Exploring Bioactive Compounds in Anaerobically Digested Slurry: Extraction, Characterization, and Assessment of Antifungal Activity

  • Jiaxin Lu
  • Atif Muhmood
  • Hongtao Liu
  • Renjie Dong
  • Sen Pang
  • Shubiao WuEmail author
Original Paper


Anaerobically digested slurry is well known for its use as a bio-fertilizer because of its high macro-nutrient content (e.g. N, P, K), which is essential for plant growth. The suppressive effect of slurry land application on pathogenic fungal growth due to the presence of bioactive and antimicrobial substances generated during anaerobic digestion has recently been reported. However, knowledge about the identification, characterization, and subsequent antimicrobial activity evaluation of various bioactive compounds in the anaerobically digested slurry is still insufficient. Ultrasound-assisted extraction using three solvents, ethyl acetate (EA), dichloromethane (DM), and n-butanol (NB), was employed for extraction of bioactive compounds. After extraction and characterization, antifungal activity against Fusarium oxysporum was assessed. EA was found to be a more efficient extractant with less evaporating time (4 min), irrespective of temperature. DM was found to be efficient in the extraction of O-heterocycles, while N-heterocycles were enriched in EA extracts. Furthermore, the bioactive compounds, 1,2-benzenedicarboxylic acid, butyl 2-methylpropyl ester (54.9%), and 9-octadecenamide, (Z)-(2.51%) were detected in the EA extract, while tetrahydro-2-furanmethanamine (1.32%), cyclic octatomic sulfur (1.17%), squalene (4.06%), and cholestan-3-ol (9.06%) were detected in the DM extract. The EA extract achieved approximately 84% inhibition of F. oxysporum, while only 63% inhibition was observed with the DM extract. Collectively, these findings indicate that in addition to its role as a fertilizer, digested slurry may also contribute to the control of phytopathogens upon land application because of the presence of various bioactive compounds. However, further work is needed to explore the diversity of bioactive compounds in digested manures and evaluate their antimicrobial potential.

Graphical Abstract


Bioactive compounds Heterocycles Phytopathogens Ultrasound Extraction 



This work was financed by grants from the project of “Research Fund for International Young Scientist (51650110489)”.

Compliance with Ethical Standards

Conflict of interest

The authors declare no conflict of interest whether financial or relational during the preparation and submission of this work.


