Humic Substaces Inhibit Calcite Crystallizn Ii

Humic Substances Inhibit Calcite Crystallization Possible Relevance to Global Carbon Dioxide Balance. D Grant (Turriff)* [email protected] Summary Water soluble organic humus components (fulvic acids) are potent inhibitors of calcite crystallization, suggesting that their augmentation in natural waters may contribute to global warming. Introduction. Soil humus, which contains environmentally stable organic polymer /colloidal materials of molecular weights up to several million and typical showing 14-C ages of ca1000 years, evidently formed by chemical and biochemical alteration of plant and microbial debris, is the major reservoir of terrestrial organic compounds and provides water and mineral holding properties which are important for plant nutrition, stimulation of root growth (Kononova 1966) and the binding of calcium carbonate in calcareous soils. Soil humus may be depleted by the effects of augmented dissolution/dispersion by the effects of humus degradation by intensive agriculture and increased precipitation effects following climatic change (cf Houghton & Woodwell 1989). Poorly-biodegradable polymethylene humic components and carbohydrate-like fractions are interlinked in humified organic matter of agricultural soils (Grant 1977 cf Potts et al 1973) but more easily degradable, more overtly polysaccharide-like structures are present in water soluble fulvic acids obtained from climatic peats. The former are likely to persist environmentally and augment 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 humic materials present in the sea. (It should be noted that humic substances remain somewhat enigmatic as regards their constituent chemical structures despite numerous attempts to achieve this knowledge, e.g., by measurement 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 "humic acid” and "fulvic acid" (cf, e.g., Schnitzer & Khan 1978 who favored an aromatic-rich model for their core structures but the consequences of a highly alkaline extraction protocol may have produced this type of artifact) seem to depend more on their physical chemistry, including hydrophobicity, than any overt chemical structural differences; a further index of the chemical structure of humus is now shown to be provided by measurement of their effects on seeded crystallization rates). Although marine biological activity involving photosynthesis and calcification is of major importance for consideration of global carbon dioxide cycling (cf Pentcost 1985), the activities of extracellular 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 are probably held in supersaturation (greatest under tropical conditions) with respect to solid phase calcium 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 he inhibitory effects of magnesium and phosphate ions which have been suggested to be insufficient to 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 carried out by the author some years ago for assessment of potential inhibitors for oilwell usage and biochemical calcification research, revealed that soil humic polymers and their derivatives were amongst the most strongly inhibitory substances available. A wider discussion of the results obtained in these studies now seems warranted owing to the environmental implication of the

retardation of calcium carbonate precipitation in the global system of seawater bicarbonate/dissolved carbon dioxide which is the major holding reservoir for potential atmospheric carbon dioxide balance. Inskeep & Bloom (1986) reported that fulvic acid from a USA soil efficiently inhibited calcite crystallization, and similar inhibitory effects of various aquatic humic fractions were reported by Hoch et al (2000). Materials & Methods Humic 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 by Glentworth & Muir 1963) had pH 5.9 and 5.9% C and a fulvic acid type of material by Soxhlet extraction 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 were obtained from local commercial sources. Infrared spectra were obtained as described by Grant et al (1987) and crystallization kinetic data obtained 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 0,9mM CaCl2 in 20mM NaHCO3 at 25oC (cf. Fiig. 1).

