INTRODUCTION
Primarily in the form of amino acids, uptake of organic N, is an important component of plant nutrition. The discovery grew out of a need to account for a gap in the N budget in ecosystems that have high concentrations of labile organic N and where plant uptake and assimilation of N exceeded available inorganic forms of N. The relative amount of amino acid uptake depends on their concentrations compared to concentrations of available inorganic N. Although the ratio of organic to inorganic N is low in agricultural soils amended with synthetic N fertilizers and the uptake of amino acids by roots is expected to contribute little to the overall N supply of an agronomic plant, several important agronomic and horticultural crops have been shown capable of amino acid uptake, including corn, some pasture species, wheat (Triticum aestivum L.), barley (Hordeum vulgare), and cucumber (Cucumis sativus). Mycorrhizae may play a role in amino acid uptake, but specific transporter molecules also mediate direct, active uptake by plant roots. Although competition for amino acids is expected between plants and microorganisms, significant plant uptake depends on spatiotemporal conditions that promote high concentrations of amino acids in soil.
MATERIALS AND METHODS
Amino acid uptake under greenhouse conditions
Corn was selected as the agronomic crop because it has been shown to take up some species of acid, basic, and neutral amino acids and is the agronomic crop that uses the largest amount of N fertilizer. The first greenhouse trial was conducted in the summer months of 2021 (April 23-July 16) and a second trial was conducted in the winter months of 2021 (October 19-January 18). For the first trial, L-alanine (C3H7NO2, 15.7% N) and L-lysine (C6H14N2O2, 19.2% N) were selected for use as amino acid fertilizers. All amino acids in this study were left-handed structures (L-), so for brevity the amino acids will be referred to by name only. Alanine was selected because it is one of the smallest molecules and might possibly be more easily taken up by the plant because of its smaller size. It is reported passive uptake of alanine by corn but concluded that amino acids are a minor source of N in agricultural crops. Alanine has a neutral charge in the agronomic pH range between pH 3 and 9. Lysine was selected because it is one of three positively charged amino acids in the agronomic pH range between 3 and 9 and so should be attracted to negatively charged clay particles and resist leaching, but it is also one of the largest amino acid molecules.
Three corn seeds were planted near the center of each bucket. Upon emergence, plants were thinned to one plant per bucket and fertigated weekly. Treatments were equal amounts of elemental N provided as alanine as the only N source, lysine as the only N source, ammonium nitrate, and a control (zero N). Each treatment was replicated three times. Each replicate consisted of one bucket containing one plant. Weekly fertigation contained a measure of stock solution containing micronutrients and masses of N, phosphorus (P), and potassium (K) approximating nutrient requirements for a single corn plant based on the five stages of growth as described in tab. 1 with an assumed plant population of 79,300 plants ha−1 (32,100 plants ac−1). For example, in the early stage of growth (0-25 days), the 21.3 kg N ha−1 (19 lb N ac−1) requirement was applied in three weekly fertigation events containing 7.1 kg N ha−1 (89 mg N for a single plant). Phosphorus and potassium requirements were applied similarly. Macronutrient application deviated by increasing N amounts by 33% in the rapid growth stage to account for the use of a shorter season corn variety and by increasing amounts during the late stages to ensure there was no N deficiency. Monopotassium phosphate (MKP) was used as the source of P. The amount of K supplied by MKP was supplemented with potassium sulfate to meet the targeted weekly application of total K.
Weekly amounts of micronutrients applied per plant were as follows: 67 mg calcium (Ca), 118 mg magnesium (Mg), 3.4 mg iron (Fe), 1 mg zinc (Zn), 0.7 mg manganese (Mn), 0.3 mg copper (Cu), and 0.1 mg boron (Bo). Sprint 330, a chelated iron containing 10% Fe, was the source for iron and borax (sodium borate) was the source for boron. Sulfate salts were the source for all other micronutrients.
