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Published online 2 February 2006
Published in J Environ Qual 35:490-494 (2006)
DOI: 10.2134/jeq2005.0276
© 2006 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
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TECHNICAL REPORTS

Plant and Environment Interactions

Does Calcite Encrustation in Chara Provide a Phosphorus Nutrient Sink?

Kian Siong* and Takashi Asaeda

Department of Environmental Science and Human Engineering, Saitama University, 255 Shimo-okubo, Sakura-Ku, Saitama 338-8570, Japan

* Corresponding author (03D5052{at}post.saitama-u.ac.jp, siongk{at}gmail.com)

Received for publication July 17, 2005.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
We studied the effect of calcite encrustation in stoneworts (Chara spp.) on P cycling in an aquatic ecosystem. Sequential fractionation was performed to quantify P fractions of the internodes of calcified (Ca-CF) and uncalcified (UCa-CF) Chara fibrosa Agardh ex Bruzelius. Our results showed that Ca-CF was able to store more P and about 14 to 23% of total P in Ca-CF was co-precipitated with encrusted calcite, while only 2 to 3% was found in UCa-CF. Furthermore, in Ca-CF, an increased amount of total P did not result in a higher release of bioavailable water-soluble and sodium hydroxide–extractable P. Extracellular calcification in Chara enhanced nutrient sink for P, provided a further bottom-up control of phytoplankton, and should be regarded as a positive feedback in stabilizing Chara dominance in lakes.

Abbreviations: AFDW, ash-free dry weight • Ca-CF, calcified Chara fibrosa Agardh ex Bruzelius • DOC, dissolved organic carbon • LOI, loss on ignition • SRP, soluble reactive phosphorus • TDP, total dissolved phosphorus • TOC, total organic carbon • TP, total phosphorus • UCa-CF, uncalcified Chara fibrosa Agardh ex Bruzelius


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
THE PRESENCE of Charophyceae (stoneworts) is often associated with clean and rather hard water with a low P concentration. Forsberg (1964) reported that soluble reactive phosphorus (SRP) concentration as low as 15 µg P L–1 inhibited the plant growth and the sensitivity to P explained the absence of charophytes in very eutrophic waters and their disappearance from polluted localities. Contrary to earlier findings, later studies have shown that the plant can grow well in water with a high P concentration, for example 1.0 mg P L–1 (Blindow, 1988) and 0.8 mg P L–1 (Kufel and Ozimek, 1994) and the disappearance of the plant is caused primarily by shading through phytoplankton and other algae (Blindow, 1992). Kufel and Ozimek (1994) also reported the enhancement of P content in biomass when the concentration of SRP in culture media increased. Furthermore in a review by Kufel and Kufel (2002), dense Chara beds in hard water may act as nutrient sinks in several ways, such as nutrient incorporation in plant biomass, nutrient withdrawal from decomposing detritus, release of allelophatic compounds, reduced sediment resuspension, slow decomposition rate on plant senescence, and the co-precipitation of P with calcite.

Most photosynthetic aquatic plants in hard water are capable of precipitating calcite (Hutchinson 1957; Wetzel, 1960) and P co-precipitation with photosynthetically induced calcite in marl lakes (Otsuki and Wetzel, 1972). However, in most cases, the calcite is dispersed and not associated with the plants themselves. Charophytes, on the other hand, have calcite encrusted on the plant cell wall; therefore, the efficiency of the plant in reducing P bioavailability in the water column can be assessed. Moreover, the plant can be heavily calcified-more than 50% (as CaCO3) of the total plant biomass dry weight has been reported (Hutchinson, 1975; Królikowska, 1997). Thus, the objective of this study was to elucidate the relationship between calcite encrustation in Chara and P cycling in aquatic ecosystems. We tested the hypothesis that calcite encrustation in charophytes enhanced P stored in the plant biomass and increased the redox-insensitive forms of Ca-bound P that have potential as a P nutrient sink.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Materials
Uncalcified Chara fibrosa Agardh ex Bruzelius (UCa-CF) was collected from Myall Lake (152°20' E, 32°25' S), a fresh–brackish shallow lake located in New South Wales, Australia, in January, May, September, and December 2004. The plant was collected at a water depth of 1 to 1.5 m, while the maximum water depth of Myall Lake is 5 to 6 m. The plant grew on the sediment, composed of noncalcareous gyttja (Table 1). Soluble reactive phosphorus of the lake water ranged from undetectable to 4 µg L–1, while the total dissolved phosphorus (TDP) was 10 to 60 µg L–1. Magnesium concentration was between 92 and 98 mg L–1, while Ca concentration was four to five times lower than Mg (Table 2).


