Humic Substaces Inhibit Calcite Crystallizn II

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Humic Substances Inhibit Calcite CrystallizationPossible Relevance to Global Carbon Dioxide Balance.

D Grant (Turriff)*[email protected]

SummaryWater soluble organic humus components (fulvic acids) are potent inhibitors of calcitecrystallization, suggesting that their augmentation in natural waters may contribute to globalwarming.

Introduction.Soil humus, which contains environmentally stable organic polymer /colloidal materials ofmolecular weights up to several million and typical showing 14-C ages of ca1000 years, evidentlyformed by chemical and biochemical alteration of plant and microbial debris, is the majorreservoir of terrestrial organic compounds and provides water and mineral holding propertieswhich are important for plant nutrition, stimulation of root growth (Kononova 1966) and thebinding of calcium carbonate in calcareous soils.Soil humus may be depleted by the effects of augmented dissolution/dispersion by the effects ofhumus degradation by intensive agriculture and increased precipitation effects following climatic

change (cf Houghton & Woodwell 1989).Poorly-biodegradable polymethylene humic components and carbohydrate-like fractions areinterlinked in humified organic matter of agricultural soils (Grant 1977 cf Potts et al 1973) butmore easily degradable, more overtly polysaccharide-like structures are present in water solublefulvic acids obtained from climatic peats. The former are likely to persist environmentally andaugment marine humic materials. It is also commonly believed (e.g. Hoch et al 2000)that lignin-derived phenolic groups could be suitable markers of land-plant derived humicmaterials present in the sea.

(It should be noted that humic substances remain somewhat enigmatic as regards theirconstituent chemical structures despite numerous attempts to achieve this knowledge, e.g., bymeasurement of cation exchange capacity (e.g. Pleysier et al 1986), by mass spectroscopy(e.g.Nagar et al 1975), ir spectroscopy (e.g., Shurukhina et al 1973) and nmr spectrosopy (e.g.,Grant 1977; Wilson 1984). Defining properties of traditional humus fractions termed "humicacid and "fulvic acid" (cf, e.g., Schnitzer & Khan 1978 who favored an aromatic-rich model fortheir core structures but the consequences of a highly alkaline extraction protocol may haveproduced this type of artifact) seem to depend more on their physical chemistry, includinghydrophobicity, than any overt chemical structural differences; a further index of the chemicalstructure of humus is now shown to be provided by measurement of their effects on seededcrystallization rates).

Although marine biological activity involving photosynthesis and calcification is of majorimportance for consideration of global carbon dioxide cycling (cf Pentcost 1985), the activities ofextracellular calcification inhibitors present in natural waters may also be relevant to this process(Berner et al (1978); Morse (1983). Dissolved oceanic calcium ion and carbon dioxide areprobably held in supersaturation (greatest under tropical conditions) with respect to solid phasecalcium carbonate crystallization, at least in part, by the influence of such natural humic inhibitors

which are highly efficient deactivators of crystallization nuclei. This activity is additional to heinhibitory effects of magnesium and phosphate ions which have been suggested to be insufficientto account for current oceanic calcium carbonate supersaturation levels (Suess 1970, 1973;Berner et al 1978; Kitano 1983, cf Morse 1983).

Studies of the controlled seeded inhibition of crystallization of calcite and barium sulphate carriedout by the author some years ago for assessment of potential inhibitors for oilwell usage andbiochemical calcification research, revealed that soil humic polymers and their derivatives wereamongst the most strongly inhibitory substances available. A wider discussion of the resultsobtained in these studies now seems warranted owing to the environmental implication of the


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retardation of calcium carbonate precipitation in the global system of seawaterbicarbonate/dissolved carbon dioxide which is the major holding reservoir for potentialatmospheric carbon dioxide balance. Inskeep & Bloom (1986) reported that fulvic acid from aUSA soil efficiently inhibited calcite crystallization, and similar inhibitory effects of various aquatichumic fractions were reported by Hoch et al (2000).