Water volume was adjusted throughout the study as the corn grew and matured to provide adequate water but not allow leaching from the bottom drain. Fertigation volumes were 500 mL in stage 1 (early), 1000 mL in stage 2 (rapid growth), 1200 mL in stage 3 (silk), and 1500 mL in stages 4-5 (grain and mature). Beginning week 6, these same volumes of deionized water were applied twice more weekly to meet evaporation demand of plants growing similarly to those of the ammonium nitrate treatment and half volumes were applied twice weekly to those plants growing similarly to those of the control treatment. Greenhouses were maintained at 26.7°C (80 °F) and sodium lights were positioned 2.1 m (7 ft) above the sand surface of each bucket and switched on from 6 a.m. to 6 p.m. each day. Ambient light was allowed to enter the greenhouse. Plant heights were recorded weekly, and total dry biomass of the three plants per treatment (shoot and root weighed separately) was measured at the end of the study when plants reached maturity and began to dry.
A second greenhouse trial, conducted in the winter months of 2021, followed the same protocol. Treatments were lysine (a repeat of trial 1 to confirm positive results), histidine (C6H9N3O2, 27.1% N), arginine (C6H14N4O2, 32.2% N), and ammonium nitrate. Histidine and arginine are the other two positively charged amino acids in the agronomic pH range and are similar in molecular size to lysine. Following the second greenhouse trial, whole plant above ground tissue was dried, ground, and sent to the Ajinomoto Health and Nutrition North America, Inc. Amino Acid Laboratory in Eddyville, IA, for analysis to determine whether growing corn with amino acid as the N source results in any beneficial change in nutritional value.
The two trials were analyzed separately because ambient conditions were very different between summer months and winter months even though artificial light and heat were the same. The study was a randomized complete block design with treatments blocked by location in the greenhouse (northeast, center, and southwest) to account for differences in greenhouse growing conditions as affected by external light and temperature. All significance tests were performed at α < 0.05 using PROC GLM for a single-factor ANOVA with type 3 tests of fixed effects and Tukey-Kramer honestly significant difference (HSD) test for differences among means. A Shapiro-Wilk test was used to confirm a normal distribution prior to testing for differences among means.
Testing liquid lysine-N fertilizer under field conditions
Positive results for lysine prompted field trials at the Russell E. Larson Agricultural Research Center at Rock Springs, Pennsylvania, in 2022 and 2023 to demonstrate corn growth with lysine as the only N source under field conditions. Whereas it is well known that corn grown with manure as the N source benefits from microbial decomposition of organic compounds to release ammonium and nitrate, microbial decomposition of the relatively small lysine molecule in the natural soil environment to release ammonium and nitrate for corn uptake is expected. Therefore, the field trial does not confirm lysine uptake by corn, but demonstrates how liquid lysine, which is commercially available in bulk quantities, could be used as fertilizer and extends the results from corn grown in buckets in the greenhouse to real-world agronomic conditions.
A composite soil sample, consisting of 10 soil cores taken to a depth of 15 cm using a two cm diameter probe, was collected prior to the field trial and submitted to the Penn State Agricultural Analytical Services Lab for soil test analysis. Nitrogen application rate was determined in accordance with the Penn State Agronomy Guide. No soil test is used to make nitrogen recommendations. Rather, recommended N application is 7.2 kg N per Mg corn ha−1 (1 pound N bushel−1 corn ac−1) of expected corn grain yield. During the 2022 field season, a corn trial compared commercially available liquid lysine (10.15% N) as the only N source and liquid urea ammonium nitrate (UAN, 32% N) as the only N source at a rate of 200 kg N ha−1 (180 lb N ac−1), representing the recommended N application for a yield goal of 11.3 Mg corn grain ha−1 (180 bushels ac−1). The trail included a zero N rate (control).
At the milk stage (18-22 days after silking) on September 29, a 15 m (50 ft) length of rows three and four of the six row plots was harvested for silage using a Wintersteiger plot harvester (Wintersteiger Holding Ag) with a Kemper harvesting header (Maschinenfabrik KEMPER GmbH & Co. KG). Biomass weights were determined by the Wintersteiger’s onboard scale, and a subsample was dried and weighed to determine moisture content. Biomass yield was reported as wet weight in order to compare with yield reported by producers but has been corrected to 65% moisture to allow for comparison between years within this study. On November 22, ears from a 6 m (20 ft) length of rows two and five were hand harvested, shelled, and weighed. Corn ears were shelled with a Haban corn sheller (Haban Manufacturing Company; no longer in operation). A subsample was dried and weighed to determine moisture content. The trial was repeated in 2023 in the same field; the only difference was that the N rate was adjusted downward to better match the yield potential of the field as observed in 2022. In 2023, the trial consisted of zero N (control) and liquid lysine and liquid urea applied at the rate of 170 kg N ha−1 (150 lb N ac−1), representing the recommended N application for a yield goal of 9.4 Mg ha−1 (150 bushels ac−1). Corn was planted on May 22, 2023; silage was harvested on September 21, 2023; and grain was harvested on November 7, 2023.