  1. 1.
    Akhaja, T.N., Raval, J.P.: 1,3-Dihydro-2H-indol-2-ones derivatives: design, synthesis, in vitro antibacterial, antifungal and antitubercular study. Eur. J. Med. Chem. 46(11), 5573–5579 (2011)Google Scholar
  2. 2.
    Altarejos, J., Salido, S., Pérez-Bonilla, M., Linares-Palomino, P.J., Beek, T.A.V., Nogueras, M.: Preliminary assay on the radical scavenging activity of olive wood extracts. Fitoterapia. 76(3–4), 348–335 (2005)Google Scholar
  3. 3.
    Argade, N.D., Bhujade, V.K., Deshpande, A.A., Bachute, R.T., Karale, B.K., Gill, C.H.: Synthesis of some 5-(1,3-diphenyl)-1H-pyrazol-4-yl)-3-(2-hydroxyphenyl)-4,5- dhiydro-pyrazolines and 4-(1,3-diphenyl-1H-pyrazol-4-yl)-6-(2-hydroxyphenyl)-2-thionopyrimidines. Indian J. Heterocycl. Chem. 12(1), 53–56 (2002)Google Scholar
  4. 4.
    Badoil, L., Benanou, D.: Characterization of volatile and semivolatile compounds in waste landfill leachates using stir bar sorptive extraction-GC/MS. Anal. Bioanal. Chem. 393(3), 1043 (2009)Google Scholar
  5. 5.
    Bagavathi, P.E., Ramasamy, N.: GCMS analysis of phytocomponents in the ethanol extract of Polygonum chinense L. Pharmacogn. Res. 4, 11–14 (2012)Google Scholar
  6. 6.
    Balachandran, C., Lakshmi, R.S., Duraipandiyan, V., Ignacimuthu, S.: Antimicrobial activity of Streptomyces sp. (ERI-CPDA-1) isolated from oil contaminated soil from Chennai, India. Bioresour. Technol. 112, 83–90 (2012)Google Scholar
  7. 7.
    Benzohra, I., Megateli, M., Berdja, R.: Bayoud disease of date palm in Algeria: history, epidemiology and integrated disease management. Afr. J. Biotechnol. 14, 542–550 (2015)Google Scholar
  8. 8.
    Blok, W.J., Coenen, T.C.M., Termorshuizen, A.J., Lamers, J.G.: The potential of biological soil disinfestation to manage Fusarium foot and root rot in Asparagus. Acta Hort. 776(776), 135–144 (2008)Google Scholar
  9. 9.
    Bustamante, M.A., Alburquerque, J.A., Restrepo, A.P., De la Fuente, C., Paredes, C., Moral, R., Bernal, M.P.: Cocomposting of the solid fraction of anaerobic digestates to obtain added-value materials for use in agriculture. Biomass Bioenergy. 43, 26–35 (2012)Google Scholar
  10. 10.
    Cao, C., Zhang, N., Dang, D., Cheng, Y.: Modeling syngas production for miniaturized gas-to-liquids application: extending operation window by process improvement from different scales. Paper presented at the Aiche Meeting/aiche: 2016, at San Francisco, US (2016)Google Scholar
  11. 11.
    Cao, J.P., Zhao, X.Y., Morishita, K., Wei, X.Y., Takarada, T.: Fractionation and identification of organic nitrogen species from bio-oil produced by fast pyrolysis of sewage sludge. Bioresour. Technol. 101, 7648–7652 (2010)Google Scholar
  12. 12.
    Cao, Y., Chang, Z., Wang, J., Ma, Y., Fu, G.: The fate of antagonistic microorganisms and antimicrobial substances during anaerobic digestion of pig and dairy manure. Bioresour. Technol. 136(3), 664 (2013)Google Scholar
  13. 13.
    Cao, Y., Wang, J., Wu, H., Yan, S., Guo, D., Wang, G., Ma, Y.: Soil chemical and microbial responses to biogas slurry amendment and its effect on Fusarium wilt suppression. Appl. Soil. Ecol. 107, 116–123 (2016)Google Scholar
  14. 14.
    Cole, D.P., Smith, E.A., Dalluge, D., Wilson, D.M., Heaton, E.A., Brown, R.C., Lee, Y.J.: Molecular characterization of nitrogen-containing species in switch grass bio-oils at various harvest times. Fuel. 111, 718–726 (2013)Google Scholar
  15. 15.
    Dávila, E.L., Hurtado, A.C., León, Y.G., Unday, Z.G., Henderson, D., Jimenez, J.: Efecto agronómico del biosólido en cultivo de tomate (Solanum lycopersicum): control biológico de Rhizoctonia solani. Cultivos Tropicales 38(1), 13–23 (2017)Google Scholar
  16. 16.
