Open access

Soybean as a Nitrogen Supplier

Written By

Matsumiya Yoshiki, Horii Sachie, Matsuno Toshihide and Kubo Motoki

Submitted: 15 June 2012 Published: 02 January 2013

DOI: 10.5772/51017

Chapter metrics overview

3,108 Chapter Downloads

View Full Metrics

1. Introduction

Soybean has been cultivated all over the world since ancient times for its high protein and lipid content. It is one of the most important agricultural products in the world and its global production is more than 220 million tons per year [1]. Vegetable oil production from soybean is the highest among plant oils (30%) [2].

Soybean is used directly as food in Japan and several Asian countries. Recently, soybean protein was recognized as both healthy and tasty and is used in food such as Tofu and soy sauce. Soybean-meal, which remains after extraction of the vegetable oil, contains about 50% protein with well balanced amino acids. Therefore, soybean-meal is often re-utilized as animal foodstuff.

Soybean waste was utilized as an organic fertilizer prior to the 1940s [3-6]. However, a chemical fertilizer took the place of the organic fertilizer because it produced faster results. Organic fertilizers are now gradually being used again for increased food production safety and the protection of the environment.

Soybean cultivation is well known for improving soil fertility [3, 7, 8]. Root-nodules are formed by the soybean plant, and atmospheric N2 is fixed by the nitrogen fixing bacteria in the root-nodule [9]. N2 is converted to NH4+ by nitrogenase from these nitrogen fixing bacteria, and this NH4+ is supplied to the soil environment.

Recently, investigations into the utilization of proteins from soybean waste have been carried out for the development of high quality foods. Protein fractions, such as soy protein isolates and whey protein are industrially produced, and these fractions are used as additives for the improvement of food nutrition [10]. Moreover, several soybean proteins and peptides have been purified and utilized as medicines for hypotension, rheumatism, and cholesterol control [11-13]. The bioactive peptides of soybean protein have also been investigated [5, 6].

This chapter explains how soybean cultivation and soybean protein are nitrogen suppliers and describes the production of novel bioactive peptides from soybean and legumes.


2. Nitrogen supply by soybean cultivation

2.1. Nitrogen fixing bacteria

N2 is fixed by nitrogen fixing bacteria in the soil environment [14-17]. These bacteria convert N2 to NH4+. The biological reduction of atmospheric N2 to NH4+ (nitrogen fixation) provides about 65% of the biosphere's available nitrogen [18].

As long ago as 1890, a nitrogen fixing bacteria was isolated from a root nodule and identified as Rhizobium leguminosarum [19, 20]. Shortly after this, Clostridium pasteurianum and Azotobacter sp. were also isolated as nitrogen fixing bacteria in the soil environment [21-23]. Now, more than 100 genera have been isolated and identified as nitrogen fixing bacteria. Among them, genera Rhizobium, Bradyrhizobium, Azorhizobium, and Frankia lead to the formation of root-nodules in legumes [16].

Nitrogenase (EC from nitrogen fixing bacteria catalyzes N2 to NH4+ (N2 + 8H2 + 8e- + 16ATP + 16H2O → 2NH3 + H2 + 16ADP + 16Pi). NH4+ is further converted to NO2- and NO3- by ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB).

Figure 1.

Soybean root nodule

2.2. Relationship between nitrogen fixing bacteria and soybean cultivation

The roots of soybean secrete flavonoids and enhance the growth of nitrogen fixing bacteria around the root [24]. The nitrogen fixing bacteria infect the soybean root, and the root-nodule is formed. Bacteroids in the root-nodule fix and provide nitrogen from the air [25]. Bradyrhizobium japonicum, B. elkanii, B. lianigense, and Sinorhizobium fredii have been identified as the root-nodule forming bacteria in soybean cultivation [16, 26, 27].

The change in soil microbial diversity after soybean cultivation has been analyzed by PCR-DGGE. Root-nodules were shown to be formed and specific bacteria were increased during cultivation (Figures 1 and 2) but not the total number of bacteria in the soil. Soybean cultivation caused nitrogen accumulation in the soil environment.

Figure 2.

PCR-DGGE profiles of soybean cultivated soil, 1: Before cultivation, 2: after cultivation.


