

Received 12 February 1982 and in revised form 10 September I982

Keywords:

humic

substances;

humic

acids;

diatoms;

decomposition;

sediments

This work has investigated the possible formation of humic and fulvic acids, particularly high molecular weight moieties, in degrading diatom debris. The diatom debris was collected, freshly sedimented, from a well characterized natural field diatom population and allowed to degrade under conditions similar to those found at the sediment-water interface of many marine, organic-rich sediments. Samples of the detritus and overlying water were taken regularly over a period of 4 months and analysed for the presence of humic compounds. In the case of the detrital material, a sequential series of extractions were used. Molecular weight fractionation of the extracted humic material was carried out using Diatlow Ultrafiltration Membranes and two different fractionation methods have been compared. It appears that the choice of method may have an important effect on the determined distribution of the humic material, particularly when humic acid concentrations are low. High molecular weight ( > 300 000- > 100 000) humic and fulvic acids were found in all the detrital samples, the content increasing with the period of decomposition. No evidence of any humic material was found in extracts of the living diatoms, the conclusion being that this material had heenformed in situ during the degradation of the diatom debris. Most (65-90%) of the humic material was extracted from the detritus using a mild 0.05 M NaOH extraction at room temperature. Fulvic acids comprised approximately 90% of the total 0.05 M NaOH extraction, being composed mainly of either low molecular weight compounds (40-50% < 10000) or high molecular weight compounds (38-4845 > 100 000). Levels of humicacidswere muchlower and includeda wide range

of molecularweight fractions. The relevanceof thesefindings to the possibleorigin of humic materialin certain marinesedimentsis discussed. Introduction The occurrence of substantial proportions of humic and fulvic acidsin organic-rich marine surface sedimentshas been reported by several workers (e.g. Nissenbaum& Kaplan, 1972; Brown et al., 1972; Nissenbaumet al., 1972; Simoneit et al., 1979; Morris & Calvert, 1977; MacFarlane, 1978). Some of the organic-rich marine sedimentsinvestigated included very young sediments which contained high levels of humic material (e.g. Morris & Calvert, ‘Present address: Institute of Marine Research, Box 166, 00141 Helsinki, bReprint

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Finland,

to R. J. Morris.

189 0272-7714/83/080189+08803.00/0

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Press Inc. (London)

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190

E.-L. Poutanen &r R. J. Mani>

1977). This suggested that the relatively rapid formation of high molecular weight compounds may be a very important step, in certain sedimentary areas, during the incorporation of planktonic organic matter into sediments. Cronin & Morris (1982s) have found that high molecular weight ( > 300 000) humic acids form the major part of organic carbon in a young organic-rich diatomaceous sediment, while the deeper, older sediment contained relatively less humic acids and relatively more fulvic acids. There was also a significant age difference between the high and low molecular weight humic fractions; the lower molecular weight fraction being older than the higher molecular weight fraction. These workers concluded that the planktonic input (primarily diatoms) at this sedimentary site was rapidly complexed and converted to high molecular weight humic compounds. Cronin & Morris (19826) have also studied the rapid formation of high molecular weight humics from two degradation experiments, designed to simulate sedimentary conditions where there is a large input of diatoms. One of these samples was a pure, cultured phytoplankton species and the other a field phytoplankton population from a large, enclosed experimental ecosystem. Also a sample from a sediment trap under the experimental ecosystem was analysed. The results clearly indicate very rapid formation of high molecular weight humic complexes, which appear to be easily broken down by chloroform-methanol treatment suggesting extremely weak, labile bonds/associations between the various components. The objective of the present work was to investigate the possible formation of humic and fulvic acids, particularly high molecular weight moieties, in degrading diatom debris freshly sedimented from a well characterized natural field diatom population. It was hoped that the results would supplement work on diatomaceous-rich sediments and lead to a clearer understanding of the formation of humic material in such sedimentary environments. Material Samples

Samples were taken from the D.A.F.S. experimental ecosystem bags moored in Loch Ewe, N.W. Scotland. The bags are 5 m in diameter and 20 m deep. During a study of the initial spring bloom in one of the enclosed water columns (March-April 1981), freshly sedimented particulate matter (i.e. material sedimented in 24 hours) was collected by pump from the bottom of the experimental bag. A microscopic examination showed the particulate matter consisted of phytoplankton remains (> 90%) and amorphous organic matter. The phytoplankton debris was dominated by the diatoms Sheletonema costatum, Nitzschia seviata and Thalassiosira sp. The sedimented material (approximately 200 g wet wt) was quickly transferred to a clean 211 glass jar, filled with filtered seawater from the experimental bag, placed in the dark and nitrogen bubbled through the mixture for f hour. The experiment was thus carried out under anoxic conditions, similar to those found at the sediment-water interface of most marine, organic-rich sedimentary environments. The jar was then sealed and stored in the dark at 8-10 “C, the temperature of the Loch at the time of the experiment. Both water samples and samples of the sediments for subsequent analysis were then regularly taken from the jar by means of a clean glass, wide bore pipette. On the same day that the detrital sample was taken, a collection was made of living phytoplankton. Twenty litres of water, taken from a depth of 3 m in the experimental bag, was pressure filtered through a pre-cleaned GF/F filter. This sample was taken at the

