Biology and Fertility of Soils

, Volume 50, Issue 4, pp 583–592 | Cite as

A novel phosphorus biofertilization strategy using cattle manure treated with phytase–nanoclay complexes

  • Daniel Menezes-Blackburn
  • Milko A. Jorquera
  • Liliana Gianfreda
  • Ralf Greiner
  • María de la Luz Mora
Original Paper


The aim of this work was to evaluate the treatment of cattle manure with phytases stabilized in allophanic nanoclays as a potential novel phosphorus (P) biofertilization technology for crops grown in volcanic soils (Andisol). Furthermore, because the optimal pH for commercial phytase catalysis does not match the natural pH of manure, a complementary experiment was set up to evaluate the effect of manure inoculation with an alkaline phytase-producing bacterium. Finally, phytase-treated soil, manure, and soil–manure mixtures were evaluated for their P-supplying capacity to wheat plants grown under greenhouse conditions. Treating cattle manure with phytases stabilized in nanoclays resulted in a significant (P ≤ 0.05) increase of inorganic P in soil extracts (NaOH-EDTA and Olsen). The use of phytase-treated cattle manure increased dry weights by 10 % and the P concentration by 39 % in wheat plants grown under greenhouse conditions, which is equivalent to a P fertilizer rate of about 150 kg of P per hectare. The inoculation of cattle manure with β-propeller phytase-producing bacteria led to an ∼10 % increase in inorganic P in the manure extracts. However, applying inoculated manure to soil did not significantly increase wheat yield or P acquisition responses. Our results suggest that the novel approach of incubating cattle manure with phytases stabilized in nanoclay enhances the organic P cycling and P nutrition of plants grown in P-deficient soils.


Phytase Phytate Phosphorus Organic phosphorus Biofertilization Volcanic soils Cattle manure Wheat 



This work was financed by FONDECYT research projects no 1100625 and no 1120505. D. Menezes-Blackburn acknowledges to the following financial supports: UFRO and CONICYT Doctoral Scholarships, and Georg Forster Research Postdoctoral Fellowship (Humboldt Foundation). MA Jorquera also thanks to the support from International Cooperation Research CONICYT-BMBF code 2009-183.