    De La Fuente, C., Alburquerque, J.A., Clemente, R., Bernal, M.P.: Soil C and N mineralization and agricultural value of the products of an anaerobic digestion system. Biol. Fertil. Soils 49(3), 313–322 (2013)Google Scholar
  17. 17.
    Desai, N.C., Harsora, J.P., Patel, B.Y., Jadeja, K.A.: Synthesis of a novel series of imines containing nitrogen heterocycles as promising antibacterial and antifungal agents. Indian J. Chem. 56, 976–983 (2017)Google Scholar
  18. 18.
    Duan, N., Lin, C., Wang, Z., Meng, J., Chen, H., Li, X.Cong: Ecological analysis of a typical farm-scale biogas plant in China. Front. Earth Sci. 8(3), 375–384 (2014)Google Scholar
  19. 19.
    Eichlerloebermann, B., Bachmann, S., Uptmoor, R.: Biogas residues—aspects of nutrient management and soil fertility. World Fertilizer Congress: (2014)Google Scholar
  20. 20.
    El-Azab, I.H.: Synthesis of some new benzo[b][1,4]diazepine based heterocycles. J. Heterocycl. Chem. 50(S1), E178–E188 (2013)Google Scholar
  21. 21.
    Elfstrand, S., Hedlund, K., Martensson, A.: Soil enzyme activities, microbial community composition and function after 47 years of continuous green manuring. Appl. Soil. Ecol. 35(3), 610–621 (2007)Google Scholar
  22. 22.
    Gautam, A.K., Sharma, S., Avasthi, S., Bhadauria, R.: Diversity, pathogenicity and toxicology of A. niger: an important spoilage fungi Res. J. Microbiol. 6, 270–280 (2011)Google Scholar
  23. 23.
    Gil, A., Pabon, A., Galiano, S., Burguete, A., Perez-Silanes, S., Deharo, E., Aldana, I.: Synthesis, biological evaluation and structure–activity relationships of new quinoxaline derivatives as anti-Plasmodium falciparum agents. Molecules 19(2), 2166–2180 (2014)Google Scholar
  24. 24.
    Gong, H., Yan, Z., Liang, K.Q., Jin, Z.Y., Wang, Z.Y.: Concentrating process of liquid digestate by disk tube-reverse osmosis system. Desalination. 326, 30–36 (2013). K. J.Google Scholar
  25. 25.
    Guo, X., Huang, J., Lu, Y., Shan, G., Li, Q.: The influence of flue gas desulphurization gypsum additive on characteristics and evolution of humic substance during co-composting of dairy manure and sugarcane press mud. Bioresour. Technol. 219, 169–174 (2016)Google Scholar
  26. 26.
    Hameed, I.H., Abdulzahra, A.I., Jebor, M.A., Kqueen, C.Y., Ommer, A.J.: Haplotypes and variable position detection in the mitochondrial DNA coding region encompassing nucleotide positions 10,716–11,184. Mitochondrial DNA 26(4), 544–549 (2015)Google Scholar
  27. 27.
    Ighachane, H., Sedra, M.H., Lazrek, H.B.: Synthesis and evaluation of antifungal activities of (3H)-quinazolin-4-one derivatives against tree plant fungi. J. Mater. Environ. Sci. 8(1), 134–143 (2017)Google Scholar
  28. 28.
    Insam, H., Gomez-Brandon, M., Ascher, J.: Manure-based biogas fermentation residues—friend or foe of soil fertility? Soil Biol. Biochem. 84, 1–14 (2015)Google Scholar
  29. 29.
    Jin, H.M., Chang, Z.Z., Ye, X.M., Yan, M., Jin, Z.: Physical and chemical characteristics of anaerobically digested slurry from large-scale biogas project in Jiangsu Province. Trans. Chin. Soc. Agric. Eng. 27(1), 291–296 (2011)Google Scholar
  30. 30.
    Komarla, K.R., Sivakumar, P., Shemsu, S.A., Tadesse, M.Y.: Synthesis, characterization and in silicobiological activity of some 2-(N,N-dimethyl guanidinyl)-4,6-diaryl pyrimidines. J. Pharm. Bioallied Sci. 8(3), 181–187 (2016)Google Scholar
  31. 31.
    Lee, K.J., Song, N.Y., Oh, Y.C., Cho, W.K., Ma, J.Y.: Isolation and bioactivity analysis of ethyl acetate extract from Acer tegmentosum using in vitro assay and on-line screening HPLC-ABTS + System. J. Anal. Methods Chem. 2014, 1–15 (2014)Google Scholar
  32. 32.
    Li, X., Guo, J., Pang, C., Dong, R.: Anaerobic digestion and storage influence availability of plant hormones in livestock slurry. ACS Sustain. Chem. Eng. 4(3), 719–727 (2016)Google Scholar