Forma&ion of high molecular weight compounds

191

height of the spring phytoplankton bloom and consisted almost entirely of diatoms, Skeletonemacostatum, Nitzschia seviata and Thalassiosirasp. being the dominate species. The filter plus phytoplankton was then immediately stored at -30 “C under nitrogen until analysis. Sampling of the degradation experiment Water sampleswere taken for spectrophotometric measurementsafter 1, 2, 3 and 7 days, then after 24, 3 and 31weeks. They were filtered through Whatman GF/F glassfibre filters and analysed immediately. Sediment sampleswere taken after 4, 5, 6 and 19 weeks, respectively. The pore waters were immediately separated by centrifugation and measured spectrophotometrically and the residue extracted by meansof a sequential seriesof extractants of gradually increasing harshness(see Methods) so as to take out first the labile, loosely bound material and then the lesslabile, more strongly bound material. Dry weight/wet weight ratios were obtained (from separated sample) by drying an aliquot of the sediment samplesafter pore waters had been separated.

Methods Optical densities for filtered water sampleswere measured at a wavelength of 465 nm, which is in the region of maximum absorbancefor humic material in the visible spectrum (Schnitzer & Khan, 1972), and at 665 nm with a Beckman DU spectrophotometer. The sediment samples(equivalent to 0.4-O. 5 g dry weight) were first suspendedin distilled water (24 hours, under nitrogen). The suspensionwas centrifuged and the water decanted off and kept. The residue wasthen extracted once with a mixture (1 : 1) of 0.05 M Na,P20, and 0.05 M NaOH and then repeatedly with 0.05 M NaOH until the extract wascolourless. This was followed by separate extractions with 0.1 M NaOH and 0.5 M NaOH until the respective supernatants were colourless. For each extraction the mixture was agitated in an ultrasonic bath for one hour under nitrogen and then allowed to stand for several hours in the dark. The residue wasfinally refluxed with 0.5 M NaOH under nitrogen for 20 hours, washedseveral times with distilled water, the supernatant being added to the 0.5 M NaOH extract, and the residue dried and weighed. A separate extraction was performed on the frozen phytoplankton sample‘by sonicating the GF/F filter in a solution 0.5 M in NaOH and Na,P,O, for one hour then allowing to stand for three hours in the dark at room temperature. The extract wasthen immediately examined spectroscopically for the presence of humic material. All extraction operations were performed under nitrogen and all extracts stored under nitrogen at 5 “C to minimize auto-oxidation of the humic material. Care was taken that identical procedures were followed with each sampleso that results could be compared. Distilled water extracts were found to have somecolour in their visible spectrum, but low absorbance values indicated very small amounts of humics. No further work was performed on these extracts. The 0.05 M, 0.1 M and 0.5 M alkaline extracts, after separation from the residual sediments by centrifugation, were acidified to pH 2 with 6 M HCl and allowed to stand for 24 hours at room temperature. The precipitated humic acid was purified by recycling between acid and basic solutions and it wasfinally left in 0.05 M NaOH solution. The fulvic acids remained in the acid (pH 2) solution.