Infrared spectra

Infrared spectra of soil extracts [(1-Countesswells agricultural soil; 2-climatic peat 3-(1)sulphated and lignin derivatives] were obtained on disposable aluminium foil mirrors by a specular reflection technique (Grant et al 1987). Table I Infrared Spectra of Humic and Fulvic Acids Studied Humic acid (1) Fulvic acid (1) Fulvic acid (2) Fulvic acid (3) REAX 88B REAX 100M 498 w 532 w 533 w 521 m 533 w 511 w 513 m 558 w 551 m 554 w 557 w 624 w,b 616 m 627 w 616 w 615 m 642 s,b 640 m 644 m,b 716 w 764 w 760 w 760 w 743 m 742 m 783 s 783 s 807 m 805 m 882 m 874 w 892 m 872 s,b 855 m 964 s,b 954 m 941 m 1020 sh 1007 sh 996 m 1046 m 1049 n 1040 vs,b 1051 m 1034 sh 1107 s.b 1110 s,b 1061 sh 1052 vs 1152 s,sh 1132 m,b 1176 sh 1160 sh 1226 sh 1219 w,b 1276 w,sh 1237 s,b 1250 vs 1241 vs 1268 m 1266 sh 1386 s 1381 w,sh 1358 sh 1433 sh 1414 s,b 1431 s 1410 vs 1431 m 1436 m 1456 m 1556 w 1506 s 1503 m 1588 vs 1602 vs 1602 vs 1629 sh 1632 sh 1620 s 1609 m 1640 sh 1659 s 1686 sh 1707 sh 1716 sh 1730 sh 1737 sh 1746 sh 2110 w 2544 w 2544 m,b 2574 w 2626 sh 2940 m 2846 m, 2908 s 2959 w 2932 m 2969 m 3264 sh 3229 vs 3256 vs 3369 vs 3376 vs 3420 vs 3400 vs 3529 vs w: weak; m: medium; s: strong; b: broad; sh: shoulder; v: very

Results Calcium carbonate (calcite) crystallization.

Accurate second order kinetic curves were obtained for seeded calcite crystallization of fulvic acid solutions after a short initiation period. The second order rate constant declined to zero over the range 1 -100 ppm of added fulvic acid. Table II Reaction conditions: [HCO3-] 10mM, 25C Fulvic acid ppm 0 2.7 5.5 70 100

Secondary reaction , % uninhibited rate 100.0 21.0 3.5 (ca 0.0) 0.0

Fig. 1 Effect of Additives on Rates of Seeded Crystallization of CaCO3 (Calcite) a - Fulvic acid (Countesswells) 100µg/ml b -Sulphated/sulphonated humic acid 20µg/ml c - Sulphated fulvic acid 20µg/ml d - Fulvic acid (Countesswells) 5.5µg/ml e - Fulvic acid (Countesswells) 2.8µg/ml f - Fulvic acid (Climatic pear) 15µg/ml g - Lignin derivative REAX 88B 20µg/ml h- No additive i Lignin derivative REAX 100M 20µg/ml The dependence of inhibitor concentration on crystallization rate obeyed the Freundlich (1922) isotherm (1) log (go/g) = kc

l/n (1)

(where go is the relative rate of crystallization in the absence of inhibitor and g is the relative rate of crystallization in the presence of inhibitor) is characteristic of surface adsorption of inhibitors on crystallization nuclei. The value of n = 2 in the above isotherm described seeded calcite crystallization from 10-20 mM NaHCO3 solutions for the fulvic acids (25C) as well as heparin, heparan sulphate and Chondroitin-4 sulphate (studied at 25 and 37C). The relative slopes linear Freundlich are listed in Table III.

Effect of Additives on Rates of Seeded Crystallization of CaCO3 (Calcite) 70ppm marine algal anionic polysaccharides and heparin

No additive Carrageenans kappa iota iota lambda Heparin

100 (a) (b) (c) (d) (e)

[Further details of the anionic polysaccharides studied MW sulphate half ester/ disacch. (a) 380000, 0.98 (b) 750000 1.21 (c) 610000 1.28 (d) 500000 1.6 (e) 20000 2.75

104 87 74 47

anhydrogalactose/ disacch. 0.82 0.84 0.60 0.13 0.00 ]

----------------------------------------------------------------------------------------------------Table III Slopes of Freundlich Isotherm Plots. Fulvic acids from various agricultural soils Sulphated humic acid

10-20 (a)(b) ca 10

(b)

Fulvic acid (Countesswells)

9.6 (b)

Fulvic acid (climatic peat)

4.5 (d)

Phosphate Ethane, hydroxy, 1,1-diphosphonate

ca 4 ca20

(f) (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) __________________________________________________________________

(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 studied were similarly treated by plotting Freundlich isotherms with similar conclusions (results not discussed here). The data listed in Table III reflect on a log(10) scale, the relative anti-crystallization effects of the substances 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 sulphonated soluble form, and the Freundlich isotherm of this material was apparently similar to that of native fulvic 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 commercial polyphosphate calcification inhibitors, but were up to some two orders of magnitude more effective than animal polysaccharides inhibitors such as heparin or heparan sulphate and analogous algal coccolith sulphated polysaccharides for which their in vivo roles likely include an inhibition/control of biological calcification (cf. Grant et al. 1992).