Materials & MethodsHumic acid and fulvic acid were extracted by the traditional method (e.g. as described by Ogner(1973)) from a non-calcareous agricultural soil (Countesswells, Aberdeenshire described byGlentworth & Muir 1963) had pH 5.9 and 5.9% C and a fulvic acid type of material by Soxhletextraction with water from climatic peat (Cairn O'Mount, Aberdeenshire) had pH 3.7 and 56%C.Various commercial calcification inhibitors including phosphonates and lignin derivatives wereobtained from local commercial sources.Infrared spectra were obtained as described by Grant et al (1987) and crystallization kinetic dataobtained using methods developed by Nancollas et al., as described by Grant al (1989).The variation of the conductivity was recorded using a Philips PW9514160 electrode fior 0,9mMCaCl2 in 20mM NaHCO3 at 25oC (cf. Fiig. 1).

Infrared spectra


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Infrared spectra of soil extracts [(1-Countesswells agricultural soil; 2-climatic peat 3-(1)sulphatedand lignin derivatives] were obtained on disposable aluminium foil mirrors by a specular reflectiontechnique (Grant et al 1987).

Table IInfrared Spectra of Humic and Fulvic Acids StudiedHumic acid (1) Fulvic acid (1) Fulvic acid (2) Fulvic acid (3) REAX 88B REAX 100M

498 w532 w 533 w 521 m 533 w 511 w 513 m

558 w 551 m 554 w 557 w624 w,b 616 m 627 w 616 w 615 m

642 s,b 640 m 644 m,b716 w764 w 760 w 760 w 743 m 742 m

783 s 783 s807 m 805 m

882 m 874 w 892 m 872 s,b 855 m964 s,b 954 m 941 m

1020 sh 1007 sh 996 m1046 m 1049 n 1040 vs,b 1051 m 1034 sh

1107 s.b 1110 s,b 1061 sh 1052 vs1152 s,sh 1132 m,b 1176 sh 1160 sh

1226 sh 1219 w,b 1276 w,sh 1237 s,b 1250 vs 1241 vs1268 m 1266 sh1386 s 1381 w,sh 1358 sh1433 sh 1414 s,b 1431 s 1410 vs 1431 m 1436 m

1456 m1556 w 1506 s 1503 m

1588 vs1602 vs 1602 vs 1629 sh 1632 sh 1620 s 1609 m

1640 sh 1659 s 1686 sh1707 sh 1716 sh 1730 sh 1737 sh 1746 sh

2110 w2544 w 2544 m,b 2574 w 2626 sh2940 m 2846 m, 2908 s 2959 w 2932 m 2969 m

3264 sh 3229 vs 3256 vs3369 vs 3376 vs 3420 vs 3400 vs 3529 vs

w: weak; m: medium; s: strong; b: broad; sh: shoulder; v: very

ResultsCalcium carbonate (calcite) crystallization.


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Accurate second order kinetic curves were obtained for seeded calcite crystallization of fulvicacid solutions after a short initiation period. The second order rate constant declined to zero overthe range 1 -100 ppm of added fulvic acid.Table II

Reaction conditions: [HCO3-] 10mM, 25C

Fulvic acid Secondary reaction ,ppm % uninhibited rate

0 100.02.7 21.05.5 3.570 (ca 0.0)

100 0.0


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Fig. 1Effect of Additives on Rates of Seeded Crystallization of CaCO3 (Calcite)a - Fulvic acid (Countesswells) 100g/mlb -Sulphated/sulphonated humic acid 20g/ml

c - Sulphated fulvic acid 20g/mld - Fulvic acid (Countesswells) 5.5g/mle - Fulvic acid (Countesswells) 2.8g/mlf - Fulvic acid (Climatic pear) 15g/mlg - Lignin derivative REAX 88B 20g/mlh- No additivei Lignin derivative REAX 100M 20g/ml

The dependence of inhibitor concentration on crystallization rate obeyed the Freundlich (1922)isotherm (1)

l/nlog (go/g) = kc (1)

(where go is the relative rate of crystallization in the absence of inhibitor andg is the relative rate of crystallization in the presence of inhibitor) is characteristic of surfaceadsorption of inhibitors on crystallization nuclei.The value of n = 2 in the above isotherm described seeded calcite crystallization from 10-20 mMNaHCO3 solutions for the fulvic acids (25C) as well as heparin, heparan sulphate andChondroitin-4 sulphate (studied at 25 and 37C).The relative slopes linear Freundlich are listed in Table III.