192

E.-L. Poutanen & R. J. Morris

The extracts were made up to a standard volume (50 ml) with distilled water then fractioned by passing them through DiafIo ultrafltration membranes. Such membranes have been used for the study of aquatic humic substances for several years (e.g. Gjessing, 1970). Diaflo ultrafiltration membranes XM-300, XM-lOOA, PM-30 and PM-16 were used together with an Amicon Ultrafiltration Cell (Model 52). Fractionation was achieved by use of the membranes in decreasing nominal molecular weight cut-off (300 000, 100 000, 30 000, 10 000). The sample solution was pushed through the membrane (XM-300) under nitrogen pressure (10 psi) until 10-15 ml remained in the cell. This was then washed with distilled water and the filtration continued until a total of 50 ml of titrate was collected. Then a further 40 ml of wash water was passed through the sample. The hnal pH of the filtrate and retentate from the humic acid solutions was 8, the fulvic acid solutions giving a final pH of 5. This procedure was adopted to minimize any possible effect of pH on the size of the humic molecules (Gjessing, 1971), or on the molecular weight permeability of the ultrafiltration membranes (see later). The filtrate was then passed through the other membranes in decreasing molecular weight cut-off, and the corresponding molecular weight fractions collected. Optical densities were measured from all molecular weight fractions at wavelengths 465 nm and 665 run. The light absorbance properties of humic extracts are known to be highly pH dependent, but according to Gjessing (1976) changes of colour at the pH levels of these fractions (pH 5-g) should be small. The amount of humic and fulvic acid in each fraction was estimated by comparing the light absorption values with standard curves constructed by use of humic and fulvic acid solutions extracted from marine sediments, after correction for the appropriate blanks. These values were checked by freeze-drying the residual solutions and by weighing the residues. The filtrates from the 10 000 molecular weight filter (< 10 000 fractions) were dialysed in distilled water to remove all salts prior to freeze-drying. Goh & Williams (1979) have suggested that the separation of humic extractions into molecular size fractions, prior to the acid fractionation into fulvic and humic acids, minimizes changes in the nature of the extracts during acid fractionation. As a direct comparison of the two methods the weak alkali extract (O-05 M NaOH) from 19 week old sediment (sample 4) was divided into two portions, one of which was handled as above and the other by the method given in Goh i? Williams (1979). In the latter case after separation of the molecular size fraction ( > 300 000, > 100 000. > 30 000, > 10 000 and < 10 000) the humic and fulvic acids were then separated and subjected to spectrophotometric and gravimetric analysis as before. Results and discussion

No evidence of any humic material was detected in the visible spectrum of the phytoplankton extract; however, significant amounts of high molecular weight humics were extracted from all the other samples. Our assumption therefore is that the majority of this humic material must have been found in situ during the degradation of the diatom debris. The absorbance values measured in the samples of overlying water and pore water from the degradation experiment increased during the fhst few days (0~016-0~090). For the remainder of the experiment they were surprisingly constant (0.080-O .090). The spectra of all samples were featureless curves of increasing absorbance with decreasing wavelength which are typical for soil and marine humic substances (Schnitzer & Khan, 1972). The ratio of absorptivities at two wavelengths (EJE,& can be used to characterize the featureless spectra of humic substances (Schnitzer & Khan, 1972; Nissenbaum & Kaplan,

Formation of high molecular weight compounds

193

1972). The values of E465/E665ratios were very similar in all water samplesand they varied between 1.7 and 4 .O which indicates a relatively low degree of condensation. The water soluble organic matter measured here appeared to be in a very unstable state and upon storage (after filtration) there wasa marked production of turbidity. The samephenomenon was found by Sieburth & Jensen(1969) in their exudation study of Fucus. As their clear exudates becamecoloured they also becameturbid and a buff-coloured precipitate formed. They concluded that there is a ‘disappearanceor masking of the phenols’ and an increase in the amount of Gelbstoff. A similar tendency to form a precipitate was seen by Craigie & McLachlan (1964). We believe that these unstable water soluble compounds could represent the very earliest association/polymerization products en route to the humic complexes, unfortunately their unstable nature rendered further work impossible. Significant amounts of high molecular weight material were extracted from all sediment samples.The data characterizing the extraction efficiency of humic material by the three alkali solutions are presented in Table 1. In all cases,most (65~90%) of the humic material was extracted by the 0.05 M NaOH solution, which indicates the predominantly labile, loosely bound nature of these compoundsin the samples.Much smaller amounts of humic material resulted from the other, harsher extractions. The distribution of the various molecular weight humic and fulvic acid fractions in the 0.05 M NaOH extraction are given in Table 2. The fulvic acid fractions make up 87-959/o of the total humic extract and are mainly composedof two groups of compounds: one low molecular weight ( < 10000) which constitutes 50, 39, 52 and 41% of the total fulvic acids in each sample, and the other high molecular weight (> 100000) comprising 38, 42, 48

TABLE

sediment)

1. The extraction efficiency of humic by the three alkali solutions Sediment sample

Sample 1 Sample 2 Sample 3 Sample 4

(4 (5 (6 (19

‘V 5 M NaOH

0.05~NaoH0.1~NaOH extract weeks) weeks) weeks) weeks)

extract

(mg g-r

42 36 0.5

M

NaOH

dry

0.5~Na0H extract

extract

216 180 193 252 and refluxed

compounds

Residue

48 65 48 30 extract

85 166 131 39

measured

together.