  1. Abelson PH (1999) A potential phosphate crisis. Science 283:2015PubMedCrossRefGoogle Scholar
  2. Acuña JY, Jorquera MA, Martínez OA, Menezes-Blackburn D, Fernández MT, Marschner P, Mora ML (2011) Indole acetic acid and phytase activity produced by rhizosphere bacilli as affected by pH and metals. J Soil Sci Plant Nutr 11:1–12Google Scholar
  3. Ashley K, Cordell D, Mavinic D (2011) A brief history of phosphorus: from the philosopher’s stone to nutrient recovery and reuse. Chemosphere 84:737–746PubMedCrossRefGoogle Scholar
  4. Borie F, Rubio R (2003) Total and organic phosphorus in Chilean volcanic soils. Gayana Bot 60:69–73CrossRefGoogle Scholar
  5. Briceño M, Escudey M, Galindo G, Borchardt D, Chang A (2004) Characterization of chemical phosphorus forms in volcanic soils using 31P-NMR spectroscopy. Commun Soil Sci Plant Anal 35:1323–1337CrossRefGoogle Scholar
  6. Brinch-Pedersen H, Sorensen LD, Holm PB (2002) Engineering crop plants: getting a handle on phosphate. Trends Plant Sci 7:118–125PubMedCrossRefGoogle Scholar
  7. Cade-Menun BJ, Preston CM (1996) A comparison of soil extraction procedures for 31P NMR spectroscopy. Soil Sci 161:770–785CrossRefGoogle Scholar
  8. Calabi-Floody M, Bendall JS, Jara AA, Welland ME, Theng BK, Rumpel C, Mora ML (2011) Nanoclays from an Andisol: extraction, properties and carbon stabilization. Geoderma 161(3):159–167CrossRefGoogle Scholar
  9. Calabi-Floody M, Velásquez G, Gianfreda L, Saggar S, Bolan N, Rumpel C, Mora ML (2012) Improving bioavailability of phosphorous from cattle dung by using phosphatase immobilized on natural clay and nanoclay. Chemosphere 89:648–655PubMedCrossRefGoogle Scholar
  10. Dao TH (2003) Polyvalent cation effects on myo-inositol hexakis dihydrogenphosphate hnzymatic dephosphorylation in dairy wastewater. J Environ Qual 32:694–701PubMedCrossRefGoogle Scholar
  11. Dao TH (2004) Ligands and phytase hydrolysis of organic phosphorus in soils amended with dairy manure. Agron J 96:1188–1195CrossRefGoogle Scholar
  12. FAO (2006) Food and Agriculture Organization yearbook. Rome, ItalyGoogle Scholar
  13. Fuentes B, Bolan N, Naidu R, Mora ML (2006) Phosphorus in organic waste–soil systems. J Soil Sci Plant Nutr 6:64–83Google Scholar
  14. Fuentes B, Mora ML, Bolan NS, Naidu R (2008) Assessment of phosphorus bioavailability from organic wastes in soil. Dev Soil Sci 32:363–411CrossRefGoogle Scholar
  15. Fuentes B, Jorquera M, Mora ML (2009) Dynamics of phosphorus and phytate-utilizing bacteria during aerobic degradation of dairy cattle dung. Chemosphere 74:325–331PubMedCrossRefGoogle Scholar
  16. Garrido-Ramírez EG, Theng BK, Mora ML (2010) Clays and oxide minerals as catalysts and nanocatalysts in Fenton-like reactions—a review. Appl Clay Sci 47:182–192CrossRefGoogle Scholar
  17. George T, Simpson R, Gregory P, Richardson A (2007a) Differential interaction of Aspergillus niger and Peniophora lycii phytases with soil particles affects the hydrolysis of inositol phosphates. Soil Biol Biochem 39:793–803CrossRefGoogle Scholar
  18. George TS, Simpson RJ, Hadobas PA, Marshall DJ, Richardson AE (2007b) Accumulation and phosphatase-lability of organic phosphorus in fertilised pasture soils. Aust J Agric Res 58:47–56CrossRefGoogle Scholar
  19. Giaveno C, Celi L, Richardson AE, Barberis E (2010) Interaction of phytases with minerals and availability of substrate affect the hydrolysis of inositol phosphates. Soil Biol Biochem 42:491–498CrossRefGoogle Scholar
  20. Glick BR (1995) The enhancement of plant growth by free-living bacteria. Can J Microbiol 41:109–117CrossRefGoogle Scholar