3. Enhancement of nitrogen circulation by soybean cultivation and soybean protein

3.1. Evaluation of nitrogen circulation in soil environment

The nitrogen cycle is illustrated in Figure 3. Organic forms of nitrogen such as protein are degraded to peptides and amino acids by soil microorganisms, and these peptides and amino acids are then converted to NH4+. Subsequently, NH4+ is further converted to NO2- and NO3- (nitrification). NO2- is denitrified to N2 by denitrifying bacteria and this N2 is converted to NH4+ by the nitrogen fixing bacteria, and NH4+ is accumulated in the soil environment again.

Figure 3.

The soil nitrogen cycle

The nitrification process is the rate limiting step in the nitrogen cycle [28]. To further investigate the soil nitrogen cycle, a new method for the evaluation of nitrogen circulation activity was constructed based on bacterial number, ammonium oxidizing activity (AOA), and nitrite oxidizing activity (NOA) (Figure 4) [29]. These three indices were used to construct a radar chart of nitrogen circulation in the soil. The area of the radar chart was calculated, and then the value was treated as a nitrogen circulation activity (0–100 points).

3.2. Enhancement of nitrogen circulation

A database of nitrogen circulation activity was constructed using 155 agricultural soils (Figure 5). The nitrogen circulation activity of agricultural soil ranges from 0 to 99.6 points with an average of 26 points.

Figure 4.

Values of nitrogen circulation activity in soil environments

Figure 5.

Database of nitrogen circulation activity in 155 agricultural soils

Soybean cultivation leads to nitrogen accumulation in the soil environment, and therefore nitrogen circulation activity should be enhanced by soybean cultivation. This enhancement was further analyzed (Figure 6) and activity was shown to be enhanced 26 to 95 points after soybean cultivation.

Soybean waste is also rich in nitrogen (Table 1), and is often used as an organic fertilizer. Soil nitrogen is increased by using soybean waste as fertilizer, and consequently nitrogen circulation is increased. Soybean waste is also rich in carbon (C/N values; 5.1), and therefore soil bacteria and bacterial activity may also be increased by the addition of soybean waste.

Figure 6.

Effect of soybean cultivation on nitrogen circulation activity in soil

Component Value
Total carbon 450,000 mg/kg
Total nitrogen 87,500 mg/kg
Total phosphorous 6,100 mg/kg
Total potassium 18,900 mg/kg
C/N ratio 5.1

Table 1.

Components of soybean meal


4. Bioactive peptides from soybean protein

4.1. Plant growth promoting peptides from soybean waste

For efficient use of soybean waste, it is treated with an alkaline protease from Bacillus circulans HA12 (degraded soybean meal products; DSP) [4, 6]. Plant growth promotion by DSP has been investigated using various plant species [30]. The fresh weight of Brassica rapa was shown to be increased by 25% through the addition of DSP (12 mg-peptides/kg-soil) (Figure 7). The growth of Solanum tuberosum L., Solanum lycopersicum, and Brassica juncea were also promoted by addition of DSP. Moreover, DSP also produced thicker roots than a chemical fertilizer, indicating that DSP contains bioactive peptides for plant growth.

Figure 7.

Plant growth-promoting effect of DSP, A: Chemical fertilizer, B; DSP.

4.2. Root hair promoting peptide in DSP

The number of root hairs in B. rapa was increased and each was elongated when DSP (30 µg/ml) was added (Figure 8) to the soil. In order to analyze the root hair promoting effect by DSP, the structure of the root hair promoting peptide (RHPP) in DSP was investigated [6]. Degraded products of Kunitz trypsin inhibitor (KTI) in soybean protein showed higher root hair promoting activity, and the RHPP was purified by several chromatographic steps from degraded products of KTI.

Figure 8.

Root hair promoting effect of DSP, A: Root of Brassica rapa grown in plant growth medium, B: root of B. rapa grown with DSP in plant growth medium. Bar denotes 1 mm.

The molecular mass of RHPP was analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) [6]. The molecular weight of the bioactive peptide was 1,198.2 Da (Figure 9), and the molecular weight of the amino acid sequence in KTI was searched. Positions 27–38 in KTI (Gly-Gly-Ile-Arg-Ala-Ala-Pro-Thr-Gly-Asn-Glu-Arg) were identical to this molecular weight, and this peptide was thus designated as the RHPP (Figure 9). The RHPP that was chemically synthesized was also shown to have root hair promoting activity (data not shown).

Figure 9.

Amino acid sequence of RHPP in Kunitz trypsin inhibitor, the RHPP amino acid sequence is shown by gray box.