Discussion The present studies were conducted with methodologies (due to Nancollas and co-workers (cf Nancollas 1979)) believed to allow crystallization kinetic results to be optimally obtained with regard to high reproducibility and significance. It was established that the natural polyanionic fulvic acid derived from agricultural soil is a highly effective 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

of calcite crystallization by some humic acids inhibition than that indicated by our studies which may have been partly due to differences in experimental technique (lower carbonate concentration and different mixing procedures used)). The present study suggested that a similar high degree of calcite crystallization inhibition efficiency was achieved with a sulphated (otherwise difficult to quantify by this procedure) humic acid derived from the same soil as the native fulvic acid studied. Similar conductometric techniques were used to more briefly study (results not given here) the relative efficiency of the range of chemical inhibitors identified as inhibitors of calcite crystallization. The results of these studies indicated that the crystallization inhibitors also inhibited calcite dissolution but with altered relative inhibitory efficiencies. The natural, relatively well-defined polyanionic biopolymer, heparan sulphate is also an efficient inhibitor of calcification when measured on a molar basis, in which role (amongst numerous other protein 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 of the soil-derived humic polymers is considerably (ca. two orders of magnitude) greater than is that of 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 by complexation could not be correlated with inhibition of crystallization which process which, however, obeyed a Freundlich isotherm, which is thought to be characteristic of the surface adsorption of the inhibitor molecules at crystallization nuclei surfaces. Soil-derived polymers also effectively inhibit barium sulphate crystallization but, in this case, less effectively than do heparin-like polymers. Sulphation of the soil-derived polymers however improves their barium sulphate inhibitor effectiveness. Although land-derived soil humus fractions are thought likely to contribute to oceanic carbon dioxide balance, especially under conditions of humus depletion through intensive agricultural practices, knowledge of the relevant quantities and oceanic distribution of such polymers as well as of other industrially produced calcification inhibitors (stable calcification inhibitor input analysis should include poorly biodegradable phosphonates) is currently uncertain but should be assessed for gaining an acceptable scientific basis for international legal frameworks to limit global warming (cf Lasho & Ahuja 1990). BaSO4 Crystallization While the role of polyanionic inhibitors of BaSO4 crystallization is of interest to a fuller understanding of the marine supersaturation of these inorganic ions it can also provide a rough indicator 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-crystallization activities (for barium sulphate) and a rational dependence upon polysaccharide microstructure evidenced. (These results, are summarised in Table V, were obtained by the author and have previously been presented as a poster by M.I. Tait at the XIIIth International Seaweed Symposium, Vancouver,1989; since they are of more general interest in regard to marine chemistry the results are now further reported here. Table IV Barium sulphate crystallization (method used - Nancollas 1979 (cf Grant et al 1989)) Inhibitor 20 ppm Crystallization rate (from second order rate plot) Control 100 REAX 88B (lignin deruived) 86.4 Climatic peat polysaccharide-rich humus extract 13.3 Ethane hydroxy 1,1 diphosphonic acid (Grant 1979) 4.2 Scaletreat 206 1.8 Baker ML 1559 1.2 Nalfloc NAL 1285 0.9

