Non-thermal plasma and ultrasound-assisted open lactic acid fermentation of distillery stillage

  • Aleksandra Djukić-VukovićEmail author
  • Saša Lazović
  • Dragana Mladenović
  • Zorica Knežević-Jugović
  • Jelena Pejin
  • Ljiljana Mojović
Advances & Prospects in the field of Waste Management


Stillage is the main by-product of bioethanol production and the cost of its treatment significantly affects the economy of bioethanol production. A process of thermal sterilization before lactic acid fermentation (LAF) is energy demanding and is causing deterioration of valuable compounds in stillage. In this study, ultrasound (UT) and plasma (PT) treatments were used for microbial inactivation, and a significant reduction in the number of viable microorganisms in the stillage after PT and UT was observed. After application of treatment, LAF by Lactobacillus rhamnosus ATCC 7469 was initiated. The concentration of LA is used to quantify the efficiency of the stillage revalorization. The highest LA productivity of 1.21 g/Lh and yield of 0.82 g/g were obtained after PT, while UT of 10 min provided productivity of 1.02 g/Lh and LA yield of 0.69 g/g. The results were benchmarked against closed LAF. Around 20% better revalorization of stillage by PT was achieved when compared with conventional sterilization. In addition, an excellent L (+) LA stereoselectivity of 95.5% was attained after PT. From the aspect of energy efficiency, that of PT was three times lower than UT and almost ten times lower than thermal sterilization, but it is the most expensive due to the high consumption of gas which could reduce application of closed Ar atmosphere on larger scales. This way, a simpler and energy efficient process for LA production on stillage was accomplished by “open” fermentation.


Biorefinery Pretreatments Microbial inactivation Sterilization Lactic acid bacteria 



The authors want to acknowledge Milica Carević, PhD and Prof. Dejan Bezbradica, PhD, Faculty of Technology and Metallurgy, University of Belgrade, for their help in HPLC analysis of samples and Stevan Jovanović, ing., Insitute of Physics Belgrade, University of Belgrade, for his help in calculation of capital costs.