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  		Table 1. Mean of total phosphorus (TP), total phosphorus ash-free dry weight (AFDW), calcium, loss on ignition (LOI), and percentage of calcium as calcium carbonate in ash residue.{dagger}

 

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  		Table 2. Water chemistry of Myall Lake and laboratory culture water.

 
Calcified C. fibrosa (Ca-CF) was obtained by growing young UCa-CF on the sediment taken from Myall Lake in two fish tanks (length = 35 cm, width = 25 cm, and height = 30 cm). City tap water with TDP in the range 20 to 30 µg L–1 was used; calcium as CaCl2 concentration was added and the concentration was maintained between 15 and 20 mg Ca L–1 during the experiment (Wetzel and McGregor, 1968). Salinity was adjusted to 2 PSU using NaCl, and water chemistry was checked every week. Illumination was supplied using 2 x 20 W fluorescent lamps several centimeters above the surface water with a photoperiod of 12 h light and 12 h dark. The plants were harvested 4 mo after planting and calcification was observed in most of the plant thalli. For chemical analysis and P fractionation, only mature plant internodal cell of Ca-CF and UCa-CF was used.

Methods
Water Chemistry
Water pH, temperature, and salinity were recorded using a calibrated water checker (U-20; Horiba, Kyoto, Japan). Soluble reactive phosphorus was determined by the molybdenum blue colorimetric method (Murphy and Riley, 1962). Total dissolved phosphorus was determined with the same procedure as the SRP after digestion with K2S2O8 in an autoclave (120°C) for 30 min (American Public Health Association, 1995). Both Ca and Mg were determined using a spectrophotometer, based on the Calmagite colorimetric method (Hardness Method 8030 DR/4000; Hach, Loveland, CO). Dissolved organic carbon (DOC) and total organic carbon (TOC) were determined using a TOC analyzer (TOC-5000; Shimadzu, Kyoto, Japan) based on procedure given in American Public Health Association (1995).

Plant Analysis
Total P and Ca of plant internodes were analyzed following dry ashing at 550°C for 1 h; the same procedure as for water was used to determine total phosphorus (TP) and Ca after the residue was dissolved in 1.0 mol L–1 HCl. Percentage loss on ignition was calculated, based on weight remaining after the dry ashing.

Phosphorus Fractionation
Three fractionation procedures were performed to quantify P fractions of the internodes of UCa-CF and Ca-CF. Fractionation A was developed based on Dou et al. (2000) for characterizing manure phosphorus and Kuo (1996) for inositol P extraction in soil. The procedure included (i) extraction of sample (50–75 mg) with 50 mL distilled water for 30 min to obtain water-soluble P (H2O-P), (ii) extraction with 50 mL of 1.0 mol L–1 NaOH for 20 h to extract organic P (NaOH-P), and (iii) extraction with 50 mL of 1.0 mol L–1 HCl for 30 min to release Ca-bound P (HCl-P).

Fractionation B was a modification of procedure for inositol P extraction in soil developed by Kuo (1996). The fractionation procedure comprised (i) extraction of sample (50–75 mg) with 50 mL of 0.05 mol L–1 HCl to remove simultaneously H2O-P and Ca-bound P [HCl (0.05 mol L–1)-P]; the difference between this fraction and H2O-P of Fractionation A represents the P associated with calcite, (ii) extraction with 50 mL of 1.0 mol L–1 NaOH for 20 h to extract organic P (NaOH-P), and (iii) extraction with 50 mL of 1.0 HCl for 30 min to release remaining Ca-bound P (HCl-P).

Fractionation C was a modification fractionation of inorganic phosphate in calcareous lake sediment developed by Hieltjes and Lijklema (1980). The procedure included (i) two consecutive extractions of sample (50–75 mg) with 50 mL of 1.0 mol L–1 NH4Cl for 2 h to remove P adsorbed on carbonates and loosely bound Ca ions (NH4Cl-P); (ii) extraction with 50 mL of 1.0 mol L–1 NaOH for 20 h to extract organic P (NaOH-P), and (iii) extraction with 50 mL of 1.0 mol L–1 HCl for 30 min to release remaining Ca-bound P (HCl-P). Recovery for each fractionation scheme was calculated from the sum of fractions divided by the mean of TP of tested sample. Calcium was measured in the extract of H2O-P and HCl-P of Fractionation A, HCl (0.05 mol L–1)-P of Fractionation B, and NH4Cl-P of Fractionation C.