Fulvic acid (Countesswells) sulphated oligosaccharide 10 Donmarn actipol

0.4 0.1

heparin

30

4

sodium tripolyphosphate

-

6

Aquarite

-

9.5

Table V Alginate microstructure dependent BaSO4 crystallization Alginates are anionic linear glycuronans that are major structural components of the cell wall and intercellular regions of marine brown algae (Phaeophyceae). Post-polymerization enzymatic C-5 epimerization of D-mannuronic acid [M] residues generates L-guluronic [G] acid units. The distribution within the polymer of β, 1-4-linked, 4C1, D-mannuronic and α, 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 similar procedure to that used for the humic matter derived and related samples discussed above suggested that a progressive alteration of absorptions in the 630-1190cm-1 region occurred according to the degree of potency of the alginates as inhibitors of BaSO4 crystallization. Alginate microstructure

Disaccharide chain length

Control

(None present)

Relative second order rate constant

Attained after initiation period, min

100.0a

0

Random

25

59.6

0

Poly G blocks

80

36.4

ca. 0

F387b

24

18.8

2

4000

15.2

2

Poly M blocks

22

14.6

1

Poly alternating MG blocks

22

6.0

Poly M

0.5

The uninhibited approx. second order unhibited crystallization rate was 3.2.103xmol-1.min-1.dm3 (mg of seed)-1.100cm3 b A commercial product obtained from Ascophyllum nosoum described by Grasdalen et al. (1981) a

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

References Anon 1992 New Scientist 16 May p7 Soils spoilt by farming and industry (24% of soils are seriously degraded and two thirds of soils in Africa and Asia) Berner RA Westrich JP Graber R Smith J Martens CS (1978) Amer J Soil Sci 278 816-837 CHECK (reviewed by Morse (1983) Freundlich H (1922) (English translation 1926) Colloid and Capillary Chemistry p336-341 Methuen, London Glentworth R Muir JW (1963) The Soils of the Country Round Aberedeen, Inverurie and Fraserburgh Mem Soil Surv Gt Br Scot Edinburgh HMSO Grant D (1977) Chemical structure of humic substances Nature 270 709-710 Molecular composition of soil organic matter components using proton and 13-C NMR techniques International Peat Society was also discussed by Grant Proc International Workshop (Braunschweig, Germany) on Properties of Organic Peat Components, Abstract No 11. [1-H and 13-C NMR spectra allowed classification of humus-derived organic polymers e.g. following removal of paramagnetic ions by sodium pyrophosphate chelation. Humus polymers were originally thought to contain condensed aromatic core structures e.g. derived from lignin or consisting of hydrogen-bonded aggregates of low molecular weight phenolic compounds analogous to phenol formaldehyde resins; a non-aromatic core stucutre similar to polymaleic anhydride had also been postulated. Use of NMR however indicated a degraded carbohydrate type core associated with higly branched polymethylene structures and generally smaller amounts of aromatic structures than previously thought. The amounts and microstructures of thse oligomeric polymeric organic components which are considered to be linked by hydrogen bonds and metal ion crosslinks onto colloidal size particles including clays varies systematically between soil types]. Grant (1979) The rearrangement polymerization of phosphorus acid with acetic anhydride Eur Polym J 15 1161-1165 Grant D Long WF Williamson FB (1987) Infrared spectra of heparin-cation complexes Biochem J 244 143-149 Grant D Long WF Williamson FB (1989) Inhibition of glycosaminoglycans of CaCO3(calcite) crystallization Biochem J 259 41-45 Grant D Long WF Williamson FB (1992) Degenerative and inflammatory diseases may result form defects in antimineralization mechanisms afforded by glycosaminoglycans Medical Hypotheses 38 49-55

Grasdalen H Larsen B Smidsrod O (1981) 13C-N.M.R. studies of monomeric composition and sequences in alginate Carbohydr Res 89 179-181 Hoch AR Reddy MM Aiken GR (2000) Calcite crystal growth inhibition by humic substances with emphasis on hydrophobic acids from the Florida Everglades Geochim Cosmochim Acta 64 61-72 Houghton RA Woodwell GM (1989) Global climatic change Scientific American 260 (4) 18-26 Howarth WN Pinkard FN Stacey M (1946) Function of bacterial polysaccharides in the soil nature 158 836 Inskeep WP Bloom PR (1986) Kinetics of calcite precipitation in the presence of water-soluble organic ligands (additional index words calcium carbonate, crystal growth, fulvic acid, calcite, supersaturation) Soil Sci Soc Am J 50 1431-1437; cf ibid 1167-1172

