- Email: [email protected]
Characterization of organic phosphorus in marine sediments by 31P NMR
220
GEOCHEMISTRY OF THE EARTH'S SURFACE AND OF MINERAL FORMATION 2nd INTERNATIONAL SYMPOSIUM, July, 2-8, 1990, Alx en Provence, F r a n c e .
C H A R A C T E R I Z A T I O N OF O R G A N I C P H O S P H O R U S IN M A R I N E S E D I M E N T S BY 31p N M R
INGALL E. D.,. SCHROEDER P. A. and BERNER R. A. Department of Geology and Geophysics, Yale University Box 6666, New Haven, Connecticut 06511 U.S.A.
INTRODUCTION The burial of organic phosphorus in marine sediments is quantitatively an important sink for phosphorus in the oceans. Furthermore, the burial of organic phosphorus compounds resistant to diagenetic breakdown (refractory organic P) may play a significant role in the overall marine phosphorus budget (Ingall and Van Cappellen, 1990; Froelich et al., 1982). Despite the importance of organic phosphorus burial, little is known about the speciation of organic phosphorus compounds in marine sediments. As a direct method of characterizing organic phosphorus in sediment organic matter, 31p nuclear magnetic resonance (NMR) techniques were employed. S A M P L E S AND M E T H O D S Samples used in this study were chosen to represent a wide range of marine depositional environments in terms of both water depth and bottom water oxygenation. Specifically, sediments from the Carmen Basin and Walvis Bay were deposited in low oxygen bottom waters (<0.5 ml/1 O2) and sediments from the SEEP and DOMES sites were deposited in normal marine environments (bottom waters >> 0.5 ml/1 02). Core sample locations, water depths and references containing site information are summarized in Table 1. In addition, a sample of freeze dried phytoplankton was analyzed in order to characterize one of the many possible organic inputs to the sediments. Both solution and solid-state magic angle spinning(MAS) NMR techniques were used. All samples were pre-treated with a citrate- dithonite-
bicarbonate solution buffered to pH 7.6 to remove paramagnetic iron phases which strongly interfere with the collection of NMR spectra. Samples were prepared for solution N M R by placing approximately 30 grams of dry sediment in 200 ml of 0.5 N NaOH and stirring for 24 hours. The resulting solution was centrifuged, to remove suspended solids > 0.2 micrometers, and then concentrated by evaporation to a volume of approximately 10 ml. Samples for solid-state MAS NMR measurements were prepared by treating the sediments several times with 20% HF in 1 N HC1. This method was effective in removing the bulk of the mineral phases and concentrating the organic matter. Solution samples were analyzed at 11.7 T using a Bruker NMR spectrometer. Solid samples were analyzed at 7 T using a homebuilt spectrometer equipped with a Doty Scientific high-speed spinning probe. RESULTS Solution and solid-state NMR spectra are presented in figures 1 and 2, respectively. Chemical shifts are reported in ppm using 85% phosphoric acid as the standard. The most intense peak at 6 ppm on all solution spectra (fig 1) is characteristic of free inorganic orthophosphate in a strong NaOH solution. This peak most likely represents phosphate liberated from mineral and organic phases during base extraction. Spectra for the SEEP A, SEEP C, DOMES and Carmen Basin extracts, contain a small peak at approximately 20 ppm. In addition, chemical shifts of 18.2 and 16.6 ppm are observed in the
GEOCHEMISTRY OF THE EARTH'S SURFACE AND OF MINERAL FORMATION 2nd INTERNATIONAL SYMPOSIUM, July, 2-8, 1990, Alx en Provence, France.