5. Novel plant bioactive peptides from other legume

Many other legumes form root-nodules with nitrogen fixing bacteria. The nitrogen fixing bacteria related to legume cultivation are classified into 13 genera (Rhizobium, Ensifer, Mesorhizobium, Bradyrhizobium, Methylobacterium, Azorhizobium, Devosia, Burkholderia, Phyllobacterium, Microvirga, Ochrobactrum, Cupriavidus, and Shinella) and 98 species [31].

Legumes such as Astragalus sinicus, Trifolium repens, and Arachis hypogaea are cultivated as green manure for the improvement of soil fertility. The host specificity of the nitrogen fixing bacteria, M. huakuii, R. trifolii, and Bradyrhizobium sp., are very high, infecting A. sinicus, T. repens, and A. hypogaea, respectively [32]. These legumes are rich in proteins and form root-nodules via the same mechanisms as soybean.

In order to find novel bioactive peptides, attempts to degrade protein biomass from A. hypogaea by various proteases (thermolysin, subtilisin, proteinaseK, and trypsin) were made. Bioactivities of root hair and lateral root formation were found by degradation with proteinaseK (Figure 10). Degraded products of A. hypogaea by proteinase K (30 µg/ml) showed strong root hair promoting activity at the same level as DSP. Moreover, degraded products of A. hypogaea promoted lateral root growth in B. rapa, suggesting that degradation of legume proteins has a possibility to produce new bioactive peptides.

Figure 10.

Bioactive effect of degraded products of A. hypogaea on root of B. rapa, A: Root of Brassica rapa grown in plant growth medium, B: root of B. rapa grown with degraded products of A. hypogaea. Bar denotes 1 mm.


6. Conclusion

Soybean supplies nitrogen into the soil environment by forming root nodules and accumulating protein in its seed. Soybean cultivation has been shown to enhance nitrogen circulation by about 3.6 times accompanied with increases in nitrogen fixing bacteria.

DSP has been shown to increase the fresh weight of plants, and a peptide from DSP promoted root hair formation in B. rapa. Moreover, other bioactive peptides were found by degradation of proteins from A. hypogaea with proteinaseK treatment. The proteins of legumes will also become nitrogen sources for plant growth and the soil environment.