Reliability of the fractionation procedure was checked by including in Fractionation A and B a sample of phosphate co-precipitated with calcium carbonate (CaCO3–P). The CaCO3–P was prepared by bubbling 1 L of saturated Ca(OH)2(aq) solution containing 1.0 mg P L–1 with CO2(g); the precipitated CaCO3(s) was washed with distilled water, filtered, and dried at 60°C for 1 wk. In all cases, P concentration in the extract was determined by molybdenum blue colorimetric method (Murphy and Riley, 1962). Acid or alkaline extract was neutralized before the determinations. Phosphorus in NaOH-P extract was measured following digestion with K2S2O8 in an autoclave (120°C) for 30 min (American Public Health Association, 1995).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
Calcite encrustation appeared as a white band in the plant internodes and the calcification increased with the age of the plant. The plant was initially encrusted with calcite in the alkaline region of internodal cell, however when the cell grew older, the whole thalli became encrusted. Calcium content of the internodes increased significantly from 20 to 35 mg g–1 in UCa-CF to 276 mg g–1 in Ca-CF (Table 1). The percentage loss on ignition decreased from about 80% in UCa-CF to about 30% in Ca-CF, and the percentage of CaCO3 in residual ash of UCa-CF (<50%) was lower than that of Ca-CF (97%). The mean of total P increased from 0.48 to 0.85 mg g–1 in UCa-CF to 0.92 mg g–1 in Ca-CF or from 0.58 to 1.00 mg g–1 in UCa-CF to 3.16 mg g–1 in Ca-CF, based on ash-free dry weight (AFDW). This implies that calcification in Chara enhanced the amount of P stored in the plant. Furthermore, to understand whether this P enhancement also changes P speciation in the plant and increases potential for P nutrient sink on plant senescence and decomposition, phosphorus fractionation was performed. Our result showed that calcification in charophytes increased redox insensitive Ca-bound P in the plant; moreover an increase of TP in Ca-CF did not result in a higher percentage of H2O-P or NaOH-P (Fig. 1).


Figure 1
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  		Fig. 1. Percentage of P fractions relative to the mean total P following (A) Fractionation A, (B) Fractionation B, and (C) Fractionation C with error bars representing standard deviation (attachment).

 
Under Fractionation A, UCa-CF showed a higher release of H2O-P for samples with a higher TP content, that is, up to 60% (SD 1%) for January 2004 samples (Fig. 1A). The sum of H2O-P and NaOH-P fractions accounted for more than 96% in all UCa-CF samples, while HCl-P, representing the Ca-bound P, was small (2–3%). Unlike UCa-CF, high TP content in Ca-CF did not result in a higher release of H2O-P; the mean of H2O-P was 47% (SD 2%). While the percentage of NaOH-P in Ca-CF was generally lower than those of UCa-CF samples, the HCl-P fraction increased significantly and ranged 14 to 23%. Analysis of this last fraction of Ca-CF also showed that the Ca concentration, when converted to a dry weight basis, ranged between 220 and 240 mg g–1 or equal to 80 to 86% of total Ca in the sample (Table 3). In addition to the Ca release, CO2 gas was also observed as a reaction product of calcite and HCl. This result confirmed that P measured in the 1.0 mol L–1 HCl fraction was associated with encrusted calcite of Ca-CF.


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  		Table 3. Mean of phosphorus and calcium concentration extracted in various fractions for calcified (Ca-CF) and uncalcified (UCa-CF) Chara fibrosa.

 
McConnaughey (1991) showed that P co-precipitated with the encrusted calcite in Chara while Kiyosawa (2001) reported that the Ca bands of calcified Chara cell walls were composed mainly of calcite and CaHPO4. Both of these Ca compounds are redox-insensitive and can still bind P even under anoxic conditions which release Fe-bound P to the overlying water column (Kufel and Kufel, 2002). Thus, these Ca-bound P forms have potential as a P nutrient sink because they are not considered to be very bioavailable. Moreover, charophytes often grow in rather alkaline and hard water, where marl is often found as bottom sediment (Vymazal, 1995, p. 257–284). These marl sediments are likely produced by the detritus of calcified charophytes and other carbonate precipitations, and have been very useful in paleobotany and paleoecology studies (e.g., Graham, 1993; Anadón et al., 2000). Under the condition outlined above, this suggests that both calcite and Ca-bound P will function as long-term storage of Ca and P in sediment on the plant senescence and decomposition.