  33. 33.
    Lichtenthaler, F.W.: Unsaturated O- and N-heterocycles from carbohydrate feedstocks. Acc. Chem. Res. 35(9), 728–737 (2002)Google Scholar
  34. 34.
    Liu, A., Xu, S., Lu, C., Peng, P., Zhang, Y., Feng, D., Liu, Y.: Anaerobic fermentation by aquatic product wastes and other auxiliary materials. Clean Tech Environ Policy 16, 415–421 (2014)Google Scholar
  35. 35.
    Liu, Y., Zhao, E., Zhu, W., Gao, H., Zhou, Z.: Determination of four heterocyclic insecticides by ionic liquid dispersive liquid–liquid micro extraction in water samples. J. Chromatogr. A. 1216, 885–891 (2009)Google Scholar
  36. 36.
    López-Robles, J., Olalla, C., Rad, C., Díez-Rojo, M.A., López-Pérez, J.A., Bello, A., Rodríguez-Kábana, R.: The use of liquid swine manure for the control of potato cyst nematode through soil disinfestation in laboratory conditions. Crop Prot. 49(7), 1–7 (2013)Google Scholar
  37. 37.
    Lu, Y., Wei, X.Y., Zong, Z.M., Lu, Y.C., Cao, J.P., Fan, X., Zhao, W., Rong, L.C., Zhao, Y.P., Li, L., Yan, H.L., Peng, Y.L.: Organonitrogen compounds identified in degraded wheat straw by oxidation in a sodium hypochlorite aqueous solution. Fuel 109, 61–67 (2013)Google Scholar
  38. 38.
    Majumdar, P., Pati, A., Patra, M., Behera, R.K., Behera, A.K.: Acid hydrazides.: potent reagents for synthesis of oxygen-, nitrogen-, and/or sulfur-containing heterocyclic rings. Chem. Rev. 114, 2942–2977 (2014)Google Scholar
  39. 39.
    Mallikarjunaswamy, C., Mallesha, L., Bhadregowda, D.G.: Pinto. Studies on synthesis of pyrimidine derivatives and their antimicrobial activity. Arab. J. Chem. 10, 484–490 (2017)Google Scholar
  40. 40.
    Martins, F.J., Caneschi, C.A., Senra, M.P., Carvalho, G.S.G., Da Silva, A.D., Raposo. N. R. B.: In vitro antifungal activity of hexahydropyrimidine derivatives against the causative agents of dermatomycosis. Hindawi. Sci. World J. 2017, 1–8 (2017)Google Scholar
  41. 41.
    Mohammd, G.J., Al-Jassani, M.J., Hameed, I.H.: Anti-bacterial, antifungal activity and chemical analysis of Punica grantanum (pomegranate peel) using GC–MS and FTIR spectroscopy. Int. J. Pharmacogn. Phytochem. Res. 8(3), 480–494 (2016)Google Scholar
  42. 42.
    Möller, K., Stinner, W.: Effects of organic wastes digestion for biogas production on mineral nutrient availability of biogas effluents. Nutr. Cycline Agroecosyst. 87, 395–413 (2010)Google Scholar
  43. 43.
    Möller, K.: Effects of anaerobic digestion on soil carbon and nitrogen turnover, N emissions, and soil biological activity. A review. Agron. Sustain. Dev. 35(3), 1021–1041 (2015)Google Scholar
  44. 44.
    Monlau, F., Sambusiti, C., Ficara, E., Aboulkas, A., Barakat, A., Carrère, H.: New opportunities for agricultural digestate valorization: current situation and perspectives. Energy Environ. Sci. 8(9), 2600–2621 (2015)Google Scholar
  45. 45.
    Nataraj, K.S., Rao, J.V., Jayaveera, K.N.: Synthesis and antimicrobial activity of new indole derivatives. Int. J. Chem. Sci. 8(1), 609–616 (2017)Google Scholar
  46. 46.
    Pérez-Bonilla, M., Salido, S., van Beek, T.A., et al.: Isolation and identification of radical scavengers in olive tree (Olea europaea) wood. J. Chromatogr. A. 1112(1–2), 311–318 (2006)Google Scholar
  47. 47.
    Pérez-Bonilla, M., Salido, S., Sánchez, A., Beek, T.A., Altarejos, J.: Effect of extraction conditions on the antioxidant activity of olive wood extracts. Int. J. Food Sci. 2013, 1–13 (2013)Google Scholar
  48. 48.
    Pintać, D., Majkić, T., Torović, L., Orčić, D., Beara, I., Simin, N., Dukić, N.M., Lesjak, M.: Solvent selection for efficient extraction of bioactive compounds from grape pomace. Ind. Crops Prod. 111, 379–390 (2018)Google Scholar
  49. 49.
