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<strong>Nitrate</strong> <strong>Uptake</strong> <strong>by</strong> <strong>Terrestrial</strong> <strong>and</strong> <strong>Aquatic</strong> <strong>Plants</strong><br />

Richard A. Larson, Marina Montez-Ellis, Karen Marley, <strong>and</strong> Gerald K. Sims<br />

University of <strong>Illinois</strong> at Urbana-Champaign<br />

Abstract<br />

Fertilizer-derived nitrate has been the subject of scrutiny among environmental scientists <strong>and</strong><br />

regulatory agencies concerned about the ecological <strong>and</strong> public health effects of elevated nitrate<br />

levels in surface water, groundwater, <strong>and</strong> drinking water. An approach that has not received<br />

much attention is to remove or convert excess nitrate from at or near the point of application<br />

using plants. In this project, we demonstrated the kinetics of nitrate uptake from water <strong>and</strong> soil <strong>by</strong><br />

plants of potential management value. Special attention was paid to potential cover crop species<br />

that might be planted in the autumn after fertilizer application, <strong>and</strong> plowed under or killed in the<br />

spring to act as “green manure.” In addition, we examined the activity of the key enzyme, nitrate<br />

reductase, in a variety of aquatic <strong>and</strong> terrestrial plants. Switchgrass (Panicum virgatum) <strong>and</strong><br />

hornwort (Ceratophyllum demersum) appeared to be the most promising nitrate-removing<br />

species in preliminary studies.<br />

Introduction<br />

Nitrogen fertilization is an established practice in production agriculture for the maintenance of<br />

high yields in crops such as corn <strong>and</strong> soybeans. <strong>Nitrate</strong> is a highly soluble species, <strong>and</strong> is readily<br />

transported from the point of application, as surface runoff as well as leachate to subsurface<br />

waters. In central <strong>Illinois</strong> nitrate N losses at the field <strong>and</strong> watershed scales range from 15-40 kg<br />

N/hectare/year. High nitrate levels in groundwarer <strong>and</strong> drinking water have been linked to health<br />

disorders such as methemoglobinemia, gastric cancer, goiters, <strong>and</strong> birth malformations. In<br />

addition, some researchers have suggested that increases in midwestern N fertilizer application<br />

has led to the development of “dead zones” caused <strong>by</strong> overgrowth <strong>and</strong> dieoff of algae in the Gulf<br />

of Mexico. Suggestions that growers use lower nitrogen application levels, however, have been<br />

resisted <strong>by</strong> both farmers <strong>and</strong> industry.<br />

Literature Review<br />

<strong>Nitrate</strong> transformations. Nitrogen exists in several forms of importance to agriculture.<br />

The forms are interconverted in soil, water, <strong>and</strong> air in the geochemical nitrogen cycle.<br />

Atmospheric nitrogen, N2, is taken up or “fixed” <strong>by</strong> a few kinds of microorganisms, whose<br />

+<br />

enzymes convert it to ammonium, NH4 . <strong>Plants</strong> then convert ammonium to organic N, initially in<br />

the form of amino acids, which are eventually largely converted to proteins. In aerobic soil,<br />

ammonium is also nitrified, that is, oxidatively converted to nitrite (NO2�) <strong>and</strong> nitrate (NO3�) <strong>by</strong><br />

_________________________________<br />

Address: Department of Natural Resources <strong>and</strong> Environmental Sciences, Urbana, IL 61801.<br />

Address correspondence to Richard Larson.


microorganisms. <strong>Nitrate</strong>, if it descends out of the aerobic root zone, is typically denitrified <strong>by</strong><br />

bacteria to reduced forms such as nitrite <strong>and</strong> N 2. A minor product of denitrification is nitrous<br />

oxide, N 2O, which is an important atmospheric greenhouse gas. The recent increase in<br />

tropospheric N 2O concentration has been attributed to increased fertilizer use. Other nitrogen<br />

oxides are products of fuel combustion <strong>and</strong> play important roles in the photochemistry <strong>and</strong> ozone<br />

chemistry of the atmosphere (Graedel <strong>and</strong> Crutzen, 1993).<br />

<strong>Nitrate</strong> transport. <strong>Nitrate</strong> is a highly oxidized <strong>and</strong> soluble form which exists in aerated<br />

environments such as surface waters <strong>and</strong> surface soils. Because all nitrate salts are water-soluble,<br />

the anion is exceptionally mobile, unlike, for example, sulfide, which tends to be bound in<br />

insoluble forms within a few centimeters of its generation. Accordingly, nitrate may migrate great<br />

distances from its point of application, carried <strong>by</strong> surface or subsurface water over or through the<br />

soil. <strong>Nitrate</strong> leaching is especially pronounced in cornfields in the period following harvest<br />

