Physiology of microalgae and their application to sustainable agriculture: A mini-review.

Concern that depletion of fertilizer feedstocks, which are a finite mineral resource, threatens agricultural sustainability has driven the exploration of sustainable methods of soil fertilization. Given that microalgae, which are unicellular photosynthetic organisms, can take up nutrients efficiently from water systems, their application in a biological wastewater purification system followed by the use of their biomass as a fertilizer alternative has attracted attention. Such applications of microalgae would contribute to the accelerated recycling of nutrients from wastewater to farmland. Many previous reports have provided information on the physiological characteristics of microalgae that support their utility. In this review, we focus on recent achievements of studies on microalgal physiology and relevant applications and outline the prospects for the contribution of microalgae to the establishment of sustainable agricultural practices.

Prediction of protein and oil contents in soybeans using fluorescence excitation emission matrix.

To investigate the potential of fluorescence spectroscopy in evaluating soybean protein and oil content, excitation emission matrix (EEM) was measured on 34 samples of soybean flours using a front-face measurement, and the accuracy of the protein and oil content prediction was evaluated. The EEM showed four main peaks at excitation/emission (Ex/Em) wavelengths of 230/335, 285/335, 365/475, and 435/495 nm. Furthermore, second derivative synchronous fluorescence (SDSF) spectra were extracted from the EEMs, and partial least square regression and support vector machine models were developed on each of the EEMs and SDSF spectra. The R2 values reached 0.86 and 0.74 for protein and oil, respectively. From the loading spectra, fluorescence at Ex/Em of 230-285/335 nm and 350/500 nm mainly contribute to the protein and oil content prediction, respectively. Those results revealed the potential of fluorescence spectroscopy as a tool for a rapid prediction of soybean protein and oil content.

Application of Nitrate, Ammonium, or Urea Changes the Concentrations of Ureides, Urea, Amino Acids and Other Metabolites in Xylem Sap and in the Organs of Soybean Plants (Glycine max (L.) Merr.).

Soybean (Glycine max (L.) Merr.) plants form root nodules and fix atmospheric dinitrogen, while also utilizing the combined nitrogen absorbed from roots. In this study, nodulated soybean plants were supplied with 5 mM N nitrate, ammonium, or urea for 3 days, and the changes in metabolite concentrations in the xylem sap and each organ were analyzed. The ureide concentration in the xylem sap was the highest in the control plants that were supplied with an N-free nutrient solution, but nitrate and asparagine were the principal compounds in the xylem sap with nitrate treatment. The metabolite concentrations in both the xylem sap and each organ were similar between the ammonium and urea treatments. Considerable amounts of urea were present in the xylem sap and all the organs among all the treatments. Positive correlations were observed between the ureides and urea concentrations in the xylem sap as well as in the roots and leaves, although no correlations were observed between the urea and arginine concentrations, suggesting that urea may have originated from ureide degradation in soybean plants, possibly in the roots. This is the first finding of the possibility of ureide degradation to urea in the underground organs of soybean plants.

Effects of Different Chemical Forms of Nitrogen on the Quick and Reversible Inhibition of Soybean Nodule Growth and Nitrogen Fixation Activity.