  21. Godoy S, Meschy F (2001) Utilisation of phytate phosphorus by rumen bacteria in a semi-continuous culture system (Rusitec) in lactating goats fed on different forage to concentrate ratios. Reprod Nutr Dev 41:259–265PubMedCrossRefGoogle Scholar
  22. Godoy S, Chicco C, Meschy F, Requena F (2005) Phytic phosphorus and phytase activity of animal feed ingredients. Interciencia 30:24–28Google Scholar
  23. Green VS, Dao TH, Stone G, Cavigelli MA, Baumhardt RL, Devine TE (2007) Bioactive phosphorus loss in simulated runoff from a phosphorus-enriched soil under two forage management systems. Soil Sci 172:721CrossRefGoogle Scholar
  24. Greiner R, Konietzny U, Jany KD (1993) Purification and characterization of two phytases from Escherichia coli. Arch Biochem Biophys 303:107–113PubMedCrossRefGoogle Scholar
  25. Gujar PD, Bhavsar KP, Khire JM (2013) Effect of phytase from Aspergillus niger on plant growth and mineral assimilation in wheat (Triticuma estivum Linn.) and its potential for use as a soil amendment. J Sci Food Agric 93:2242–2247PubMedCrossRefGoogle Scholar
  26. Hayes JE, Richardson AE, Simpson RJ (2000) Components of organic phosphorus in soil extracts that are hydrolysed by phytase and acid phosphatase. Biol Fertil Soils 32:279–286CrossRefGoogle Scholar
  27. He ZQ, Honeycutt CW, Griffin TS (2003) Enzymatic hydrolysis of organic phosphorus in extracts and resuspensions of swine manure and cattle manure. Biol Fertil Soils 38:78–83CrossRefGoogle Scholar
  28. He Z, Cade-Menun BJ, Toor GS, Fortuna AM, Honeycutt CW, Sims JT (2007) Comparison of phosphorus forms in wet and dried animal manures by solution phosphorus-31 nuclear magnetic resonance spectroscopy and enzymatic hydrolysis. J Environ Qual 36:1086PubMedCrossRefGoogle Scholar
  29. Hinsinger P, Plassard C, Tang C, Jaillard B (2003) Origins of root-mediated pH changes in the rhizosphere and their responses to environmental constraints: a review. Plant Soil 248:43–59CrossRefGoogle Scholar
  30. Jorquera MA, Crowley DE, Marschner P, Greiner R, Fernández MT, Romero D, Menezes-Blackburn D, Mora ML (2011) Identification of β-propeller phytase-encoding genes in culturable Paenibacillus and Bacillus spp. from the rhizosphere of pasture plants on volcanic soils. FEMS Microbiol Ecol 75:163–172PubMedCrossRefGoogle Scholar
  31. Koenig R, Johnson C (1942) Colorimetric determination of phosphorus in biological materials. Ind Eng Chem Anal Ed 14:155–156CrossRefGoogle Scholar
  32. Lott JNA, Ockenden I, Raboy V, Batten GD (2000) Phytic acid and phosphorus in crop seeds and fruits: a global estimate. Seed Sci Res 10:11–33Google Scholar
  33. Menezes-Blackburn D, Jorquera M, Gianfreda L, Rao M, Greiner R, Garrido E, Mora ML (2011) Activity stabilization of Aspergillus niger and Escherichia coli phytases immobilized on allophanic synthetic compounds and montmorillonite nanoclays. Bioresour Technol 102:9360–9367PubMedCrossRefGoogle Scholar
  34. Menezes-Blackburn D, Jorquera M, Greiner R, Gianfreda L, Mora ML (2013) Phytases and phytase-labile organic phosphorus in manures and soils. Crit Rev Environ Sci Technol 43:916–954CrossRefGoogle Scholar
  35. Mora ML, Escudey M, Galindo GG (1994) Sintesis y caracterización de suelos alofánicos. Bol Soc Chil Quim 39:237–243Google Scholar
  36. Nannipieri P, Bollag J-M (1991) Use of enzymes to detoxify pesticide-contaminated soils and waters. J Environ Qual 20:510–517CrossRefGoogle Scholar
  37. Nannipieri P, Giagnoni L, Renella G, Puglisi E, Ceccanti B, Masciandaro G, Marinari S (2012) Soil enzymology: classical and molecular approaches. Biol Fertil Soils 48:743–762CrossRefGoogle Scholar
  38. Neumann G, Römheld V (1999) Root excretion of carboxylic acids and protons in phosphorus-deficient plants. Plant Soil 211:121–130CrossRefGoogle Scholar