(Owens et al., 1995). Agricultural operations such as improperly managed feedlots may also lead<br />

to unusually high nitrate levels in groundwater or drinking water, especially in areas where high<br />

population densities coexist with large-scale production agriculture (Goulding <strong>and</strong> Poulton, 1992).<br />

<strong>Uptake</strong> <strong>by</strong> plants <strong>and</strong> microorganisms. Some, but not all, plants are able to take up nitrate<br />

<strong>and</strong> reduce it to usable forms, mostly ammonium, <strong>by</strong> the action of a key enzyme, nitrate reductase,<br />

an unusual protein that employs several electron-transfer mediators, including riboflavin,<br />

molybdenum ions, <strong>and</strong> NADH/NADPH. Its synthesis in the plant is induced <strong>by</strong> the presence of its<br />

substrate, nitrate, in the environment, <strong>and</strong> its activity tends to be greatest under high-light <strong>and</strong><br />

high-temperature conditions. <strong>Nitrate</strong> reductase is also found in many other organisms, such as<br />

soil fungi. Denitrifying activity tends to be highest in the rhizosphere of some plants, <strong>and</strong> it also<br />

appears that certain plant species also enhance the growth of microbial communities capable of<br />

degrading a variety of organic compounds (Jordahl et al., 1997). Accordingly, phytoremediation<br />

of nitrate may be a desirable practice not only because of direct removal of nitrate <strong>by</strong> the plant, but<br />

also because the presence of the plant could alter the population of soil microflora in a desirable<br />

direction for removal not only of nitrate but of other contaminant forms.<br />

A few plant species have been noted for their ability to accumulate nitrate in their roots or aboveground<br />

parts; members of the genera Beta, Borago, Chenopodium <strong>and</strong> Menyanthes are such<br />

nitrate-storing plants (Salisbury <strong>and</strong> Ross, 1978). Borago officinalis (borage) is being<br />

increasingly grown <strong>by</strong> specialty producers since its seed oil is a rich source of gamma-linolenic<br />

acid, a compound of rising interest as a natural pharmaceutical (Redden et al., 1995). Borage can<br />

also be sown in the autumn <strong>and</strong> persist over the winter. Swiss chard (Beta vulgaris), in addition<br />

to being an edible vegetable, also has the virtue of accumulating very high amounts of nitrate (up<br />

to 3.8 g/kg fresh weight) in its leaves (Santamaria et al., 1999). In addition, it is not uncommon<br />

for Swiss chard to remain viable past the first snowfall, or until temperatures fall well below<br />

freezing.<br />

Several species of grasses have been shown to display increased nitrate reductase activity in the<br />

presence of N fertilizers. Timothy (Phleum pratense), for example, had increased total N, nitrate


N, <strong>and</strong> amino N in leaves <strong>and</strong> stems which correlated with the amount of calcium nitrate added<br />

(Friedrich et al., 1977); at the highest levels of fertilization, leaf nitrate was at a toxic level.<br />

Switchgrass (Panicum virgatum) showed a similar response, but to a lesser degree.<br />

Cover cropping techniques. “Green manuring” is the use of a cover crop which is sown<br />

either together with a cash crop, or applied between growing seasons, which then is either plowed<br />

under or killed with herbicide before the next cash crop is planted. In other instances, N-fixing<br />

crops such as legumes are planted to increase N levels in the soil. (In some cases, it is feasible to<br />

use the cover crop as a cash crop, or for on-farm use as animal feed.) This procedure is related to<br />

“biofiltration” or “phytoremediation,” practices in which strips of plants are permanently planted<br />

adjacent to fields in order to intercept <strong>and</strong> take up agrochemicals before they can enter<br />

grooundwater or surface water.<br />

Organic farmers, tropical farmers, <strong>and</strong> practitioners of conservation tillage are especially likely to<br />

engage in cover cropping. It has been practiced for some time (Pieters, 1927), although seldom<br />

with the specific objective of nitrate interception. Most often, erosion control, weed control, or<br />

soil moisture retention are the incentives for the practice. In favorable cases, weed suppression <strong>by</strong><br />

cover crops is great enough to allow decreased herbicide applications (Dyck et al., 1995).<br />