It has been reported that supply of nitrate to culture solution rapidly and reversibly inhibits nodule growth and nitrogen fixation activity of soybean. In this study, the effects of ammonium, urea, or glutamine on nodule growth and nitrogen fixation activity are compared with that for nitrate. Soybean plants were cultivated with a nitrogen-free nutrient solution, then 1 mM-N of nitrate, ammonium, glutamine, or urea were supplied from 12 DAP until 17 DAP. Repression of nodule growth and nitrogen fixation activity at 17 DAP were observed by ammonium, urea, and glutamine like nitrate, although the inhibitory effects were milder than nitrate. The removal of nitrogen from the culture solutions after nitrogen treatments resulted in a recovery of the nodule growth. It was found that the glutamine treatment followed by N-free cultivation gave highest nitrogen fixation activity about two times of the control. Tracer experiments with 15N and 13C were performed to evaluate the translocation of N and C to the different tissues. Culture solutions containing a 15N-labeled nitrogen source were supplied from 21 DAP, and the whole shoots were exposed to 13CO2 for 60 min on 23 DAP, and plants were harvested on 24 DAP. The percentage distribution of 15N in nodules was highest for ammonium (1.4%) followed by glutamine (0.78%), urea (0.32%) and nitrate (0.25%). The percentage distribution of 13C in the nodules was highest for the control (11.5%) followed by urea (5.8%), glutamine (2.6%), ammonium (2.3%), and nitrate (2.3%). The inhibitory effects of nitrogen compounds appeared to be related to a decrease in photoassimilate partitioning in the nodules, rather than 15N transport into the nodules. The free amino acid concentrations after nitrogen treatments were increased in the nodules and leaves by nitrate, in the roots by ammonium, in the stems by urea, and the roots, stems, and leaves by glutamine treatment. The concentrations of asparagine, aspartate, and glutamine were increased after nitrogen treatments. By the long-term supply of nitrogen for 2-weeks, nitrate significantly increased the lateral roots and leaf growth. The long-term supply of urea and glutamine also promoted the lateral roots and leaf growth, but ammonium suppressed them.

Transcriptome and Metabolome Analyses Reveal That Nitrate Strongly Promotes Nitrogen and Carbon Metabolism in Soybean Roots, but Tends to Repress It in Nodules.

Leguminous plants form root nodules with rhizobia that fix atmospheric dinitrogen (N2) for the nitrogen (N) nutrient. Combined nitrogen sources, particular nitrate, severely repress nodule growth and nitrogen fixation activity in soybeans (Glycine max [L.] Merr.). A microarray-based transcriptome analysis and the metabolome analysis were carried out for the roots and nodules of hydroponically grown soybean plants treated with 5 mM of nitrate for 24 h and compared with control without nitrate. Gene expression ratios of nitrate vs. the control were highly enhanced for those probesets related to nitrate transport and assimilation and carbon metabolism in the roots, but much less so in the nodules, except for the nitrate transport and asparagine synthetase. From the metabolome analysis, the concentration ratios of metabolites for the nitrate treatment vs. the control indicated that most of the amino acids, phosphorous-compounds and organic acids in roots were increased about twofold in the roots, whereas in the nodules most of the concentrations of the amino acids, P-compounds and organic acids were decreased while asparagine increased exceptionally. These results may support the hypothesis that nitrate primarily promotes nitrogen and carbon metabolism in the roots, but mainly represses this metabolism in the nodules.

Effect of nitrate on nodule and root growth of soybean (Glycine max (L.) Merr.).

The application of combined nitrogen, especially nitrate, to soybean plants is known to strongly inhibit nodule formation, growth and nitrogen fixation. In the present study, we measured the effects of supplying 5 mM nitrate on the growth of nodules, primary root, and lateral roots under light at 28 °C or dark at 18 °C conditions. Photographs of the nodulated roots were periodically taken by a digital camera at 1-h intervals, and the size of the nodules was measured with newly developed computer software. Nodule growth was depressed approximately 7 h after the addition of nitrate under light conditions. The nodule growth rate under dark conditions was almost half that under light conditions, and nodule growth was further suppressed by the addition of 5 mM nitrate. Similar results were observed for the extending growth rate of the primary root as those for nodule growth supplied with 5 mM nitrate under light/dark conditions. In contrast, the growth of lateral roots was promoted by the addition of 5 mM nitrate. The 2D-PAGE profiles of nodule protein showed similar patterns between the 0 and 5 mM nitrate treatments, which suggested that metabolic integrity may be maintained with the 5 mM nitrate treatment. Further studies are required to confirm whether light or temperature condition may give the primary effect on the growth of nodules and roots.

