Simple solvatochromic spectroscopic quantification of long-chain fatty acids for biological toxicity assay in biogas plants

  • Takuro KobayashiEmail author
  • Hidetoshi Kuramochi
  • Kai-Qin Xu
  • Kouji Maeda
Resource Recovery from Wastewater, Solid Waste and Waste Gas: Engineering and Management Aspects


Oily organic waste is a promising feedstock for anaerobic co-digestion. Free long-chain fatty acids (LCFAs) produced from lipids can inhibit methanogenic consortia, so optimal control of LCFA concentration is the key to successful operation of co-digestion. Most LCFAs are present in the solid phase, making them difficult to be detected and monitored. This study proposes a simple and easy method for detecting LCFAs in both the liquid and solid phases of anaerobic digestate by combining liquid–liquid extraction followed by solid-phase extraction (SPE) and spectrophotometric analysis. The extraction procedure successfully removed impurities that interfere with the absorbance spectrum and ensured high recovery rates of LCFAs. The utility of the pretreatment used for the extraction was discussed using thermodynamic analysis and calculations of phase equilibrium for the solvent extraction system. The absorbance spectrum shift of pyridinium N-phenolate betaine (PNPB) dye-stained solution showed a good correlation with LCFA concentration and enabled highly sensitive measurements. Good quantification was demonstrated in experiments using various digestate samples obtained from the laboratory, pilot, and full-scale reactors.


Anaerobic digestion Grease trap waste Inhibitory substance Monitoring Simplified analysis method 



We would like to thank Mr. Yuji Yamazaki, Tomoyuki Nara, and Toshitaka Kato of Takenaka Corporation, Japan, for their valuable help in the collection of digestate samples.

Supplementary material

11356_2019_6532_MOESM1_ESM.docx (138 kb)
ESM 1 (DOCX 137 kb)