221
TABLE I. Sample information. Site Name & Description
Location
Water Depth (m)
Core Interval Drganic Reference Analyzed (cm) Carbon %
Carmen Basin
Gulf of Califomia 26°28.3~N110°00.0'W
527
40-50
3.6
DeMasterand Turekian,1987
DOMES SiteC57-58
N.EquatorialPacific 15°9.5'N125°54.4'W
4638
7-9
0.4
Cochran and Kfishnaswami, 1980
SEEP A 83 Gyre 9 Sta. 1
ConfinentalShelf Mid Atlantic Bight 40°28.1'N70°54. rw
80
15.5-17.5
1.6
Andersonet al., 1988
SEEP C 83 Gyre 9 Sta 5
Continental Slope Mid Atlantic Bight 39°10.9q,,170°42.9"vV
2700
13.5-15.5
0.8
Andersonet al., 1988
Walvis Bay AII-93/3-15
Namibian Shelf 22°36.0'S14°07.0E
106
40-48
7.0
DeMaster,1979
spectra of SEEP A. Chemical shifts in this region are generally representative of phosphonic compounds. For example, the chemical shift of ethylaminophosphonic acid in a 1N NaOH is 19.8 ppm (Newman and Tate, 1980). Based on the similar chemical shifts of our samples, the peaks at 20 p p m are interpreted as representing alkyl-phosphonic acids. Chemical shiftS at 18.2 and 16.6 ppm are also representative of phosphonic compounds but with more shielding of the P nuclei compared to the alkyl-phosphonic acids. Likely candidates for these c o m p o u n d s are the phosphonolipids in which more shielding of the P nuclei results from the ester-linkage. To our knowledge, these results document for the first time the occurrence of phosphonic compounds in marine sediments. In phosphonic compounds phosphorus is bonded into an organic molecule via a direct carbonphosphorus bond. The chemical stability of the C-P bond makes release of phosphorus from phosphonic c o m p o u n d s difficult even after prolonged acid and base hydrolysis (Kittredge and Roberts, 1969). This property, along with their wide occurrence in nature, makes phosphonic compounds a reasonable candidate for the proposed refractory organic P buried in marine sediments. Phosphonic compounds have been shown to
comprise approximately 3 percent of the total phosphorus in an undiferentiated sample of marine phytoplankton and microzooplankton (Kittredge et al., 1969). In addition, phosphonates have been found in a wide range of marine phyla (Hilderbrand, 1983) and have been identified in soil samples (Newman and Tate, 1980). The absence of a 20 ppm peak in our marine phytoplankton sample suggests that either phosphonic compounds are absent or that their concentration in the extract was too low for detection. It is possible that during early diagenesis in marine sediments preferential decomposition of the orthophosphate-containing organic compounds enriches the sedimentary organic matter with the more resistant phosphonic compounds. It has been shown that while certain marine microorganisms have the capability to break down aminophosphonic acids, this process is inhibited in the presence of orthophosphatecontaining compounds. Peaks in the range 5.5 to -4 ppm on the solution and solid-state spectra are generally representadve of orthophosphate esters and diesters. The second largest peak in all the solution spectra is attributed primarily to orthophosphate monoesters such as inositol, sugar and mononucleotide which have been shown to appear in the region from 5 to 3.5 ppm in basic solutions (Newman and Tate,
222
GEOCHEMISTRY OF THE EARTH'S SURFACE AND OF MINERAL FORMATION SYMPOSIUM, July, 2-8, 1990, Aix en Provence, France.
2nd INTERNATIONAL
JE
,iii
i11
SEEP A Plankton i'l
Walvis Bay
SEEP C
I
' vr--1~-v~,,'w",em~lle~r~l'v
-or • tl,,tv),-,
L ,!
Carmen Basin
DOMES
J
3b
2'o
o
!
-lb
I
lb
30
I
2o
!
lb
o
-lb
PPM
PPM
Figure 1 : 31p solution spectra For NaOH extracts of marine phytoplankton and sediment samples listed in Table I.
Walvis Bay /!i '
~l~
SEEP A 'J
~
t
,
I
100
I
0 PPM
/
!,
,
,,
".,J
I
-lifO 100
I
0 PPM
f
t
-lifO
Figure 2 : 31P MAS NMR spectra of Walvis Bay and SEEP A organic concentrates at spinning frequencies of 9 kHz and 8 kHz, respectively. Data collected over 750 scans, using a 90 sec recycle time, 4-phased cycled 4 lasec 90 ° pulse time and 1024 data points.