Rapid quantification of cyanamide by ultra-high-pressure liquid chromatography in fertilizer, soil or plant samples.

A rapid and simple method for determination of cyanamide in fertilizer, soil and plants has been developed. In this method, cyanamide is extracted with 2% acetic acid and the extract separated by centrifugation. It is then purified by passing through a membrane filter. The extract was derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate and the derivatized compound separated by ultra-high-pressure liquid chromatography. It is then detected with a UV detector at 260 nm by the same method as is used for amino acid analysis. The proposed method is fast, simple and cheap and also has good selectivity and sensitivity for the determination of cyanamide in a wide range of biotic and abiotic materials.

Nitrogen Fixation and Translocation in Young Sugarcane (Saccharum officinarum L.) Plants Associated with Endophytic Nitrogen-Fixing Bacteria.

The tracer (15)N(2) was used to investigate sites of N(2) fixation and the possible translocation of the fixed N. Young sugarcane plants (Saccharum officinarum L.) from a stem cutting were exposed to (15)N(2)-labeled air in a 500 mL plastic cylinder. Plants fed (15)N(2) for 7 days were grown in normal air for a further chase period. After 21 days, about half of the N originating in the stem cutting had been transported to the shoot and roots, suggesting that the cutting played a role in supplying N for growth. After 3 days of feeding, the percentage of N derived from (15)N(2) was higher in the roots (2.22%) and stem cutting (0.271%) than the shoot (0.027%). Most of the fixed N was distributed in the 80% ethanol-insoluble fractions in each plant part, and the (15)N fixed either in the roots or in the stem cutting remained there and was not appreciably transported to the shoot. The results were quite different from the fate of fixed N in soybean nodules, which is rapidly transported from nodules to roots and shoots.

The autoregulation of nodulation mechanism is related to leaf development.

To understand the autoregulation of nodulation (AON) system, in which leguminous plants control the nodule number, we examined the details of the characteristics of hypernodulation soybean mutants NOD1-3 and NOD3-7. A microscopic study showed that NOD1-3 and NOD3-7 produced small-size leaves due to the smaller number of leaf cells, compared with the Williams parent. These phenotypes were not affected by inoculation with bradyrhizobia or nitrate supply. The AON signaling might be related to the control system of leaf cell proliferation. This hypothesis was strongly supported by the finding that activation of AON in wild types by inoculation leads to an increase in the cell number of leaves.

Quick and reversible inhibition of soybean root nodule growth by nitrate involves a decrease in sucrose supply to nodules.

The upper part of a nodulated soybean root hydroponically cultured in a glass bottle was monitored using a computer microscope under controlled environmental conditions, and the diameter of individual nodules was measured from 10-24 d after planting. The diameter of a root nodule attached to the primary root increased from 1 mm to 6 mm for 2 weeks under nitrogen-free conditions. The increase in diameter of the nodules was almost completely stopped after 1 d of supplying 5 mM nitrate, and was due to the cessation of nodule cell expansion. However, nodule growth quickly returned to the normal growth rate following withdrawal of nitrate from the solution. The reversible depression of nodule growth by nitrate was similar to the restriction of photoassimilate supply by continuous dark treatment for 2 d followed by normal light/dark conditions. In addition, the inhibitory effect of nitrate was partially alleviated by the addition of 3% (w/v) sucrose to the medium. Plant leaves were exposed to (11)C or (14)C-labelled carbon dioxide to investigate the effects of 5 mM nitrate on the translocation and distribution of photosynthates to nodules and roots. Supplying 5 mM nitrate stimulated the translocation rate and the distribution of labelled C in nitrate-fed parts of the roots. However, the (14)C partitioning to nodules decreased from 9% to 4% of total (14)C under conditions of 5 mM nitrate supply. These results indicate that the decrease in photoassimilate supply to nodules may be involved in the quick and reversible nitrate inhibition of soybean nodule growth.