Notably, however, such cover crops do take up inorganic nitrogen <strong>and</strong> convert it to organic forms<br />

as they grow. In an isotopic 15 N-labeling study with lysimeters, it was demonstrated that autumnsown<br />

cover crops diminished the amount of N lost to leaching in the spring <strong>by</strong> about one-half<br />

compared to the loss rate without plants. In addition, the researchers found that 10-15% of the<br />

labelled N was incorporated into the biomass of the subsequent barley crop (Thomsen <strong>and</strong><br />

Christensen, 1996). In another example, early growing season nitrate leaching from sites<br />

inFinl<strong>and</strong> was reduced when cover crops (hairy vetch, red clover, ryegrass) were planted in<br />

October (Kankanen eta l., 1998). Furthermore, crop yield does not generally appear to suffer<br />

because of the fact that the form of the N in the field at the time of planting is largely organic,<br />

rather than inorganic. Residual soil nitrate declined significantly when winter rye (Secale cereale)<br />

was used as a fall-planted cover crop for soybean production, but the immobilized N was readily<br />

released when the rye was chemically killed in the spring, <strong>and</strong> yields were mostly unaffected<br />

(Kessavalou <strong>and</strong> Walters, 1997, 1999). Ogren et al. (1998) demonstrated that vegetable yields<br />

actually increased following green manuring with either fall- or spring-applied Persian, white, or<br />

yellow clover as a cover crop.<br />

Procedures<br />

<strong>Nitrate</strong> Analysis Method. -- <strong>Nitrate</strong> (<strong>and</strong> nitrite) were determined using a method<br />

modified from that of Rizzo et al. (1998). A Beckman HPLC instrument with a C-18 column <strong>and</strong><br />

a UV-visible detector was used; nitrate was detected at a wavelength of 220 nm.. The mobile<br />

phase consisted of 1 part acetonitrile to 4 parts of a 10 mM octylamine solution, pH- adjusted to<br />

~6.0 with 1 M HCl <strong>and</strong> then filtered. A flow rate of 1 ml/min was used.<br />

- <strong>Nitrate</strong> <strong>and</strong> nitrite were quantitatively determined using 1-10ppm st<strong>and</strong>ards of either NO2 <strong>and</strong><br />

- - . - NO3 , obtained <strong>by</strong> diluting a stock solution of 1000ppm Ca (NO3 )2 4H2O, K NO3 or 1M Na NO2.


<strong>Nitrate</strong> determination in soil (with plants present or absent) was performed <strong>by</strong> extracting weighed<br />

(50 g) s<strong>and</strong>-soil mixtures (moistened with Hoagl<strong>and</strong>’s nutrient solution) three times <strong>and</strong> injecting<br />

the successive (filtered) extracts onto the HPLC. Subsequent extractions did not remove<br />

significant quantities of nitrate.<br />

Plant species tested. – As a “positive control,” we utilized sweet corn (Zea mays), the crop<br />

plant most widely fertilized with nitrogen in <strong>Illinois</strong>. Other plants examined were hornwort<br />

(Ceratophyllum demersum), Swiss chard (Beta vulgaris), reed canarygrass (Phalaris<br />

arundinacea), borage (Borago officinalis) annual ryegrass (Lolium multiflorum), perennial<br />

ryegrass (Lolium perenne), <strong>and</strong> switchgrass (Panicum virgatum).<br />

<strong>Nitrate</strong> uptake from hydroponic containers. – In initial experiments, each plant species<br />

.<br />

was placed in a flask with the roots submerged in approximately 20 ppm Ca(NO3) 2 4H2O<br />

solution, while the foliage was held above with a foam stopper. The plants were grown in a 50 %<br />

Hoagl<strong>and</strong>’s nutrient solution (Hoagl<strong>and</strong> <strong>and</strong> Arnon, 1938), which contained this concentration of<br />

nitrate. (Hornwort, a floating aquatic plant, was kept in a open container because of its lack of<br />

roots.) A control with the same solution but without plants was also monitored. The plants <strong>and</strong><br />

control were kept under ambient laboratory conditions of ~ 21 degrees Celsius <strong>and</strong> under lamp<br />

light of ~ 7.5μm photosynthetically active radiation (PAR), as measured with an Li-1000<br />