GEOCHEMISTRY OF THE EARTH'S SURFACE AND OF MINERAL FORMATION 2nd I N T E R N A T I O N A L SYMPOSIUM, July, 2-8, 1990, Aix en Provence, France.
1980). Orthophosphate diesters, such as many of the phospholipids, are expected to have chemical shifts at lower ppm values relative to monoesters due to increased shielding of the P nuclei by ester linkages and thus could explain the peaks upfield from 3.5 ppm. A standard o f phosphatidyinositol, a phospholipid, run on solid-state NMR has a shift of -1.7 ppm. A standard of phosphatidyl choline, another phospholipid, in strong base run on solution NMR has observable shifts at 6 and 4.5 ppm. Peaks in the expected region for a phospholipid (ca. -2 ppm) were not observed in this solution. This is most likely due to the hydrolyzation of ester linkages in the strongly basic solution which produces phosphate monoesters and free orthophosphate in solution. Solid-state N M R spectra for SEEP A and Walvis Bay contain only one peak in the region of -2 ppm which is believed to reflect the presence of phosphate diesters such as phospholipids. This result is consistent with organic geochemical work for SEEP A and Walvis Bay that shows that sediment organic matter at these sites contains a high proportion of lipid material. The differences between the solution and solid-state spectra for the Walvis Bay and SEEP A sites can be attributed to two factors. First, due to sample size limitations, the phosphorus concentration in solid-state samples is barely above detection, therefore phases in low abundance like the phosphonic compounds are missed. Second, the strong base treatment used to extract sediment organic matter for solution NMR results in the hydrolyzation of ester linkages, as was observed in similarly treated standard lipid material. CONCLUSIONS Two major classes of phosphorus compounds have been identified in marine sediments. Phosphonic compounds are a small but identifiable
223
component in spectra of sediment extracts from pelagic (DOMES), lower slope (SEEP C) and shelf (SEEP A) normal marine environments. In addition, phosphonic compounds have been identified in a low oxygen depositional environment (Carmen Basin). The general occurrence of the chemically resistant phosphonic compounds makes them a good candidate for the proposed diagenetically resistant organic P phase. In all sediment extracts and in the extract of phytoplankton, phosphate monoesters dominate the spectra of organic phosphorus compounds. Solid-state spectra indicate that much of the organic P in the SEEP A and Walvis Bay sediments is comprised of phosphate diesters, possibly phospholipids. Comparison of solid-state and solution spectra of samples from the same site indicates that the phosphate monoesters observed in the solution spectra most likely result from base hydrolysis of phosphate diesters during extraction of organics from the sediments. REFERENCES
Anderson R.F., Bopp R.F., Buesseler K.O., and Biscaye P.E.(1988) Cont. Shelf Res. 8, 925-946 Cochran J.K. and Krishnaswami S. (1980) Amer. J. Sci. 280, 849-889. DeMaster D.J. (1979) The marine budgets of silica and 32Si. Ph.D. dissertation, Yale Univ. DeMaster D.J. and Turekian K.K. (1987) Paleoceanography 2, 249-254. Froelich P.N., Bender M.L., Luedtke N.A., Heath G.R., and DeVries T. (1982) Amer. J. Sci. 282, 474-5 1 1. Hilderbrand R.L. (1983) The Role of Phosphonates in Living Systems. CRC Press, 207p. Ingall E.D. and Van Cappellen P. (1990) Geochim. Cosmochim. Acta. 54, in press. Kittredge J.S. and Roberts E. (1969) Science 164, 37-42. Kittredge J.S., Horiguchi M., and Williams P.M. (1969) Comp. Biochem. Physiol. 29, 859-863. Newman R.H. and Tate K.R. (1980) Commun. in Soil and Plant Analysis. 11,835-842.