Funding information

This work was supported by Serbian Ministry of Education, Science and Technological Development, project number TR 31017, project no. I-1/2018 of Scientific and Technological Collaboration of Republic of Serbia and PR China and III 43007.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Abdel-Rahman MA, Tashiro Y, Sonomoto K (2013a) Recent advances in lactic acid production by microbial fermentation processes. Biotechnol Adv 31:877–902. CrossRefGoogle Scholar
  2. Abdel-Rahman MA, Tashiro Y, Zendo T, Sonomoto K (2013b) Improved lactic acid productivity by an open repeated batch fermentation system using Enterococcus mundtii QU 25. RSC Adv 3:8437–8445. CrossRefGoogle Scholar
  3. Anwar Saeed M, Ma H, Yue S, Wang Q, Tu M (2018) Concise review on ethanol production from food waste: development and sustainability. Environ Sci Pollut Res 25:28851–28863. CrossRefGoogle Scholar
  4. Baral NR, Shah A (2017) Techno-economic analysis of utilization of stillage from a cellulosic biorefinery. Fuel Process Technol 166:59–68. CrossRefGoogle Scholar
  5. Boudam MK, Moisan M, Saoudi B, Popovici C, Gherardi N, Massines F (2006) Bacterial spore inactivation by atmospheric-pressure plasmas in the presence or absence of UV photons as obtained with the same gas mixture. J Phys D Appl Phys 39:3494–3507. CrossRefGoogle Scholar
  6. Caderby E, Baumberger S, Hoareau W, Fargues C, Decloux M, Maillard M-N (2013) Sugar cane stillage: a potential source of natural antioxidants. J Agric Food Chem 61:11494–11501. CrossRefGoogle Scholar
  7. Djukić-Vuković AP, Mojović LV, Jokić BM, Nikolić SB, Pejin JD (2013) Lactic acid production on liquid distillery stillage by Lactobacillus rhamnosus immobilized onto zeolite. Bioresour Technol 135:454–458. CrossRefGoogle Scholar
  8. Djukić-Vuković AP, Mojović LV, Semenčenko VV, Radosavljević MM, Pejin JD, Kocić-Tanackov SD (2015) Effective valorisation of distillery stillage by integrated production of lactic acid and high quality feed. Food Res Int 73:75–80. CrossRefGoogle Scholar
  9. Djukić-Vuković A, Mladenović D, Radosavljević M, Kocić-Tanackov S, Pejin J, Mojović L (2016) Wastes from bioethanol and beer productions as substrates for l(+) lactic acid production - a comparative study. Waste Manag 48:478–482. CrossRefGoogle Scholar
  10. Du J, Chen L, Li J, Zuo R, Yang X, Chen H, Zhuang X, Tian S (2018) High-solids ethanol fermentation with single-stage methane anaerobic digestion for maximizing bioenergy conversion from a C4 grass (Pennisetum purpereum). Appl Energy 215:437–443. CrossRefGoogle Scholar
  11. European Parliament (2015) Directive (EU) 2015/1513. Accessed 5 February 2019
  12. Fuess LT, Garcia ML (2014) Implications of stillage land disposal: a critical review on the impacts of fertigation. J Environ Manag 145:210–229. CrossRefGoogle Scholar
  13. Gorbanev Y, O’Connell D, Chechik V (2016) Non-thermal plasma in contact with water: the origin of species. Chem Eur J 22:3496–3505. CrossRefGoogle Scholar
  14. Graves DB (2012) The emerging role of reactive oxygen and nitrogen species in redox biology and some implications for plasma applications to medicine and biology. J Phys D Appl Phys 45:263001–263042. CrossRefGoogle Scholar
  15. Herceg Z, Jambrak AR, Vukušić T, Stulić V, Stanzer D, Milošević S (2015) The effect of high-power ultrasound and gas phase plasma treatment on Aspergillus spp. and Penicillium spp. count in pure culture. J Appl Microbiol 118:132–141. CrossRefGoogle Scholar
  16. Hulsmans A, Joris K, Lambert N, Rediers H, Declerck P, Delaedt Y, Ollevier F, Liers S (2010) Evaluation of process parameters of ultrasonic treatment of bacterial suspensions in a pilot scale water disinfection system. Ultrason Sonochem 17:1004–1009CrossRefGoogle Scholar
  17. International Council on Clean Transportation (ICCT) (2018) Final recast RED II: Renewable Energy Directive for 2021–2030 in the European Union. Accessed 5 February 2019
  18. Issa-Zacharia A, Kamitani Y, Miwa N, Muhimbula H, Iwasaki K (2011) Application of slightly acidic electrolyzed water as a potential non-thermal food sanitizer for decontamination of fresh ready-to-eat vegetables and sprouts. Food Control 22:601–607. CrossRefGoogle Scholar