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Effect of Additives on Rates of Seeded Crystallization of CaCO3 (Calcite)

70ppm marine algal anionic polysaccharides and heparin

No additive 100Carrageenans

kappa (a) 104iota (b) 87iota (c) 74

lambda (d) 47Heparin (e)

[Further details of the anionic polysaccharides studiedMW sulphate half ester/ anhydrogalactose/

disacch. disacch.(a) 380000, 0.98 0.82(b) 750000 1.21 0.84(c) 610000 1.28 0.60(d) 500000 1.6 0.13(e) 20000 2.75 0.00 ]

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Table IIISlopes of Freundlich Isotherm Plots.

Fulvic acids from various agricultural soils 10-20 (a)(b)

Sulphated humic acid ca 10 (b)

Fulvic acid (Countesswells) 9.6 (b)Fulvic acid (climatic peat) 4.5 (d)

Phosphate ca 4 (f)Ethane, hydroxy, 1,1-diphosphonate ca20 (f)

Dequest-2041(N,N,N,N ethylenediaminetetramethylenephosphonate) ca22 (g)

_______________________________________________________________

Heparan sulphate 1.15 (e)

Heparin 1.06 (e)

Chondroitin 4 sulphate 0.35 (e)__________________________________________________________________


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(a) Calculated from results reported by Inskeep & Bloom (1986)) ;(b) Calculated from present studies (data obtained at 25C)(c) Derivatized by sulphation to achieve required solubility; (cf sulphuric acid extracts soluble

humic matter from soils))(e) Reported by Grant et al (1989); (data obtained at 25C)(f) Nancollas et al 1981(g) Nancollas 1979

The results of calcite dissolution experiments in the presence of inhibitors more briefly studiedwere similarly treated by plotting Freundlich isotherms with similar conclusions (results notdiscussed here).

The data listed in Table III reflect on a log(10)scale, the relative anti-crystallization effects of thesubstances studied; numerically higher values indicate greater inhibitor effectiveness.

Humic acid was less easily studied since it dispersed as a colloid rather than being soluble,however it demonstrated a high degree of anti-crystallization activity when in a sulphonatedsoluble form, and the Freundlich isotherm of this material was apparently similar to that of nativefulvic acid from the same soil.

The soil organic matter (polysaccharide-like) inhibitors were of similar degree of inhibitory activity(on a weight basis, but considerably more effective on a molar basis) to commercialpolyphosphate calcification inhibitors, but were up to some two orders of magnitude moreeffective than animal polysaccharides inhibitors such as heparin or heparan sulphate andanalogous algal coccolith sulphated polysaccharides for which their in vivo roles likely include aninhibition/control of biological calcification (cf. Grant et al. 1992).

DiscussionThe present studies were conducted with methodologies (due to Nancollas and co-workers (cfNancollas 1979)) believed to allow crystallization kinetic results to be optimally obtained withregard to high reproducibility and significance.

It was established that the natural polyanionic fulvic acid derived from agricultural soil is a highlyeffective inhibitor of calcification (in agreement with reports by Berner et al 1978, Morse 1983.Inskeep & Bloom 1986 and Hoch et al 2000; the latter workers had found an even higher degree


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of calcite crystallization by some humic acids inhibition than that indicated by our studies whichmay have been partly due to differences in experimental technique (lower carbonateconcentration and different mixing procedures used)).The present study suggested that a similar high degree of calcite crystallization inhibitionefficiency was achieved with a sulphated (otherwise difficult to quantify by this procedure) humicacid derived from the same soil as the native fulvic acid studied.

Similar conductometric techniques were used to more briefly study (results not given here) therelative efficiency of the range of chemical inhibitors identified as inhibitors of calcitecrystallization. The results of these studies indicated that the crystallization inhibitors alsoinhibited calcite dissolution but with altered relative inhibitory efficiencies.

The natural, relatively well-defined polyanionic biopolymer, heparan sulphate is also an efficientinhibitor of calcification when measured on a molar basis, in which role (amongst numerous otherprotein regulator activites) it may provide wide-range protection of blood, urinary and other tissue(cf Grant et al 1992). It is noteworthy, however, that the anti- (calcite)- calcification efficiency ofthe soil-derived humic polymers is considerably (ca. two orders of magnitude) greater than is thatof the heparin/heparan sulphate.

Inhibition of calcite crystallization likely occurs by blocking of crystal growth nuclei rather than by

sequestration of the calcium ions since conductivity changes attributable to sequestration bycomplexation could not be correlated with inhibition of crystallization which process which,however, obeyed a Freundlich isotherm, which is thought to be characteristic of the surfaceadsorption of the inhibitor molecules at crystallization nuclei surfaces.Soil-derived polymers also effectively inhibit barium sulphate crystallization but, in this case, lesseffectively than do heparin-like polymers. Sulphation of the soil-derived polymers howeverimproves their barium sulphate inhibitor effectiveness.