  1. Affes M, Aloui F, Hadrich F, Loukil S, Sayadi S (2017) Effect of bacterial lipase on anaerobic co-digestion of slaughterhouse wastewater and grease in batch condition and continuous fixed-bed reactor. Lipids Health Dis 16(1):195CrossRefGoogle Scholar
  2. Cavaleiro AJ, Salvador AF, Alves JI, Alves M (2009) Continuous high rate anaerobic treatment of oleic acid based wastewater is possible after a step feeding start-up. Environ Sci Technol 43(8):2931–2936CrossRefGoogle Scholar
  3. Dong T, Knoshaug EP, Pienkos PT, Laurens LML (2016) Lipid recovery from wet oleaginous microbial biomass for biofuel production: a critical review. Appl Energy 177:879–895CrossRefGoogle Scholar
  4. Douhal A (1994) Photophysics of Nile Blue A in proton-accepting and electron-donating solvents. Phys Chem 98(50):13131–13137CrossRefGoogle Scholar
  5. Flurkey WH (2005) Use of solid phase extraction in the biochemistry laboratory to separate different lipids. Biochem Mol Biol Educ 33(5):357–360CrossRefGoogle Scholar
  6. Fredenslund A, Jones RL, Prausnitz JM (1975) Group-contribution estimation of activity coefficients in non-ideal liquid mixtures. AICHE J 21:1086–1099CrossRefGoogle Scholar
  7. Jiney J, Kevin B (2006) Benzophenoxazine-based fluorescent dyes for labeling biomolecules. Tetrahedron. 62(48):11021CrossRefGoogle Scholar
  8. J. Hunter Long, Tarek N. Aziz, Francis L. de los Reyes, Joel J. Ducoste, (2012) Anaerobic co-digestion of fat, oil, and grease (FOG): A review of gas production and process limitations. Process Safety and Environmental Protection 90 (3):231-245Google Scholar
  9. Kanicky JR, Shah DO (2002) Effect of degree, type, and position of unsaturation on the pKa of long-chain fatty acids. J Colloid Interface Sci 256(1):201–207CrossRefGoogle Scholar
  10. Kanicky JR, Poniatowski AF, Mehta NR, Shah DO (2000) Cooperativity among molecules at interfaces in relation to various technological processes: effect of chain length on the pKa of fatty acid salt solutions. Langmuir 16(1):172–177CrossRefGoogle Scholar
  11. Kirschman HD, Pomeroy R (1949) Determination of oil in oil-field waste waters. Anal Chem 21(7):793–797CrossRefGoogle Scholar
  12. Kleyböcker A, Liebrich M, Verstraete W, Kraume M, Würdemann H (2012) Early warning indicators for process failure due to organic overloading by rapeseed oil in one-stage continuously stirred tank reactor, sewage sludge and waste digesters. Bioresour Technol 123:534–541CrossRefGoogle Scholar
  13. Kobayashi T, Kuramochi H, Maeda K, Tsuji T, Xu KQ (2014) Dual-fuel production from restaurant grease trap waste: bio-fuel oil extraction and anaerobic methane production from the post-extracted residue. Bioresour Technol 169:134–142CrossRefGoogle Scholar
  14. Kobayashi T, Kuramochi H, Xu KQ (2017a) Variable oil properties and biome thane production of grease trap waste derived from different resources. Int Biodeterior Biodegrad 119:273–281CrossRefGoogle Scholar
  15. Kobayashi T, Kuramochi H, Maeda K, Xu KQ (2017b) A simple method for the detection of long-chain fatty acids in an anaerobic digestate using a quartz crystal sensor. Energies 10:19CrossRefGoogle Scholar
  16. Kubinyi M, Brátán J, Grofcsik A, Biczók L, Poór B, Bitter I, Grün A, Bogáti B, Tóth K (2002) Proton transfer and supramolecular complex formation between Nile blue and tetraundecylcalix[4]resorcinarene—a fluorescence spectroscopic study. J Chem Soc Perkin Trans 2:1784–1789CrossRefGoogle Scholar
  17. Kuramochi H, Maeda K, Kato S, Osako M, Nakamura K, Sakai S (2009) Application of UNIFAC models for prediction of vapor-liquid and liquid-liquid equilibria relevant to separation and purification processes of crude BDF. Fuel 88:1472–1477CrossRefGoogle Scholar
  18. Lalman JA, Bagley DM (2001) Anaerobic degradation and inhibitory effects of linoleic acid. Water Res 34(17):4220–4228Google Scholar
  19. Li Y, Naghdi FG, Garg S, Adarme-Vega TC, Thurecht KJ, Ghafor WA, Tannock S, Schenk PM (2014) A comparative study: the impact of different lipid extraction methods on current microalgal lipid research. Microb Cell Factories 13:14CrossRefGoogle Scholar
  20. Linares P, Luque de Castro MD, Valcárcel M (1989) Direct automatic determination of free acidity in oils by flow-injection analysis. Anal Chim Acta 225:431–436CrossRefGoogle Scholar
  21. Luostarinen S, Luste S, Sillanpää M (2009) Increased biogas production at wastewater treatment plants through co-digestion of sewage sludge with grease trap sludge from a meat processing plant. Bioresour Technol 100:79–85CrossRefGoogle Scholar
  22. Magnussen T, Rasmussen P, Fredenslund A (1981) UNIFAC parameter table for prediction of liquid-liquid equilibria. Ind Eng Chem Process Des Dev 20:331–339CrossRefGoogle Scholar
  23. Mariotti E, Mascini M (2001) Determination of extra virgin olive oil acidity by FIA-titration. Food Chem 73:235–238CrossRefGoogle Scholar
  24. M.A. Pereira, A.J. Cavaleiro, M. Mota, M.M. Alves, (2003) Accumulation of long chain fatty acids onto anaerobic sludge under steady state and shock loading conditions: effect on acetogenic and methanogenic activity. Water Science and Technology 48 (6):33-40Google Scholar
  25. Neves L, Pereira MA, Mota M, Alves MM (2009) Detection and quantification of long chain fatty acids in liquid and solid samples and its relevance to understand anaerobic digestion of lipids. Bioresour Technol 100(1):91–96CrossRefGoogle Scholar
  26. Oren JJ, MacKay GDM (1977) Electrolyte and pH effect on emulsion stability of water-in-petroleum oils. Fuel 56(4):382–384CrossRefGoogle Scholar
  27. Paul GW, Marc de Chazal LE (1967) Interfacial tensions in ternary liquid-liquid systems. J Chem Eng Data 12(1):105–107CrossRefGoogle Scholar
  28. Pereira MA, Pires OC, Mota M, Alves MM 2005(2005) Anaerobic biodegradation of oleic and palmitic acids: evidence of mass transfer limitations caused by long chain fatty acid accumulation onto the anaerobic sludge. Biotechnol Bioeng 92(1):15–23CrossRefGoogle Scholar
  29. Reichardt C (1994) Solvatochromic dyes as solvent polarity indicators. Chem Rev 94(8):2319–2358CrossRefGoogle Scholar
  30. Salama ES, Saha S, Kurade MB, Dev S, Chang SW, Jeon BH (2019) Recent trends in anaerobic co-digestion: fat, oil, and grease (FOG) for enhanced biomethanation. Prog Energy Combust Sci 70:22–42CrossRefGoogle Scholar
  31. Schwarzenbach RP, Gschwend PM, Imboden DM (2003) Environmental organic chemistry, 2nd edn. John Wiley, HobokenGoogle Scholar
  32. Sousa DZ, Salvador AF, Ramos J, Guedes AP, Barbosa S, Stams AJ, Alves MM, Pereira MA (2013) Activity and viability of methanogens in anaerobic digestion of unsaturated and saturated long-chain fatty acids. Appl Environ Microbiol 79(14):4239–4245CrossRefGoogle Scholar
  33. Sun SY, Wang D, Yan J, Qiao W, Wang W, Zhu T (2014) Effects of lipid concentration on anaerobic co-digestion of municipal biomass wastes. Waste Manag 34:1025–1034CrossRefGoogle Scholar
  34. Wu LJ, Kobayashi T, Kuramochi H, Li YY, Xu KQ, Lv Y (2018) High loading anaerobic co-digestion of food waste and grease trap waste: determination of the limit and lipid/long chain fatty acid conversion. Chem Eng J 338:422–431CrossRefGoogle Scholar
  35. Yau YH, Rudolph V, Lo CC, Wu KC (2018) Restaurant oil and grease management in Hong Kong. Environ Sci Pollut Res.

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Center for Material Cycles and Waste Management ResearchNational Institute for Environmental StudiesTsukubaJapan
  2. 2.Department of Chemical Engineering & Materials ScienceUniversity of HyogoHimejiJapan

Personalised recommendations