  19. Jake N, Kwik J, Franko M (2016) System and process for clarifying thin stillage. US patent no. US 20170114293A1. Accessed 5 February 2019
  20. Jovanović JR, Stefanović AB, Žuža MG, Jakovetić SM, Šekuljica NŽ, Bugarski BM, Knežević-Jugović ZD (2016) Improvement of antioxidant properties of egg white protein enzymatic hydrolysates by membrane ultrafiltration. Hem Ind 70:419–428. CrossRefGoogle Scholar
  21. Kang TS, Korber DR, Tanaka T (2014) Bioconversion of glycerol to 1,3-propanediol in thin stillage-based media by engineered Lactobacillus panis PM1. J Ind Microbiol Biotechnol 41:629–635. CrossRefGoogle Scholar
  22. Kasmi M, Hamdi M, Trabelsi I (2017) Processed milk waste recycling via thermal pretreatment and lactic acid bacteria fermentation. Environ Sci Pollut Res 24:13604–13613. CrossRefGoogle Scholar
  23. Koutinas A, Vlysidis A, Pleissner D, Kopsahelis N, Lopez Garcia I, Kookos IK, Papanikolaou S, Kwan TH, Lin CSK (2014) Valorization of industrial waste and by-product streams via fermentation for the production of chemicals and biopolymers. Chem Soc Rev 43:2587–2627. CrossRefGoogle Scholar
  24. Krásný I, Lapčík L, Lapčíková B, Greenwood RWR, Šafářová K, Rowson NA (2014) The effect of low temperature air plasma treatment on physico-chemical properties of kaolinite/polyethylene composites. Compos Part B Eng 59:293–299. CrossRefGoogle Scholar
  25. Leonelli C, Mason TJ (2010) Microwave and ultrasonic processing: now a realistic option for industry. Chem Eng Process Process Intensif 49:885–900. CrossRefGoogle Scholar
  26. Li T, Chen X, Chen J, Wu Q, Chen G-Q (2014) Open and continuous fermentation: products, conditions and bioprocess economy. Biotechnol J 9:1503–1511. CrossRefGoogle Scholar
  27. Liao X, Liu D, Xiang Q, Ahn J, Chen S, Ye X, Ding T (2017) Inactivation mechanisms of non-thermal plasma on microbes: a review. Food Control 75:83–91. CrossRefGoogle Scholar
  28. Liu B, Yang M, Qi B, Chen X, Su Z, Wan Y (2010) Optimizing l-(+)-lactic acid production by thermophile Lactobacillus plantarum As. 1.3 using alternative nitrogen sources with response surface method. Biochem Eng J 52(2–3):212–219Google Scholar
  29. Ma R, Wang G, Tian Y, Wang K, Zhang J, Fang J (2015) Non-thermal plasma-activated water inactivation of food-borne pathogen on fresh produce. J Hazard Mater 300:643–651. CrossRefGoogle Scholar
  30. Mai-Prochnow A, Clauson M, Hong J, Murphy AB (2016) Gram positive and gram negative bacteria differ in their sensitivity to cold plasma. Sci Rep 6:38610. CrossRefGoogle Scholar
  31. Mladenović D, Pejin J, Kocić-Tanackov S, Djukić-Vuković A, Mojović L (2018) Enhanced lactic acid production by adaptive evolution of Lactobacillus paracasei on agro-industrial substrate. Appl Biochem Biotechnol.
  32. Moestedt J, Påledal S, Schnürer A, Nordell E (2013) Biogas production from thin stillage on an industrial scale—experience and optimisation. Energies 6:5642–5655. CrossRefGoogle Scholar
  33. Noukeu NA, Gouado I, Priso RJ, Ndongo D, Taffouo VD, Dibong SD, Ekodeck GE (2016) Characterization of effluent from food processing industries and stillage treatment trial with Eichhornia crassipes (Mart.) and Panicum maximum (Jacq). Water Resour Ind 16:1–18. CrossRefGoogle Scholar
  34. Ouyang J, Ma R, Zheng Z, Cai C, Zhang M, Jiang T (2013) Open fermentative production of l-lactic acid by Bacillus sp. strain NL01 using lignocellulosic hydrolyzates as low-cost raw material. Bioresour Technol 135:475–480. CrossRefGoogle Scholar
  35. Pejin J, Radosavljević M, Kocić-Tanackov S, Mladenović D, Djukić-Vuković A, Mojović L (2017) Fed-batch l-(+)-lactic acid fermentation of brewer’s spent grain hydrolysate. J Inst Brew 123:537–543. CrossRefGoogle Scholar
  36. Pleissner D, Demichelis F, Mariano S, Fiore S, Navarro Gutiérrez IM, Schneider R, Venus J (2017) Direct production of lactic acid based on simultaneous saccharification and fermentation of mixed restaurant food waste. J Clean Prod 143:615–623. CrossRefGoogle Scholar
  37. Puač N, Miletić M, Mojović M, Popović-Bijelić A, Vuković D, Miličić B, Maletić D, Lazović S, Malović G, Petrović ZL (2015) Sterilization of bacteria suspensions and identification of radicals deposited during plasma treatment. Open Chem 13:332–338. Google Scholar