    Popp, D., Harms, H., Sträuber, H.: The alkaloid gramine in the anaerobic digestion process—inhibition and adaptation of the methanogenic community. Appl. Microbiol. Biotechnol. 100(16), 7311–7322 (2016)Google Scholar
  50. 50.
    Riseberg, D.: Treating elderly patients with hormone receptor-positive advanced breast cancer. Clin. Med. Insights Oncol. 9, 65 (2015)Google Scholar
  51. 51.
    Santaladchaiyakit, Y., Srijaranai, S., Burakham, R.: Methodological aspects of sample preparation for the determination of carbamate residues: a review. J. Sep. Sci. 35(18), 2373–2389 (2012)Google Scholar
  52. 52.
    Scaglia, B., Pognani, M., Adani, F.: Evaluation of hormone-like activity of the dissolved organic matter fraction (DOM) of compost and digestate. Sci. Total Environ. 514, 314–321 (2015)Google Scholar
  53. 53.
    Schnitzer, M.I., Monreal, C.M., Jandl, G., Leinweber, P., Fransham, P.B.: The conversion of chicken manure to bio-oil by fast pyrolysis II. Analysis of chicken manure, bio-oils, and char by curie-point pyrolysis-gas chromatography/mass spectrometry (Cp Py-GC/MS). J. Environ. Sci. Health. 42(1), 79–95 (2007)Google Scholar
  54. 54.
    Shingare, M.S., Karale, B.K., Gill, C.H., Ganage, K.N., Bachute, M.T.: Synthesis and characterization of some 3-styryl-4-oxo-4H-1-benzopyrans. Indian J. Heterocycl. Chem. 9(2), 153–154 (1999)Google Scholar
  55. 55.
    Suárez-Estrella, F., Arcos-Nievas, M.A., López, M.J., Vargas-García, M.C., Moreno, J.: Biological control of plant pathogens by microorganisms isolated from agro-industrial composts. Biol. Control. 67(3), 509–515 (2013)Google Scholar
  56. 56.
    Sultana, B., Anwar, F., Ashraf, M.: Effect of extraction solvent/technique on the antioxidant activity of selected medicinal plant extracts. Molecules. 14(6), 2167–2180 (2009)Google Scholar
  57. 57.
    Tao, X., Shang, B., Dong, H., Chen, Y., Xin, H.: Effects of digestate from swine manure digester on in vitro growth of crop fungal pathogens: a laboratory study. Trans. ASABE 57(6), 1803–1810 (2014)Google Scholar
  58. 58.
    Tran, T.T.T.: Design and synthesis of potentially antibiofilm active compounds based on 2-aminoimidazoles and related structures (2015)Google Scholar
  59. 59.
    Vinas, P., Campillo, N., Lopez-Garcia, I., Hernandez-Cordoba, M.: Dispersive liquid–liquid micro extraction in food analysis. A critical review. Anal. Bioanal. Chem. 406(8), 2067–2099 (2014)Google Scholar
  60. 60.
    Wheelock, C.E., Severson, T.F., Hammock, B.D.: Synthesis of new carboxyl esterase inhibitors and evaluation of potency and water solubility. Chem. Res. Toxicol. 14(12), 1563–1572 (2001)Google Scholar
  61. 61.
    Xu, L., Yao, Q., Deng, J., Han, Z., Zhang, Y., Fu., Y., Guo, Q.: Renewable N-heterocycles production by thermocatalytic conversion and ammonization of biomass over ZSM-5. ACS Sustain. Chem. Eng. 3(11), 2890–2899 (2015)Google Scholar
  62. 62.
    Zhang, J., Gao, H., Peng, B., Li, S., Zhou, Z.: Comparison of the performance of conventional, temperature-controlled, and ultrasound-assisted ionic liquid dispersive liquid–liquid microextraction combined with high-performance liquid chromatography in analyzing pyrethroid pesticides in honey samples. J. Chromatogr. A 1218, 6621–6629 (2011)Google Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.College of Engineering, Key Laboratory for Clean Renewable Energy Utilization Technology, China Agricultural UniversityMinistry of AgricultureBeijingChina
  2. 2.Institute of Geographic Sciences and Natural Resources ResearchChinese Academy of SciencesBeijingChina
  3. 3.College of SciencesChina Agricultural UniversityBeijingChina
  4. 4.Aarhus Institute of Advanced StudiesAarhus UniversityAarhus CDenmark

Personalised recommendations