As described above, the trials consisted of three treatments (liquid urea, lysine, and zero N); each treatment was replicated four times. Replicate treatments were blocked by location in the field from end-to-end to account for differences in soil conditions. Silage and grain yield were analyzed for each individual year (2022 and 2023) using a two-way analysis of variance procedure using PROC GLM in SAS v9.4 (SAS Institute Inc., 2017). N rate, N fertilizer source and their interactions were treated as fixed effects with replicate as a random effect. When the F-Test showed significant differences in fixed effects of nitrogen rate or source at p ≤ 0.05, the least square means were compared using Tukey’s HSD test. Means are presented in table format; significant differences (at p ≤ 0.05) within each year are indicated with letters.
RESULTS AND DISCUSSION
Amino acid uptake under greenhouse conditions
Plant heights from the two greenhouse trials are shown from week 4 until maturity in Figure 1. In trial 1, mean plant heights (cm) at maturity were 222.5 (a), 219.4 (a), 108.7 (b), and 66.0 (b) for plants grown using ammonium nitrate, lysine, alanine, and zero N, respectively (p value = 0.0002). Clearly, corn was taking up and using lysine as the only N source. Mean plant height for corn grown with alanine as the N source was the same as the zero N control at week 9 (Figure 1). After week 9, plants in two replicates of the alanine treatment turned greener and started to grow faster than the zero N control. Plant heights (cm) at maturity were 61, 67, and 70 for the control compared to 55 (slightly less than control), 113, and 158 (double control) for the alanine treatment. One hypothesis for this behavior is that plants were not able to take up and use alanine, but by week 9 a microbial population had become established in the sand medium of two of the replicates, and alanine was being converted to inorganic N forms that were being taken up and used by corn.
In trial 2, mean plant heights (cm) at maturity were 180.3 (a), 176.9 (a), 181.1 (a), and 126.1 (b) for plants grown using ammonium nitrate, lysine, histidine, and arginine, respectively (p value = 0.0014). Plant height data show that the lysine treatment once again grew equally as well as ammonium nitrate, thereby confirming the results in trial one (Figure 1). Histidine and arginine, the only other positively charged amino acids in pH range 3-9 other than lysine that would potentially resist leaching, behaved differently. Corn receiving histidine grew similarly in height to corn receiving lysine and ammonium nitrate, but corn receiving arginine was stunted and yellowish in color. However, after week 8, corn receiving arginine began turning greener and grew more rapidly like the response that was observed in the alanine treatment in trial one. Plant heights (cm) at maturity were 102, 130, and 147 for individual replicates of the arginine treatment. As in trial 1, one hypothesis is that corn was not able to take up and use arginine directly, but by week 8 a microbial population had become established in the sand medium, and arginine was being converted to inorganic N forms that were being taken up by the corn plants.
Table 1 shows higher mean biomass values for the three plants grown with lysine compared to the three plants grown with ammonium nitrate in trial 1, but the difference was not statistically different. Corn grown with alanine produced an intermediate amount of biomass between that grown with lysine or ammonium nitrate and the control, but most of that biomass was produced after week 9. Table 1 also shows similar biomass production for ear, roots, and shoot plus ear for the ammonium nitrate and histidine treatments in trial 2. In spite of there being no difference in plant height, the lysine treatment had approximately 25% lower biomass production, but all three of these treatments significantly out produced arginine. Biomass production for the lysine treatment was double that of the arginine treatment. Trial 1 showed greater plant height and total biomass for similar treatments (lysine and ammonium nitrate) compared to trial two (Figure 1; Table 1). Trial 2 followed trial 1 and was conducted during winter months when day length was shorter and temperatures were cooler. Although the greenhouse was heated and equipped with artificial lights, these external environmental conditions resulted in less favorable growing conditions in trial 2 that resulted in less overall growth and biomass production. The lower biomass production for the lysine treatment in trial 2 is attributed to the outcome of randomization of the treatments, which may have resulted in less favorable growing conditions due to differences in external lighting and temperature. Portions of the greenhouse can become shaded by nearby trees or buildings, especially in winter months when sun angles are lower, and temperatures can be lower near the sides of the greenhouse compared to the center.