  38. Purevdorj D, Igura N, Hayakawa I, Ariyada O (2002) Inactivation of Escherichia coli by microwave induced low temperature argon plasma treatments. J Food Eng 53:341–346. CrossRefGoogle Scholar
  39. Ramazzina I, Berardinelli A, Rizzi F, Tappi S, Ragni L, Sacchetti G, Rocculi P (2015) Effect of cold plasma treatment on physico-chemical parameters and antioxidant activity of minimally processed kiwifruit. Postharvest Biol Technol 107:55–65. CrossRefGoogle Scholar
  40. Research Grand View (2017) Lactic acid market & polylactic acid (PLA) market, industry report 2025, Market research report. . Accessed 5 February 2019
  41. Sakai K, Murata Y, Yamazumi H, Tau Y, Mori M, Moriguchi M, Shirai Y (2000) Selective proliferation of lactic acid bacteria and accumulation of lactic acid during open fermentation of kitchen refuse with intermittent pH adjustment. Food Sci Technol Res 6:140–145. CrossRefGoogle Scholar
  42. Sangave PC, Pandit AB (2004) Ultrasound pre-treatment for enhanced biodegradability of the distillery wastewater. Ultrason Sonochem 11:197–203. CrossRefGoogle Scholar
  43. Sayedin F, Kermanshahi-pour A, He S(Q) (2018) Anaerobic digestion of thin stillage of corn ethanol plant in a novel anaerobic baffled reactor. Waste Manag 78:541–552. CrossRefGoogle Scholar
  44. Shan L, Liu J, Yu Y, Ambuchi JJ, Feng Y (2016) Characterization of persistent colors and decolorization of effluent from biologically treated cellulosic ethanol production wastewater. Environ Sci Pollut Res 23:10215–10222. CrossRefGoogle Scholar
  45. Srivastava A, Poonia A, Tripathi AD, Singh RP, Srivastava SK (2014) Optimization of nutritional supplements for enhanced lactic acid production utilizing sugar refinery by-products. Ann Microbiol 64:1211–1221. CrossRefGoogle Scholar
  46. Surowsky B, Schlüter O, Knorr D (2015) Interactions of non-thermal atmospheric pressure plasma with solid and liquid food systems: a review. Food Eng Rev 7:82–108. CrossRefGoogle Scholar
  47. Tang J, Wang X, Hu Y, Zhang Y, Li Y (2016) Lactic acid fermentation from food waste with indigenous microbiota: effects of pH, temperature and high OLR. Waste Manag 52:278–285. CrossRefGoogle Scholar
  48. Tresp H, Hammer MU, Winter J, Weltmann KD, Reuter S (2013) Quantitative detection of plasma-generated radicals in liquids by electron paramagnetic resonance spectroscopy. J Phys D Appl Phys 46:435401. CrossRefGoogle Scholar
  49. Uchiyama H, Zhao QL, Hassan MA, Andocs G, Nojima N, Takeda K, Ishikawa K, Hori M, Kondo T (2015) EPR-spin trapping and flow cytometric studies of free radicals generated using cold atmospheric argon plasma and X-ray irradiation in aqueous solutions and intracellular milieu. PLoS One 10:e0136956. CrossRefGoogle Scholar
  50. Vhangani LN, Van Wyk J (2016) Antioxidant activity of Maillard reaction products (MRPs) in a lipid-rich model system. Food Chem 208:301–308. CrossRefGoogle Scholar
  51. West TP (2011) Malic acid production from thin stillage by Aspergillus species. Biotechnol Lett 33:2463–2467. CrossRefGoogle Scholar
  52. Wilkie AC, Riedesel KJ, Owens JM (2000) Stillage characterization and anaerobic treatment of ethanol stillage from conventional and cellulosic feedstocks. Biomass Bioenergy 19:63–102. CrossRefGoogle Scholar
  53. Wu JM, Liu RH (2013) Cost-effective production of bacterial cellulose in static cultures using distillery wastewater. J Biosci Bioeng 115:284–290. CrossRefGoogle Scholar
  54. Yusaf T, Al-Juboori RA (2014) Alternative methods of microorganism disruption for agricultural applications. Appl Energy 114:909–923. CrossRefGoogle Scholar
  55. Zaplotnik R, Bišćan M, Kregar Z, Cvelbar U, Mozetič M, Milošević S (2015) Influence of a sample surface on single electrode atmospheric plasma jet parameters. Spectrochim Acta B At Spectrosc 103–104:124–130. CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Department of Biochemical Engineering and Biotechnology, Faculty of Technology and MetallurgyUniversity of BelgradeBelgradeSerbia
  2. 2.Institute of Physics BelgradeUniversity of BelgradeBelgradeSerbia
  3. 3.Faculty of TechnologyUniversity of Novi SadNovi SadSerbia

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