2. Molecular weight distributions of humic (HA) and fulvic (FA) acid fractions in 0.05 M NaOH sediment extracts (expressed as % of total humic extract) TABLE

>300000 >100000 >30000 >lO 000 <1ooOcl

Sample 1 (4 weeks)

Sample 2 (5 weeks)

Sample 3 (6 weeks)

Sample 4 (19 weeks)

1.7 1.0 0.7 0.7 1.7

2.8 1.2 1.7 1.4 2.3

2.8 1.0 1.3 1.4 2.1

3.3 2.5 2.1 3.0 2.1

22.1 13.8 7.0 4.6 47.7

23.0 16.5 14.1 0.4 36.6

27.5 16.1 0.4 47.3

weight

35.7 1.6 3.0 10.9 35.7

194

E.-L. Poutanen & R. J. Morris

and 43% of the total fulvic acids. The humic acids constitute only 5-13% of the total humic extract and show a wide range of molecular weight distribution. These results agree, in the case of humic acids, with the data given by Rashid & Prakash (1972) for humic compounds isolated from decomposed thalli of Pucusand L.umissuriu, but they differ in that these workers did not find any high molecular weight ( > 10 000) fulvic acids. However, it is difficult to compare reported molecular weights given in the literature, the results depending on the method used and the origin of the sample. A major factor which is likely to affect the ‘apparent’ molecular size of humic molecules is pH. The work of Gjessing (197 1) clearly indicates that either molecular size increases with increase of pH or permeability of the membranes decreases with increasing pH. This is particularly relevant if attempts are made, as in this work, to compare the molecular weight distributions of humic acids (alkali soluble, acid insoluble) and fulvic acids (acid soluble). The pH effect should tend to result in an overestimation of the high molecular weight humic acids, and possibly of the low molecular weight fulvic acids. High molecular weight fulvic acids (the major high molecular weight fraction found in this work) will, if anything, be underestimated. Thus, we consider the finding of substantial amounts of high molecular weight firlvic acids in these samples to be real and not merely an artifact of the method. A comparison of two different fractionation methods (see Methods) indicates that, for these young humic compounds, the choice of method may also have an important effect on the results (Table 3). If separation into molecular weight fractions occurs before the acid fractionation into fulvic and humic acids (pre-acid fractionation method) (Goh & Williams, 1979), then it becomes impossible to precipitate any humic acids from some molecular weight fractions. These fractions appeared, from the post-acid fractionation method (method used in this work) to definitely contain humic acids, although at low levels. The fulvic levels were also lower by the Goh & Williams method compared with the postaid fractionation method. Thus differences between the results of the two methods were significant and indicate, we believe, the instability of some of these young humic molecules towards different experimental treatments. The acid soluble fulvic acids formed the major fraction of the humic extracts, which on the face of it agrees with the hypothesis that fulvic acid is the intermediate form in the humtication process (Nissenbaum, 1973). However, Cronin & Morris (1982a) have found that the major form of organic carbon in the young (600-900 years) organic-rich diatomaceous sediments from the Namibian shelf is as high molecular weight humic acids TABLE 3. Comparison 0.05 M NaOH extract sediment)

of two different fractionation methods applied to the from sample 4 (results expressed as mg g-1 dry weight

Pre-acid fractionation (Goh & Williams, 1979)

Post-acid fractionation (this present study)

Molecular size fraction

HA

FA

HA

FA

>300000 >lOOoOO >30000 >lOOOO
11 ND ND ND ND

84 20 10 12 46

8 6 5 8 5

90 4 8 28 90

ND-not

detected.