GEOCHEMISTRY OF THE EARTH'S SURFACE AND OF MINERAL FORMATION 2nd INTERNATIONAL SYMPOSIUM, July, 2-8, 1990, Alx en Provence, F r a n c e .
C H A R A C T E R I Z A T I O N OF O R G A N I C P H O S P H O R U S IN M A R I N E S E D I M E N T S BY 31p N M R
INGALL E. D.,. SCHROEDER P. A. and BERNER R. A. Department of Geology and Geophysics, Yale University Box 6666, New Haven, Connecticut 06511 U.S.A.
INTRODUCTION The burial of organic phosphorus in marine sediments is quantitatively an important sink for phosphorus in the oceans. Furthermore, the burial of organic phosphorus compounds resistant to diagenetic breakdown (refractory organic P) may play a significant role in the overall marine phosphorus budget (Ingall and Van Cappellen, 1990; Froelich et al., 1982). Despite the importance of organic phosphorus burial, little is known about the speciation of organic phosphorus compounds in marine sediments. As a direct method of characterizing organic phosphorus in sediment organic matter, 31p nuclear magnetic resonance (NMR) techniques were employed. S A M P L E S AND M E T H O D S Samples used in this study were chosen to represent a wide range of marine depositional environments in terms of both water depth and bottom water oxygenation. Specifically, sediments from the Carmen Basin and Walvis Bay were deposited in low oxygen bottom waters (<0.5 ml/1 O2) and sediments from the SEEP and DOMES sites were deposited in normal marine environments (bottom waters >> 0.5 ml/1 02). Core sample locations, water depths and references containing site information are summarized in Table 1. In addition, a sample of freeze dried phytoplankton was analyzed in order to characterize one of the many possible organic inputs to the sediments. Both solution and solid-state magic angle spinning(MAS) NMR techniques were used. All samples were pre-treated with a citrate- dithonite-
bicarbonate solution buffered to pH 7.6 to remove paramagnetic iron phases which strongly interfere with the collection of NMR spectra. Samples were prepared for solution N M R by placing approximately 30 grams of dry sediment in 200 ml of 0.5 N NaOH and stirring for 24 hours. The resulting solution was centrifuged, to remove suspended solids > 0.2 micrometers, and then concentrated by evaporation to a volume of approximately 10 ml. Samples for solid-state MAS NMR measurements were prepared by treating the sediments several times with 20% HF in 1 N HC1. This method was effective in removing the bulk of the mineral phases and concentrating the organic matter. Solution samples were analyzed at 11.7 T using a Bruker NMR spectrometer. Solid samples were analyzed at 7 T using a homebuilt spectrometer equipped with a Doty Scientific high-speed spinning probe. RESULTS Solution and solid-state NMR spectra are presented in figures 1 and 2, respectively. Chemical shifts are reported in ppm using 85% phosphoric acid as the standard. The most intense peak at 6 ppm on all solution spectra (fig 1) is characteristic of free inorganic orthophosphate in a strong NaOH solution. This peak most likely represents phosphate liberated from mineral and organic phases during base extraction. Spectra for the SEEP A, SEEP C, DOMES and Carmen Basin extracts, contain a small peak at approximately 20 ppm. In addition, chemical shifts of 18.2 and 16.6 ppm are observed in the
GEOCHEMISTRY OF THE EARTH'S SURFACE AND OF MINERAL FORMATION 2nd INTERNATIONAL SYMPOSIUM, July, 2-8, 1990, Alx en Provence, France.