Datalogger- PAR light meter.<br />

In later studies, higher concentrations of nitrate were used. Starting with the assumption<br />

that a generous level of soil fertilization would be 100-200 lbs N/acre (110-220 kg/hectare), <strong>and</strong><br />

further assuming that this level of N would be uniformly distributed through the top 6" (15 cm) of<br />

the soil, we calculated that 300-600 ppm of nitrate (NO 3�) in water would approximate this<br />

condition.<br />

<strong>Nitrate</strong> reductase measurement. The assay procedure for nitrate reductase was modified<br />

from that of Harley (1993). A 30mM KNO3, 0.1 M K phosphate buffer, 5 % v/v isopropanol<br />

assay was added to .3 grams (wet weight) of 1 cm grass segments in a disposable test tube. One<br />

sample was immediately immersed in boiling water for 5 minutes for an initial value. A second<br />

sample was incubated in the dark for 1 hour <strong>and</strong> then boiled for 5 minutes. Nitrite st<strong>and</strong>ards were<br />

prepared with a 10 mM stock solution of 69 mg NaNO2 per 100ml solution; then 1 ml of the stock<br />

-<br />

solution was diluted in 400 ml of distilled water giving a solution containing 25 nmoles NO2 per<br />

ml.<br />

A sulfanilamide (SA) solution <strong>and</strong> a naphthylethylenediamine dihydrochloride (NEED) solution<br />

were prepared. 4ml of the sample along with 1 ml of each the SA <strong>and</strong> NEED solutions were<br />

pipetted into a disposable test tube. Together the SA <strong>and</strong> NEED solution react with the nitrite<br />

present in the sample to produce a deep pink-purplish dye that was measured spectroscopically at<br />

540 nm.<br />

Results <strong>and</strong> Discussion


<strong>Nitrate</strong> Analysis Method. – The reversephase<br />

HPLC technique gave good<br />

results with st<strong>and</strong>ard solutions (Fig.1).<br />

Analyses of natural water samples<br />

(Table 1) demonstrated that the<br />

technique was capable of detecting a<br />

wide range of concentrations.<br />

<strong>Nitrate</strong> <strong>Uptake</strong> Experiments. – Since<br />

nitrate is highly soluble, initial<br />

experiments in hydroponic containers<br />

were performed to assess the rates of<br />

nitrate uptake <strong>by</strong> several plant species.<br />

Hornwort (Ceratophyllum demersum),<br />

Swiss chard (Beta vulgaris), reed<br />

canarygrass (Phalaris arundinacea),<br />

borage (Borago officinalis) <strong>and</strong><br />

Figure 2. Initial <strong>Nitrate</strong> <strong>Uptake</strong> Rates - Screening, Low-<br />

Concentration Experiment<br />

Figure 1. St<strong>and</strong>ard Curve for <strong>Nitrate</strong> Determination<br />

in Water.<br />

switchgrass (Panicum virgatum) have been reported in the literature to take up nitrogen <strong>and</strong> to<br />

persist through low temperatures.<br />

An initial sample was taken <strong>and</strong> then each subsequent day for 3 days. Although each plant varied<br />

in weight, an overall assessment of each plant was obtained through loss/g-weight/day. Each day<br />

the water level was adjusted to compensate for loss of water due to absorption <strong>and</strong> evaporation.<br />

The results indicate that all the plants tested initially remove nitrate relatively rapidly. Borage,<br />

however, seems to maintain its uptake more consistently (Fig. 2). In later experiments, however,<br />

emphasis was directed toward the<br />

grasses; borage consistently failed to<br />

grow well over more extended<br />

periods, <strong>and</strong> Swiss chard appeared<br />

deficient in measurements of nitrate<br />

uptake per gram of plant material.<br />

Hydroponic experiments with higher<br />

concentrations of nitrate with one of<br />

the grasses (switchgrass) <strong>and</strong><br />

hornwort are summarized in Figures<br />

3-4. In an experiment with<br />

switchgrass starting at 273 ppm<br />

nitrate, the grass removed nitrate to<br />

undetectable levels within 6 days.<br />

The “half-life,” or time required to reduce the concentration to half the initial level, appeared to be<br />

about two days. It was superior even to the “positive control,” sweet corn.