Formation

of high molecular weight compounds

195

with only low levels of fulvic acids. The deeper, older sediments (3000 years) contained relatively more fulvic acids than humic acids. A similar distribution of humic material is also seen in young (10-200 years) organic-rich sedimentsfrom the Peru shelf (Poutanen & Morris, 1982). Simple degradation experiments carried out by Cronin & Morris (1982b) indicated the rapid (l-2 months) formation of humic compounds from the debris of natural and cultural diatom population. This present work, whilst being in good agreement with the work of Cronin & Morris (1982b) with respect to the quantitation of the total humic matter, shows that these humic compoundsare mainly acid soluble, fulvic acids. Humic acids are certainly present, but at much lower levels. Conclusions The results of this present work suggeststhat there may be several stagesto the early formation of humic compoundsin organic-rich, anoxic marine sediments.Our data indicate that highly unstable, water-soluble compounds, with a visible absorbancespectra similar to that of typical humic substances,are rapidly produced within days of the start of anoxic degradation of diatom debris. Unfortunately it was not possible to carry out molecular weight fractionation on these compounds but they are thought to be of intermediate molecular weight (> 10000). The formation of water insoluble, relatively unstable high molecular weight material ( > 300 000) occurs within weeks. This material is mainly ‘fulvic’ in character, although small amounts of humic acid are also formed. Taking these results together with data from young, organic-rich diatomaceoussediments (Cronin & Morris, 1982a; Poutanen & Morris, 1982) we believe the following sequenceof events is occurring in the organic-rich sedimentary environments which we have studied: first, the formation of water soluble, but very unstable, complexes of intermediate molecular weight ( > 10 000); second, the formation of water insoluble, relatively unstable high molecular weight material ( > 300 000) which is ‘fulvic’ in character; third, the formation of water insoluble, relatively unstable high molecular weight ( >300 000) material which is ‘humic’ in character; and finally the gradual formation of a more refractory lower molecular weight complex which is ‘fulvic’ in character. We believe the first three stagesoccur in a matter of weeks or months after deposition of the planktonic debris, the fourth stage taking place gradually over many hundreds of years. References Brown, F. S., Baedecker, M. J., Nissenbaum, A. & Kaplan, J. R. 1972 Early diagenesis in a reducing fjord, Saanich Inlet, British Columbia-III. Changes in organic constituents of sediment Geochimica et Cosmochimica Acta 36, 1185-1203. Craigie, J. S. & McLachlan, J. 1964 Excretion of colored ultra violet-absorbing substances by marine algae. Canadian Journal of Botany 42,23-33. Cronin, J. R. & Morris, R. J. 1982a The occurrence of high molecular weight humic material in recent organic-rich sediments from the Namibian Shelf. E&urine, Coastal and Shelf Science 15, 17-27. Cronin, J. R. & Morris, R. J. 19826 Rapid formation of humic material from diatom debris. Nato-Conference, Portugal September 1981, Nate-Conference series. Plenum (in press). Gjessing, E. T. 1970 Ultratiltration of aquatic humus. Enwironmental Scrence and Technology 4, 437438. Gjessing, E. T. 1971 Effect of pH on the filtration of aquatic humus using gels and membranes. Schweizzrische ZeitschnJt fiir Hydrologic 33, 592-600. Gjessing, E. T. 1976 Physical and Chemical Characteristics of Aquatic Humus. Ann Arbor Science, Michigan, 120 pp. Goh, K. M. & Williams, M. R. 1979 Changes in molecular weight distribution of soil organic matter during soil development. Journal of Soil Science 3, 747-755.

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& R. J. Morris

MacFarlane, R. B. 1978 Molecular weight distribution of humic and fulvic acids of sediments from a north Florida estuary. Geochimica et Cosmochimica Acta 42, 157%1582. Morris, R. J. & Calvert, S. E 1977 Geochemical studies of organic-rich sediments from the Namibian Shelf. I. The organic fractions. In A Voyage of Discowery: George Bacon 7&h Anniversary Volume (Angel, M. ed.). Pergamon Press, Oxford. pp. 647-665. Nissenbaum, A 1973 The organic geochemistry of marine and terrestrial humic substances: implications of carbon and hydrogen isotope studies. In Adwances in Organic Geochemisny 1973 (Tissot, B. & Bienner, F. eds). Edition Technip, Paris. pp. 39-52. Nissenbaum, A, Baedecker, M. J. & Kaplan, J. R. 1972 Organic geochemistry of Dead Sea sediments. Geochimica et Comwchimica Acta 36,709-727. Nissenbaum, A., Kaplan, J. R. 1972. Chemical and isotopic evidence for the in situ origin of marine humic substances. Limnology and Oceanography 17(4), 570-582. Poutanen, E. L. & Morris, R. J. 1982 The occurrence of high molecular weight humic compounds in the organicrich sediments of the Peru continental shelf. Oceanologica Acta (in press). Rashid, M. A. & P&ash, A. 1972 Chemical characteristics of humic compounds isolated from some decomposed marine algae. Journal of the Fisheries Research Board of Canada 29,55-60. Schnitxer, M. & Khan, S. U. 1972. Humic Substances in the Environment. Marcel Dekker, New York. 327 pp. Sieburth, J. McN. & Jensen, A. 1969 Studies on the algal substances in the sea. II. Exudates of phaeophyta. JournuJ of Experimental Marine Biology and Ecology 3,275289. Simoneit, B. R. T., Mazurek, M. A., Brenner, S., Crisp, P. T. & Kaplan, I. R. 1979 Organic geochemistry of recent sediments from Guaymas Basin, Gulf of California. Deep-Sea Research 26,879-982.