Phosphorus co-precipitation with calcite, induced during periods of intensive photosynthesis, occurs in hard-water lakes (Otsuki and Wetzel, 1972). Kufel and Kufel (2002) pointed out that this indirect mechanism of reducing P bioavailability in the water column may have been underestimated in assessing Chara beds acting as nutrient sinks in shallow lakes. Our result showed that besides the indirect mechanism described above, phosphorus in the water column was also directly co-precipitated with encrusted calcite along the plant internodal cell and consequently, an additional bottom-up control of phytoplankton can be expected as the bioavailability of P in the water column is reduced. Co-precipitation of P with calcite, as P storage in bottom sediment, has been reported in a charophytes-dominated shallow lake in Poland (Kufel and Kufel, 1997). Meanwhile, the inability of C. fibrosa in Myall Lake to precipitate calcite on their plant surface is not within this scope of study. However, several authors have shown that dissolved organic matter can inhibit calcite precipitation (Morse, 1974; Reynolds, 1978), while Mg, polyphenol, phosphonic acid derivatives, and fulvic acid strongly influence the kinetics of calcite formation (Reynolds, 1978). Furthermore, increasing water acidity during lake acidification, particularly in a water system with limited buffer capacity, will reduce the concentration and therefore the availability of carbonate ions that eventually inhibit the calcification process. Because the formation of Ca-bound P depends on the calcification process itself, the inability of charophytes to precipitate calcite will definitely limit the proposed nutrient P sink through the formation of Ca-bound P.

In Fractionation B (Fig. 1B), addition of 0.05 mol L–1 HCl to Ca-CF samples released both H2O-P and Ca-bound P simultaneously. Phosphorus release in this first fraction was also accompanied by CO2 generation and Ca release, which was equal to 54 to 101% of total Ca in Ca-CF (Table 3). While the percentage of NaOH-P fraction was close to the result of Fractionation A, the HCl-P in Ca-CF decreased to less than 1%. The percentage Ca-bound P itself was estimated, based on the difference of HCl (0.05 mol L–1)-P and H2O-P (Fractionation A). The mean value of 15% (SD 8.0%) was produced, which is still in the range of Ca-bound P under Fractionation A procedure.

The third fractionation method (i.e., Fractionation C) produced similar result as Fractionation A for UCa-CF samples since NH4Cl extracted as much P as distilled water in Fractionation A (Fig. 1C). Meanwhile, the NH4Cl-P in Ca-CF was significantly higher than H2O-P of Fractionation A, that is, 55% (SD 3%) vs. 47% (SD 2%). However, our further analysis showed that about 30% of the Ca in each sample was also released in the NH4Cl fraction (Table 3). This suggests that an increase of P in NH4Cl-P was due to the release of some Ca-bound P in Ca-CF. The last HCl-P fraction, which represents Ca-bound P, also decreased from 14 to 23% (Fractionation A) to 7 to 8%. Because some Ca-bound P was released in the NH4Cl fraction, Fractionation C may have underestimated the actual Ca-bound P in calcified Chara sample.

Lastly, to verify the fractionation procedure, the use of P (in form of phosphate) co-precipitated with CaCO3 was justified because calcification in Chara produces mainly CaCO3 (Table 1). In addition, calcite encrustation in Chara appears to be a by-product of the localized pH increase due to OH– efflux (Lucas and Smith, 1973) and the calcite deposits are of the crystal type expected to be produced by inorganic precipitation (Borowitzka et al., 1974). Our result showed that 99% of P co-precipitated with CaCO3 was extracted with 1.0 mol L–1 HCl in the Fractionation A procedure and 96% with 0.05 mol L–1 HCl in Fractionation B. The conclusion is that the fractionation procedures can be used to quantify Ca-bound P in Chara internodes.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
Calcite encrustation in charophytes enhanced P stored in the plant biomass. Results of sequential P fractionation showed that about 14 to 23% of total P in Ca-CF was present in redox-insensitive forms of Ca-bound P, while only 2 to 3% was found in UCa-CF. In aquatic ecosystems, an increase of Ca-bound P forms increases the potential nutrient sink of P, and hence can provide a further bottom-up control of phytoplankton. Thus the calcification in Chara should be regarded as a direct positive feedback in stabilizing Chara dominance in lakes.


   ACKNOWLEDGMENTS
 
This study was supported in part by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank Takeshi Fujino, Jagath Manatungge, H. Lalith Rajapakse, Daniel A. Shilla, Anna Redden, and Brian Sanderson for their assistance during field sampling and laboratory analysis. We thank Daniel H. Pote and two anonymous reviewers for their constructive comments.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 





This Article
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Right arrow Ecosystem Restoration
Right arrow Phosphorus
Right arrow Nutrient Cycling
Right arrow Plant and Environment Interactions

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