221
TABLE I. Sample information. Site Name & Description
Location
Water Depth (m)
Core Interval Drganic Reference Analyzed (cm) Carbon %
Carmen Basin
Gulf of Califomia 26°28.3~N110°00.0'W
527
40-50
3.6
DeMasterand Turekian,1987
DOMES SiteC57-58
N.EquatorialPacific 15°9.5'N125°54.4'W
4638
7-9
0.4
Cochran and Kfishnaswami, 1980
SEEP A 83 Gyre 9 Sta. 1
ConfinentalShelf Mid Atlantic Bight 40°28.1'N70°54. rw
80
15.5-17.5
1.6
Andersonet al., 1988
SEEP C 83 Gyre 9 Sta 5
Continental Slope Mid Atlantic Bight 39°10.9q,,170°42.9"vV
2700
13.5-15.5
0.8
Andersonet al., 1988
Walvis Bay AII-93/3-15
Namibian Shelf 22°36.0'S14°07.0E
106
40-48
7.0
DeMaster,1979
spectra of SEEP A. Chemical shifts in this region are generally representative of phosphonic compounds. For example, the chemical shift of ethylaminophosphonic acid in a 1N NaOH is 19.8 ppm (Newman and Tate, 1980). Based on the similar chemical shifts of our samples, the peaks at 20 p p m are interpreted as representing alkyl-phosphonic acids. Chemical shiftS at 18.2 and 16.6 ppm are also representative of phosphonic compounds but with more shielding of the P nuclei compared to the alkyl-phosphonic acids. Likely candidates for these c o m p o u n d s are the phosphonolipids in which more shielding of the P nuclei results from the ester-linkage. To our knowledge, these results document for the first time the occurrence of phosphonic compounds in marine sediments. In phosphonic compounds phosphorus is bonded into an organic molecule via a direct carbonphosphorus bond. The chemical stability of the C-P bond makes release of phosphorus from phosphonic c o m p o u n d s difficult even after prolonged acid and base hydrolysis (Kittredge and Roberts, 1969). This property, along with their wide occurrence in nature, makes phosphonic compounds a reasonable candidate for the proposed refractory organic P buried in marine sediments. Phosphonic compounds have been shown to
comprise approximately 3 percent of the total phosphorus in an undiferentiated sample of marine phytoplankton and microzooplankton (Kittredge et al., 1969). In addition, phosphonates have been found in a wide range of marine phyla (Hilderbrand, 1983) and have been identified in soil samples (Newman and Tate, 1980). The absence of a 20 ppm peak in our marine phytoplankton sample suggests that either phosphonic compounds are absent or that their concentration in the extract was too low for detection. It is possible that during early diagenesis in marine sediments preferential decomposition of the orthophosphate-containing organic compounds enriches the sedimentary organic matter with the more resistant phosphonic compounds. It has been shown that while certain marine microorganisms have the capability to break down aminophosphonic acids, this process is inhibited in the presence of orthophosphatecontaining compounds. Peaks in the range 5.5 to -4 ppm on the solution and solid-state spectra are generally representadve of orthophosphate esters and diesters. The second largest peak in all the solution spectra is attributed primarily to orthophosphate monoesters such as inositol, sugar and mononucleotide which have been shown to appear in the region from 5 to 3.5 ppm in basic solutions (Newman and Tate,
222
GEOCHEMISTRY OF THE EARTH'S SURFACE AND OF MINERAL FORMATION SYMPOSIUM, July, 2-8, 1990, Aix en Provence, France.
2nd INTERNATIONAL
JE
,iii
i11
SEEP A Plankton i'l
Walvis Bay
SEEP C
I
' vr--1~-v~,,'w",em~lle~r~l'v
-or • tl,,tv),-,
L ,!
Carmen Basin
DOMES
J
3b
2'o
o
!
-lb
I
lb
30
I
2o
!
lb
o
-lb
PPM
PPM
Figure 1 : 31p solution spectra For NaOH extracts of marine phytoplankton and sediment samples listed in Table I.
Walvis Bay /!i '
~l~
SEEP A 'J
~
t
,
I
100
I
0 PPM
/
!,
,
,,
".,J
I
-lifO 100
I
0 PPM
f
t
-lifO
Figure 2 : 31P MAS NMR spectra of Walvis Bay and SEEP A organic concentrates at spinning frequencies of 9 kHz and 8 kHz, respectively. Data collected over 750 scans, using a 90 sec recycle time, 4-phased cycled 4 lasec 90 ° pulse time and 1024 data points.