| Treatment | n | Shoot (g) | Ear (g) | Root (g) | Shoot plus ear (g) |
| Trial 1 | |||||
| Control | 3 | 138.3c | 0.0c | 3.2c | 138.2c |
| Alanine | 3 | 213.7b | 36.6bc | 48.0bc | 250.4b |
| Lysine | 3 | 300.5a | 96.9a | 145.5a | 397.3a |
| NH4NO3 | 3 | 258.9ab | 84.4ab | 126ab | 343.3ab |
| Trial 2 | |||||
| Arginine | 3 | 26.9c | 7.0a | 17.2b | 33.9c |
| Histidine | 3 | 77.9b | 24.4a | 73.9a | 102.3ab |
| Lysine | 3 | 69.0b | 5.6a | 40.1b | 74.6b |
| NH4NO3 | 3 | 105.2a | 4.3a | 90.3a | 109.5a |
| df | Trial 1 | ||||
| p value | 2 | 0.0056 | 0.0021 | 0.0077 | |
| Trial 2 | |||||
| p value | 2 | 0.0060 | 0.2470 | 0.0024 |
Results for crude protein and total amino acid content are shown in Table 2. There was no difference in crude protein and few differences in amino acid content between the lysine and ammonium nitrate treatments. Corn grown with lysine had lower tryptophan and tyrosine, but the amounts of these amino acids in corn are small compared to other amino acids. The histidine treatment had lower crude protein content, and this resulted in lower content of several amino acids compared to corn grown with ammonium nitrate or lysine. The most notable difference in these data is that both the lysine- and histidine-treated corn contained five to six times as much proline as did corn grown with ammonium nitrate. Proline is an osmolyte in corn and can help the plant defend against low water availability (drought) or saline soil conditions. Measurement of salinity in the N fertigation solutions showed that salinity in the amino acid solutions was about two-thirds that of the salinity of the ammonium nitrate solution, so it does not appear that the high proline content is a plant response to differences in salinity of fertilizer solutions. The reason for the higher proline content is unknown, but higher proline content would not be of significant nutritional value. Overall, there does not appear to be much difference in nutritional value among these treatments. However, if the amino acid N source is causing high proline content, then it could be imparting drought tolerance that would be a potential agronomic benefit.
| Amino acid | Lysine (mg g−1) | Histidine (mg g−1) | NH4NO3 (mg g−1) | F value | Pr > F |
| Crude protein | 138.9a | 104.5b | 158.4a | 32.39 | 0.003 |
| Alanine | 5.5 | 5.4 | 6.5 | 6.04 | 0.062 |
| Arginine | 3.2ab | 2.7b | 3.6a | 12.41 | 0.019 |
| Aspartic acid | 20.7a | 10.4b | 23.7a | 47.5 | 0.002 |
| Cysteine | 1.2ab | 1.1b | 1.3a | 9.65 | 0.030 |
| Glutamic acid | 23.8 | 18.2 | 30.3 | 4.84 | 0.085 |
| Glycine | 3.8a | 3.1b | 4.0a | 31.86 | 0.004 |
| Histidine | 1.9 | 2.1 | 2.6 | 5.44 | 0.072 |
| Isoleucine | 2.7 | 2.3 | 3.1 | 6.62 | 0.054 |
| Leucine | 4.8 | 4.7 | 5.3 | 0.22 | 0.812 |
| Lysine | 3.8a | 3.1b | 4.0a | 30.19 | 0.004 |
| Met + Cys | 2.2 | 2.0 | 2.4 | 3.14 | 0.152 |
| Methionine | 1.0 | 1.0 | 1.2 | 1.65 | 0.300 |
| Phenylalanine | 3.1 | 2.9 | 3.5 | 0.98 | 0.451 |
| Proline | 5.9a | 4.6ab | 1.0b | 8.63 | 0.035 |
| Serine | 3.8 | 3.6 | 4.4 | 4.35 | 0.099 |
| Threonine | 3.3ab | 2.9b | 3.8a | 8.19 | 0.039 |
| Tryptophan | 0.8b | 0.79b | 1.1a | 27.75 | 0.005 |
| Tyrosine | 1.3b | 1.6b | 3.0a | 31.93 | 0.004 |
| Valine | 3.8ab | 3.5b | 4.5a | 12.35 | 0.019 |
Growth response and amino acid profiles for lysine and histidine have important implications for the fate of amino acids that can be taken up by plants. One possible hypothesis is that an amino acid that crosses the cell membrane might be transported within the plant in unaltered form to a site where it could be plugged into the structure of a protein where it is needed. However, the results of this study suggest that the amino acid is being metabolized within the plant and released N is being used as building blocks for all types of amino acids or perhaps other organic molecules. The leading hypothesis for why corn did not grow in response to alanine and arginine N sources is that they could not be transported across the cell membrane. Transporter molecules for alanine and arginine are apparently absent in corn, whereas those used for lysine and histidine are apparently present.