  39. Paredes C, Menezes-Blackburn D, Cartes P, Gianfreda L, Mora ML (2011) Phosphorus and nitrogen fertilization effect on phosphorus uptake and phosphatase activity in ryegrass and tall fescue grown in a Chilean andisol. Soil Sci 176:245Google Scholar
  40. Pinochet D, Epple G, MacDonald R (2001) Organic and inorganic phosphorus fractionsin a soil transect of volcanic and metamorphic origin. J Soil Sci Plant Nutr 1:58–69Google Scholar
  41. Ramírez CA, Kloepper JW (2010) Plant growth promotion by Bacillus amyloliquefaciens FZB45 depends on inoculum rate and P-related soil properties. Biol Fertil Soils 1–10Google Scholar
  42. Rao D, Rao KV, Reddy TP, Reddy VD (2009) Molecular characterization, physicochemical properties, known and potential applications of phytases: an overview. Crit Rev Biotechnol 29:182–198PubMedCrossRefGoogle Scholar
  43. Redel YD, Rubio R, Rouanet JL, Borie F (2007) Phosphorus bioavailability affected by tillage and crop rotation on a Chilean volcanic derived Ultisol. Geoderma 139:388–396CrossRefGoogle Scholar
  44. Richardson AE, Simpson RJ (2011) Soil microorganisms mediating phosphorus availability update on microbial phosphorus. Plant Physiol 156:989–996PubMedCentralPubMedCrossRefGoogle Scholar
  45. Richardson A, Hadobas P, Hayes J (2001) Extracellular secretion of Aspergillus phytase from Arabidopsis roots enables plants to obtain phosphorus from phytate. Plant J 25:641–649PubMedCrossRefGoogle Scholar
  46. Richardson AE, George TS, Jakobsen I, Simpson RJ (2007) Plant utilization of inositol phosphates. In: Turner BL, Richardson AE, Mullaney EJ (eds) Inositol phosphates: linking agriculture and the environment. CABI, Wallingford, UK, pp 242–260CrossRefGoogle Scholar
  47. Rosas A, Mora ML, Jara AA, López R, Rao MA, Gianfreda L (2008) Catalytic behavior of acid phosphatase immobilized on natural supports in the presence of manganese or molybdenum. Geoderma 145:77–83CrossRefGoogle Scholar
  48. Sharpley A, Moyer B (2000) Phosphorus forms in manure and compost and their release during simulated rainfall. J Environ Qual 29:1462–1469CrossRefGoogle Scholar
  49. Tang J, Leung A, Leung C, Lim BL (2006) Hydrolysis of precipitated phytate by three distinct families of phytases. Soil Biol Biochem 38:1316–1324CrossRefGoogle Scholar
  50. Turner BL, Paphazy MJ, Haygarth PM, McKelvie ID (2002) Inositol phosphates in the environment. Philos Trans R Soc B 357:449–469CrossRefGoogle Scholar
  51. Wiehe W, Höflich G (1995) Survival of plant growth promoting rhizosphere bacteria in the rhizosphere of different crops and migration to non-inoculated plants under field conditions in north-east Germany. Microbiol Res 150:201–206CrossRefGoogle Scholar
  52. Wyss M, Brugger R, Kronenberger A, Rémy R, Fimbel R, Oesterhelt G, Lehman M, van Loon AP (1999) Biochemical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): catalytic properties. Appl Environ Microbiol 65(2):367–373PubMedCentralPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Daniel Menezes-Blackburn
    • 1
    • 4
  • Milko A. Jorquera
    • 2
  • Liliana Gianfreda
    • 3
  • Ralf Greiner
    • 4
  • María de la Luz Mora
    • 2
  1. 1.Chile–Italy International Doctorate in Environmental Resources ScienceUniversidad de La FronteraTemucoChile
  2. 2.Scientific and Technological Bioresource Nucleus (BIOREN)Universidad de La FronteraTemucoChile
  3. 3.Dipartimento di Scienze del Suolo, della Pianta e dell’Ambiente, e delle Produzioni AnimaliUniversità di NapoliPorticiItaly
  4. 4.Department of Food Technology and Bioprocess Engineering, Max Rubner-InstitutFederal Research Institute of Nutrition and FoodKarlsruheGermany

Personalised recommendations