GEOCHEMISTRY OF THE EARTH'S SURFACE AND OF MINERAL FORMATION 2nd I N T E R N A T I O N A L SYMPOSIUM, July, 2-8, 1990, Aix en Provence, France.
1980). Orthophosphate diesters, such as many of the phospholipids, are expected to have chemical shifts at lower ppm values relative to monoesters due to increased shielding of the P nuclei by ester linkages and thus could explain the peaks upfield from 3.5 ppm. A standard o f phosphatidyinositol, a phospholipid, run on solid-state NMR has a shift of -1.7 ppm. A standard of phosphatidyl choline, another phospholipid, in strong base run on solution NMR has observable shifts at 6 and 4.5 ppm. Peaks in the expected region for a phospholipid (ca. -2 ppm) were not observed in this solution. This is most likely due to the hydrolyzation of ester linkages in the strongly basic solution which produces phosphate monoesters and free orthophosphate in solution. Solid-state N M R spectra for SEEP A and Walvis Bay contain only one peak in the region of -2 ppm which is believed to reflect the presence of phosphate diesters such as phospholipids. This result is consistent with organic geochemical work for SEEP A and Walvis Bay that shows that sediment organic matter at these sites contains a high proportion of lipid material. The differences between the solution and solid-state spectra for the Walvis Bay and SEEP A sites can be attributed to two factors. First, due to sample size limitations, the phosphorus concentration in solid-state samples is barely above detection, therefore phases in low abundance like the phosphonic compounds are missed. Second, the strong base treatment used to extract sediment organic matter for solution NMR results in the hydrolyzation of ester linkages, as was observed in similarly treated standard lipid material. CONCLUSIONS Two major classes of phosphorus compounds have been identified in marine sediments. Phosphonic compounds are a small but identifiable
223
component in spectra of sediment extracts from pelagic (DOMES), lower slope (SEEP C) and shelf (SEEP A) normal marine environments. In addition, phosphonic compounds have been identified in a low oxygen depositional environment (Carmen Basin). The general occurrence of the chemically resistant phosphonic compounds makes them a good candidate for the proposed diagenetically resistant organic P phase. In all sediment extracts and in the extract of phytoplankton, phosphate monoesters dominate the spectra of organic phosphorus compounds. Solid-state spectra indicate that much of the organic P in the SEEP A and Walvis Bay sediments is comprised of phosphate diesters, possibly phospholipids. Comparison of solid-state and solution spectra of samples from the same site indicates that the phosphate monoesters observed in the solution spectra most likely result from base hydrolysis of phosphate diesters during extraction of organics from the sediments. REFERENCES
Anderson R.F., Bopp R.F., Buesseler K.O., and Biscaye P.E.(1988) Cont. Shelf Res. 8, 925-946 Cochran J.K. and Krishnaswami S. (1980) Amer. J. Sci. 280, 849-889. DeMaster D.J. (1979) The marine budgets of silica and 32Si. Ph.D. dissertation, Yale Univ. DeMaster D.J. and Turekian K.K. (1987) Paleoceanography 2, 249-254. Froelich P.N., Bender M.L., Luedtke N.A., Heath G.R., and DeVries T. (1982) Amer. J. Sci. 282, 474-5 1 1. Hilderbrand R.L. (1983) The Role of Phosphonates in Living Systems. CRC Press, 207p. Ingall E.D. and Van Cappellen P. (1990) Geochim. Cosmochim. Acta. 54, in press. Kittredge J.S. and Roberts E. (1969) Science 164, 37-42. Kittredge J.S., Horiguchi M., and Williams P.M. (1969) Comp. Biochem. Physiol. 29, 859-863. Newman R.H. and Tate K.R. (1980) Commun. in Soil and Plant Analysis. 11,835-842.