Liquid lysine compared to liquid urea under field conditions
Results from the greenhouse trials showed that corn can grow equally as well with lysine as the only N source as with ammonium nitrate as the N source. To extend this to field conditions and demonstrate the practical aspects of fertilizing with liquid lysine, N source trials comparing commercially available liquid lysine in the form used for feed supplement to liquid urea were conducted in 2022 and 2023. Soil test values for samples taken prior to the field trial in 2022 showed pH (6.5), Mehlich-3 P (41 mg L−1), K (94 mg L−1), Mg (336 mg L−1), Ca (1600 mg L−1), Zn (1.3 mg L−1), Cu (1.4 mg L−1), and sulfur (S) (30.6 mg L−1). Weather data collected by a station located near the field site and managed by the USDA Agricultural Research Service’s Pasture Systems and Watershed Management Research Unit in University Park, Pennsylvania, are shown in Figure 2. In 2022, rains in May provided good soil moisture at planting in early June, but the season can be characterized as having an unusually hot and dry summer from early July to mid-August. Late August and September were near normal for rainfall and temperature. In 2023, temperatures were near normal for the growing season and rainfall exceeded the norms throughout the growing season.
Weekly rainfall and average weekly temperature data for 2022 and 2023 growing seasons collected by a station managed by the USDA Agricultural Research Service’s Pasture Systems and Watershed Management Research Unit in University Park, Pennsylvania, and located near the field site at the Russell E. Larson Agricultural Research Center at Rock Springs, Pennsylvania.
Corn silage and grain yields are shown in Table 3. Both treatments receiving N were greater than the control, and there were no significant differences in yield between lysine and liquid urea treatments. As previously stated, results were not meant to prove that corn can take up lysine because soil microbes could easily convert lysine to inorganic N forms that corn might take up preferentially. However, the field trials did confirm that corn receiving lysine did grow and yield similarly to corn grown with liquid urea.
| Treatment | Silage yield | Grain yield | ||
| 2022 (Mg ha−1) | 2023 (Mg ha−1) | 2022 (Mg ha−1) | 2023 (Mg ha−1) | |
| Liquid urea | 30.9a | 31.8a | 7.3a | 7.4a |
| Lysine | 27.8a | 28.5a | 5.9a | 6.6a |
| Control | 14.2b | 14.3b | 2.2b | 3.7b |
The lysine treatment appeared to suffer from poor N placement. The drip hoses bounced from side to side between rows, and irregular plant growth observed at the early growth stage suggested that some plants had access to lysine while others did not. Compared to neutrally charged urea and negatively charged nitrate, the positively charged lysine would be much less likely to diffuse laterally to plant roots. The plants grew out of the early stunted growth, but this may have resulted in a slight yield penalty. Spraying the liquid lysine over the planting zone would require diluting the animal lysine supplement product but would more effectively deliver N to the root zone of the emerging corn plants. Injection directly below the seedbed should also be investigated as it would further concentrate the lysine in a zone where plant roots might compete more effectively with the microbial population.