  1. 1. Uchida M. 2007 Prospects for World Trade in the Soybean Sector. Journal of Nagoya Bunri University 7 97 102
  2. 2. USDA. Oilseeds: World Markets and Trade 2007
  3. 3. Okuda A. 2011 Dojou hiryou sousetu Tokyo: Yokendou Co. LTD
  4. 4. Kubo M. Okajima J. Hasumi F. 1994 Isolation and Characterization of Soybean Waste-Degrading Microorganisms and Analysis of Fertilizer Effects of the Degraded Products. Applied and Environmental Microbiology 60 1 243 247
  5. 5. Matsumiya Y. Kubo M. 2011 Soybean Peptide: Novel Plant Growth Promoting Peptide from Soybean. Soybean and Nutrition Rijeka Intech
  6. 6. Matsumiya Y. Kubo M. 2008 Utilization of Biomass Based on Biorefinery: Development of Novel Bioactive Peptides from Soybean Waste. Current Topics in Biotechnology 4
  7. 7. Cass A. Gusli S. Mac Leod. D. 1994 Sustainability of Soil Structure Quality in Rice Paddy-Soya-Bean Cropping Systems in South Sulawesi Indonesia Soil and Tillage Research 31 4 339 52
  8. 8. Tago K. Ishii S. Nishizawa T. Otsuka S. Senoo K. 2011 Phylogenetic and Functional Diversity of Denitrifying Bacteria Isolated from Various Rice Paddy and Rice-Soybean Rotation Fields. Microbes and Environments 26 1 30 5
  9. 9. Nagatani H. Shimizu M. Valentine R. 1971 The Mechanism of Ammonia Assimilation in Nitrogen Fixing bacteria. Archives of Microbiology 79 2 164 75
  10. 10. Malhotra A. Coupland J. N. 2004 The Effect of Surfactants on the Solubility, Zeta Potential, and Viscosity of Soy Protein Isolates. Food Hydrocolloids 18 1 101 8
  11. 11. Yonekura M. Yamamoto A. 2004 Isolation and Application of Physiologically Active Peptides from Soybean Whey and Okara Proteins. Soy Protein Research 7 79 84
  12. 12. Yonekura M. Tanaka A. 2003 Isolation and Application of Physiologically Active Peptides from Soybean Whey and Okara Proteins. Soy Protein Research 6 88 93
  13. 13. Farzamirad V. Aluko R. E. 2008 Angiotensin-Converting Enzyme Inhibition and Free-Radical Scavenging Properties of Cationic Peptides Derived from Soybean Protein Hydrolysates. International Journal of Food Sciences and Nutrition 59 5 428 37
  14. 14. Steenhoudt O. Vanderleyden J. 2000 Azospirillum, a Free‐Living Nitrogen‐Fixing Bacterium Closely Associated with Grasses: Genetic, Biochemical and Ecological Aspects. FEMS Microbiology Reviews 24 4 487 506
  15. 15. Orr C. H. James A. Leifert C. Cooper J. M. Cummings S.P. 2011 Diversity and Activity of Free-Living Nitrogen-Fixing Bacteria and Total Bacteria Organic and Conventionally Managed Soils. Applied and Environmental Microbiology 77 3 911 9
  16. 16. Fred E. B. Baldwin I. L. Mc Coy E. 2002 Root Nodule Bacteria and Leguminous Plants. UW-Madison Libraries Parallel Press
  17. 17. Berman-Frank I. Lundgren P. Falkowski P. 2003 Nitrogen Fixation and Photosynthetic Oxygen Evolution in Cyanobacteria. Research Microbiology 154 3 157 64
  18. 18. Lodwig E. M. Hosie A. H. Bourdes A. Findlay K. Allaway D. Karunakaran R. Downie J. A. Poole P. S. 2003 Amino-Acid Cycling Drives Nitrogen Fixation in the Legume-Rhizobium Symbiosis. Nature 422 6933 722 6
  19. 19. Nutman P.S. 1998 Biological Nitrogen Fixation. Foundations of Modern Biochemistry 4 199 216
  20. 20. Frank B. 1890 Ueber die Pilzymbiose der Leguminosen. Paul Parey Publishing
  21. 21. Winogradski S. 1895 Recherches Sur L’assimilation de L’azote Libre de L’atmosphere Par les Microbes. Archives des Sciences Biologiques 3 297 352
  22. 22. Beijerinck M. 1901 Ueber Oligonitrophile Mikroben" (in German). Zentralblatt für Bakteriologie, Parasitenkunde, Infektionskrankheiten und Hygiene. Abteilung II. 7 561 582
  23. 23. Coyne M. 1996 A Cartoon History of Soil Microbiology. Journal of Natural Resources and Life Sciences Education 25 1 30 36
  24. 24. Van de Sande K. Bisseling T. 1997 Signalling Symbiotic Root Nodule Formation, Essays in Biochemistry 32 127 42
  25. 25. Nakayama T. Botany W. E. B. 2005
  26. 26. Kaneko T. Nakamura Y. Sato S. Minamisawa K. Uchiumi T. Sasamoto S. Watanabe A. Idesawa K. Iriguchi M. Kawashima K. 2002 Complete Genomic Sequence of Nitrogen-Fixing Symbiotic Bacterium Bradyrhizobium japonicum USDA110. DNA Research 9 6 189 197
  27. 27. Scholla M.H. Elkan G.H. 1984 Rhizobium fredii sp. nov., a Fast-Growing Species that Effectively Nodulates Soybeans. International Journal of Systematic Bacteriology 34 4 484 6
  28. 28. Højberg O. Binnerup S. J. Sørensen J. 1996 Potential Rates of Ammonium Oxidation, Nitrite Oxidation, Nitrate Reduction and Denitrification in the Young Barley Rhizosphere. Soil Biology and Biochemistry 28 1 47 54
  29. 29. Tsuda H. Matsuno T. Kubota K. Matsumiya Y. Kubo M. 2010 A New Method for an Evaluation of a Nitrogen Circulation Activity in Soil. Memoirs of the Institute of Science & Engineering 69 39 46
  30. 30. Hasegawa N. Fukumoto Y. Minoda M. Plikomol A. Kubo M. 2002 Promotion of Plant and Root Growth by Soybean Meal Degradation Products. Biotechnology Letters 24 18 1483 6
  31. 31. Weir B. 2008 The Current Taxonomy of Rhizobia
  32. 32. Nuswantara S. Fujie M. Yamada T. Malek W. Inaba M. Kaneko Y. Murooka Y. 1999 Phylogenetic Position of Mesorhizobium huakuii subsp. rengei, a Symbiont of Astragalus sinicus cv. Japan. Journal of Bioscience and Bioengineering 87 1 49 55

Written By

Matsumiya Yoshiki, Horii Sachie, Matsuno Toshihide and Kubo Motoki

Submitted: 15 June 2012 Published: 02 January 2013