Although land-derived soil humus fractions are thought likely to contribute to oceanic carbondioxide balance, especially under conditions of humus depletion through intensive agriculturalpractices, knowledge of the relevant quantities and oceanic distribution of such polymers as wellas of other industrially produced calcification inhibitors (stable calcification inhibitor input analysisshould include poorly biodegradable phosphonates) is currently uncertain but should be assessedfor gaining an acceptable scientific basis for international legal frameworks to limit global warming(cf Lasho & Ahuja 1990).

BaSO4 CrystallizationWhile the role of polyanionic inhibitors of BaSO4 crystallization is of interest to a fullerunderstanding of the marine supersaturation of these inorganic ions it can also provide a roughindicator of the possible role of alginates for the inhibition of calcification (cf. Weinstein et a.l,1963)].A series of alginates of known microstructure were compared as to their anti-crystallizationactivities (for barium sulphate) and a rational dependence upon polysaccharide microstructureevidenced. (These results, are summarised in Table V, were obtained by the author and havepreviously been presented as a poster by M.I. Tait at the XIIIth International SeaweedSymposium, Vancouver,1989; since they are of more general interest in regard to marinechemistry the results are now further reported here.

Table IVBarium sulphate crystallization (method used - Nancollas 1979 (cf Grant et al 1989))Inhibitor 20 ppm Crystallization rate (from second order rate plot)Control 100REAX 88B (lignin deruived) 86.4Climatic peat polysaccharide-rich humus extract 13.3Ethane hydroxy 1,1 diphosphonic acid (Grant 1979) 4.2Scaletreat 206 1.8Baker ML 1559 1.2Nalfloc NAL 1285 0.9


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Fulvic acid (Countesswells) 0.4sulphatedoligosaccharide 10 0.1Donmarn actipol

heparin 30 4

sodium - 6tripolyphosphate

Aquarite - 9.5

Table VAlginate microstructure dependent BaSO4 crystallizationAlginates are anionic linear glycuronans that are major structural components of the cell wall andintercellular regions of marine brown algae (Phaeophyceae).Post-polymerization enzymatic C-5 epimerization of D-mannuronic acid [M] residues generatesL-guluronic [G] acid units. The distribution within the polymer of, 1-4-linked, 4C1, D-mannuronicand , 1-4-linked, 1C4, L-guluronic acid residues is dependent upon species and, it is believed,sub-cellular location and other cellular factors.

Microstructural analyses of alginates by N.M.R. (as reported in the literature e.g. Grasdalen et al(1981) suggests that the polymers contain short homopolymer sequences.A study of the infrared spectra of the alginates studied which was conducted by a similarprocedure to that used for the humic matter derived and related samples discussed abovesuggested that a progressive alteration of absorptions in the 630-1190cm-1 region occurredaccording to the degree of potency of the alginates as inhibitors of BaSO4 crystallization.

Alginate Disaccharide chain Relative second order Attained aftermicrostructure length rate constant initiation period, min

Control (None present) 100.0a 0

Random 25 59.6 0

Poly G 80 36.4 ca. 0blocks

F387b 24 18.8 2

Poly M 4000 15.2 2

Poly M blocks 22 14.6 1

Polyalternating 22 6.0 0.5MG blocks

a The uninhibited approx. second order unhibited crystallization rate was3.2.103xmol-1.min-1.dm3 (mg of seed)-1.100cm3b A commercial product obtained from Ascophyllum nosoum described by Grasdalen et al. (1981)

If seed crystals were pre-incubated with the inhibitor solutions a considerably greater apparentdegree of inhibition was achieved (The standard procedure in this work used immediatecrystallization rates measured on the addition of seed crystals which started the crystallizationreaction (Grant et al 1989)).


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References

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*Most recent affiliationUniversity of Aberdeen Department of Molecular & Cell Biology

Acknowledgements

Thanks are due to Drs FW Williamson, WF Long, Mrs M Ross and Mrs J Somers (University ofAberdeen) and DR MV Cheshire (Macaulay Institute Aberdeen) who provided data and samplesfor this study.

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Humic Substaces Inhibit Calcite Crystallizn II