In the only other known field trial using amino acids as fertilizer, isonitrogenous formulations of tryptophan and lysine byproducts and pure tryptophan showed no difference in yield compared to ammonium nitrate and ammonium sulfate in a 2-year field corn trial in Iowa. However, lysine byproduct contained 78% ammonium and only 22% lysine. It is assumed that the majority of organic N was rapidly mineralized by microorganisms following soil application and the yield response was due to uptake of inorganic forms of N.
Nitrate and lysine leaching
Given evidence that corn can be grown using lysine as the only source of N fertilizer, a core lysimeter leaching study was conducted to assess the potential water quality benefits of using the positively charged lysine as N fertilizer. Mean nitrate-N concentrations in leachate over time are shown in Figure 3. Mean nitrate-N concentrations for weeks 2 and 3 were adjusted to account for the lower volume of water added (800 mL in weeks 2-3 versus 1 L in weeks 4-10). The results show that lysine is acting like a slow-release fertilizer with respect to nitrate availability. Nitrate-N concentrations from the lysine treatments do not exceed those from the ammonium nitrate treatment until week 6 when concentrations are approximately the same. Similar to results observed for alanine and arginine in the greenhouse study, it appears that microbes are capable of mineralizing lysine, but several weeks are required to produce high nitrate concentrations that would be subject to leaching. Delayed availability of nitrate corresponds to the rapid growth and silking stages of corn growth when N demand is greater compared to earlier in the season. Meanwhile, lysine is readily available for direct uptake by corn during the early growth stage. Total-N concentration, which would have included nitrate-N, ammonium-N, and leached lysine-N, never exceeded nitrate-N concentration in the lysine treatments by more than 4 mg L−1 (data not shown). Results support the conclusion that the positively charged lysine is bound to negatively charged soil particles and is not subject to leaching. Powdered lysine was included as a treatment because it is an alternative to liquid lysine, and although it is more expensive to produce, it would be cheaper to transport. Powdered lysine behaved similarly to liquid lysine and showed no apparent advantage over the liquid form with respect to leaching.
IMPLICATIONS FOR FUTURE RESEARCH
Lysine is readily available for plant uptake but also acts as a slow-release fertilizer as microbes will eventually convert lysine to ammonium and nitrate and deliver N in inorganic form at a later growth stage when N demand is greater. Results also show that corn cannot take up all amino acids (e.g., alanine and arginine), at least not at rates that would allow them to be used as fertilizer. A study of amino acid uptake that all five of the amino acids used in their study, which did not include lysine, were taken up at low concentrations similar to what would occur in agricultural soils.
It is estimated that corn accounts for 17.8% of world fertilizer N consumption, not an insignificant potential market. Knowing the true size of the potential market for lysine as a fertilizer depends in part on knowing which agronomic and horticultural crops are capable of taking up lysine. One promising line of modern research into amino acid uptake is the identification of transporter molecules in plants and their role in the uptake and within plant transport of specific amino acids or groups of amino acids. Developing a profile of transporter molecules known to play a role in lysine uptake, as has been done in wheat, may afford a rapid screening tool for assessing the potential for an agronomic or horticultural crop to use lysine as a fertilizer.
With current technology, replacing industrially produced inorganic fertilizer with lysine is not a feasible option. However, given that there is a significant market for lysine for use as N fertilizer, what is needed is a low-cost biological production method for producing lysine using biologically fixed N, which would have the added environmental benefit of bypassing the carbon dioxide-producing Haber-Bosch process. Indeed, it has been demonstrated that heterotrophic N-fixing bacteria do produce amino acids, including lysine, but the process has not yet been developed to commercial scale. Modern genetic engineering is also a promising approach for developing N-fixing bacteria capable of producing lysine at commercial scale. Recent advances include engineering photosynthetic production of L-lysine that eliminates the need for a carbon source for the fermentation process and engineering an N-fixing Escherichia coli strain. This study barely scratches the surface of understanding how amino acids might serve as an alternative to inorganic N fertilizers, but it does provide strong incentives for research into alternative means of biosynthesizing L-lysine or other amino acids for use as fertilizer using N-fixing microorganisms.
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