Kitano Y (1983) Calcification and atmospheric CO2 Biomineralization and Biological Accumulation P Westbroek and EW de Jong (eds) Reidel Dordrecht cf pp 89-9 (cf also Garrels RM & Berner RA (1983) The global caronate-silicate sedimentary system--some feedback relations ibid 73-87);

Kononova MM (1966) Soil Organic Matter Pergamon Press Oxford Lashof DA Ahuja DR (1990) Relative contributions of greenhouse gas emissions to global warming Nature 344 529-531 Morse JW (1983) The kinetics of calcium carbonate dissolution and precipitation Reviews in Mineralogy Vol 11 Carbonates: Mineralogy and Chemistry p227et seq R J Reeder Ed PH Ribbe Series Ed Mineralogical Society of America Nagar BR Waight ES Meuzelaar HLC Kistemaker PG (1975) Studies on the structure and origin of soil humic acids by Curie point pyrolysis in direct combination with low-voltage mass spectrometry

Plant and Soil 43 681-685 Nancollas GH (1979) Advances in Colloid & Interface Science 10 215-252 (cf Reddy MM Nancollas GH (1971) The crystallization of calcium carbonate I. Isotopic exchange and kinetics J Colloid Interface Sci 36 (2) 166-172) Nancollas GH Kazmierczak TF Schuttringer E (1981) A controlled composition study of calcium carbonate crystal growth: the influence of scale inhibitors Corrosion-NACE 32 (2) 76-81 Ogner G (1973) Permanganate oxidation of methylated and unmethylated fulvic acid humic acid anmd humin isolated from raw humus Acta Chem Scand 27 1601-1612 Pentecost A (1985) _ Photosynthetic plants as intermediary agents between environmental HCO3 and carbonate deposition Chapter 29 (p459-481) in Inorganic Carbon Uptake by Aquatic Photosynthetic organisms Eds WJ Lucas JA Berry (American Society of Plant Physiologists) cf

Borowitzka MA (1987) Calcification in algae: mechanism and the role of metabolism CRC Critical Reviews in Plant Sciences 6(10) 1-45

Pleysier J Janssens J Cremers A (1986) A clay suspension stability end point titration method for measuring cation exchange capacity of soils Soil Sci Soc Am J 50 887-891 Potts JE Clendinning RA Ackart WA (1973) The effect of chemical structure on the biodegradability of plastics Proc Symp Degradability of Polymers and Plastics 27-28 Nov 1973 Plastics Inst London 12-1 - 12-10 (Studies reported herein show that the biodegradability of hydrocarbon chains in soil although rapid for smaller chains becomes very slow above C30 and this circumstance evidenty allows substantial amounts of naturally formed (partly oxidised) quasi-low-density-polyethylene humic substances to accumulate in the soil) Schnitzer M Khan SU (1978) Soil Organic matter Elsevier Suess E (1970) Interaction of organic compounds with calcium carbonate - I. Association phenomena and geochemical implications Geochim Cosmochim Acta 34 157-168 Suess E (1973) Interaction of organic compounds with calcium carbonate - II. Organo-carbonate ass ociation in Recent sediments ibid 37 2435-2447

Shurukhina SI Shurukhin VV Tarlakov Yu P (1973) Study of humus extracts by infrared spectroscopy Pochvovedeniye 146-149 Weinstein H Sachs CR Schubert M (1963) Protein polysaccharide in connective tissue: inhibition of phase separation Science 142 1073-1075

Wilson MA (1984) Soil organic matter maps by nuclear magnetic resonance J Soil Sci 35 209-315 *Most recent affiliation University 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 of Aberdeen) and DR MV Cheshire (Macaulay Institute Aberdeen) who provided data and samples for this study.