Figure 3. <strong>Uptake</strong> of <strong>Nitrate</strong> <strong>by</strong> Corn <strong>and</strong> <strong>by</strong><br />

Switchgrass.<br />

Its long fibrous roots, which<br />

extend deeper into the soil than<br />

those of most crop species, may<br />

be key for use in effective<br />

agricultural buffer zones (Huang et<br />

al., 1996).<br />

Switchgrass has also recently<br />

attracted attention as a cash crop.<br />

It is being grown in mass<br />

quantities as a potential fossil fuel<br />

replacement species. Preliminary<br />

studies have shown that<br />

switchgrass can be effectively co-<br />

Figure 5. Switchgrass<br />

(Panicum virgatum).<br />

Switchgrass (Fig. 5) is a native North<br />

American warm-season grass that can grow<br />

up to three meters in height on marginal<br />

l<strong>and</strong>s, requires little or no fertilization <strong>and</strong><br />

irrigation, <strong>and</strong> is resistant to drought. It<br />

has recently attracted attention as a<br />

biomass-filter type plant in agricultural<br />

applications. For example, a 5-M-wide<br />

strip placed below a dairy manure lagoon<br />

effluent removed 76% of total N <strong>and</strong> 47%<br />

of total P (S<strong>and</strong>erson et al., 2001). It was<br />

much more effective than cool-season filter<br />

strips in nutrient removal (Lee et al., 1998).<br />

Figure 4. <strong>Uptake</strong> of <strong>Nitrate</strong> <strong>by</strong> the Floating <strong>Aquatic</strong> Plant,<br />

Hornwort.<br />

fired with coal in power plants (McLaughlin <strong>and</strong> Walsh, 1998).<br />

Other potential uses for switchgrass are for ethanol production<br />

(Lynd et al., 1991) <strong>and</strong> incorporation into mulches, fiberboard,<br />

<strong>and</strong> paper products (Radiotis et al., 1999).<br />

The floating aquatic species, hornwort (Fig. 6), also was<br />

effective. Hornwort is a native plant that is found in ponds <strong>and</strong><br />

slow-flowing streams throughout <strong>Illinois</strong>, <strong>and</strong> is also grown for<br />

use as a decorative species for water gardens <strong>and</strong> aquariums. It<br />

can occur at considerable depths in a lake or pond, <strong>and</strong> lives<br />

throughout the winter, able to photosynthesize even under ice<br />

cover.<br />

In an experiment in which the initial nitrate concentration was<br />

350 ppm, it reduced the concentration to 75 ppm in nine days,<br />

corresponding to a half-life of 4-5 days. It removed on average


about 2 ppm nitrate per day per gram of plant. Hornwort<br />

grew well under increased nitrate concentration along with<br />

the other nutrients in the Hoagl<strong>and</strong> nutrient solution. The<br />

hornwort used in the experiment was apparently in good<br />

condition at the end of the nine days’ exposure.<br />

<strong>Nitrate</strong> reductase - preliminary data.. – In initial<br />

experiments (Fig. 7), reed canarygrass showed the most<br />

nitrate reductase activity, followed <strong>by</strong> the two ryegrasses.<br />

Switchgrass, although lowest of the four, may have<br />

potential for greater amounts of activity (the thick outer<br />

layer of the leaf blade makes complete saturation <strong>by</strong> the<br />

reagents difficult). Patches of dark <strong>and</strong> light green pigments<br />

after the boiling showed that the switchgrss leaf blade had<br />

not been completely saturated.<br />

Figure 6. Hornwort (Ceratophyllum<br />

demersum).<br />

Figure 7. <strong>Nitrate</strong> reductase activity of leaf tissue of selected grasses.<br />

Conclusions<br />

Several species of plants were tested for their ability to take up nitrate from aqueous solution.<br />

Both a terrestrial plant, switchgrass, <strong>and</strong> a floating plant, hornwort, were capable of effectively<br />

removing nitrate at concentrations similar to those that would be found in a typical nitrogenfertilized<br />

field soil.


Table 1. Determination of <strong>Nitrate</strong> Concentration in Natural Surface Water Samples.<br />

Natural Water Location Month <strong>and</strong> Year<br />

Collected<br />

Altamaha River Darien, Georgia Mar. 86 50<br />

Aucilla River Florida Feb. 86 256<br />

Busey Pond <strong>Illinois</strong> Nov. 90 24<br />

Gulf of Mexico Clearwater Beach, Florida Dec. 92 0.16<br />

Okefenokee Swamp Foster State Park, Georgia Jan. 86 0.25<br />

Suwanee River Florida Mar. 83 0.72<br />

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