Microbial Succession and Identification of Effective Indigenous Pectinolytic Yeasts From Orange Juice Processing Wastewater


Although the management of citrus residues is of concern, their valorization is a challenging alternative. In this work, raw orange juice processing wastewater was spontaneously fermented to determine the succession in microbial communities and identify new indigenous microbial strains of high pectinolytic activity. Pectinolytic strains were isolated using citrus pectin as growth substrate. A shift in fungal population observed during spontaneous fermentation, where the abundances of Hanseniaspora, Cystofilobasidium and Meyerozyma were significantly increased, whereas Pichia fermentans followed by Saccharomyces spp. became the predominant taxa in the fermented wastewater. A significant reduction in the relative abundance of homofermentative aerococci and an increase in the relative abundance of heterofermentative leuconostocs and lactobacilli occurred during fermentation, while enterococci were predominant throughout the fermentation process. Regarding ecological indices, opposite changes took place in the bacterial compared to fungal community structure, but in a less pronounced degree. Examination of beta-diversity revealed the key role of Pichia species in driving fungal community structure during fermentation. P. fermentans, the predominant taxon in the fermented wastewater, was found to be the most effective pectinolytic microorganism. Pectin hydrolysis, carbohydrate fermentation to ethanol, lactic acid and acetate, and citric acid fermentation were the most important factors, influencing microbial community structure.

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  1. 1.

    Radenkovs, V., Juhnevica-Radenkova, K., Górnaś, P., Seglina, D.: Non-waste technology through the enzymatic hydrolysis of agro-industrial by-products. Trends Food Sci. Technol. 77, 64–76 (2018)

    Article  Google Scholar 

  2. 2.

    Okino Delgado, C.H., Fleuri, L.F.: Orange and mango by-products: agro-industrial waste as source of bioactive compounds and botanical versus commercial description - a review. Food Rev. Int. 32(1), 1–14 (2016)

    Article  Google Scholar 

  3. 3.

    FAO: Citrus Fruit - Fresh and Processed, Statistical Bulletin 2016. Food and Agriculture Organization of the United Nations, Rome (2017)

    Google Scholar 

  4. 4.

    Angelidis, M.O., Markantonatos, P.O., Bacalis, NCh., Albanis, T.A.: Seasonal fluctuations of nutrients and pesticides in the basin of Evrotas river, Greece. J. Environ. Sci. Health A 31(2), 387–410 (1996)

    Google Scholar 

  5. 5.

    Lotito, A.M., De Sanctis, M., Pastore, C., Di Iaconi, C.: Biomethanization of citrus waste: effect of waste characteristics and of storage on treatability and evaluation of limonene degradation. J. Environ. Manage. 215, 366–376 (2018)

    Article  Google Scholar 

  6. 6.

    Mamma, D., Christakopoulos, P.: Biotransformation of citrus by-products into value added products. Waste Biomass Valoriz. 5(4), 529–549 (2014)

    Article  Google Scholar 

  7. 7.

    Martínez, R., Torres, P., Meneses, M.A., Figueroa, J.G., Pérez-Álvarez, J.A., Viuda-Martos, M.: Chemical, technological and in vitro antioxidant properties of mango, guava, pineapple and passion fruit dietary fibre concentrate. Food Chem. 135(3), 1520–1526 (2012)

    Article  Google Scholar 

  8. 8.

    Round, A.N., Rigby, N.M., MacDougall, A.J., Morris, V.J.: A new view of pectin structure revealed by acid hydrolysis and atomic force microscopy. Carbohydr. Res. 345(4), 487–497 (2010)

    Article  Google Scholar 

  9. 9.

    Yu, P., Zhang, Y., Gu, D.: Production optimization of a heat-tolerant alkaline pectinase from Bacillus subtilis ZGL14 and its purification and characterization. Bioengineered 8(5), 613–623 (2017)

    Article  Google Scholar 

  10. 10.

    Flutto, L.: Pectin: food use. In: Caballero, B., Finglas, P., Toldra, F. (eds.) Encyclopedia of Food Sciences and Nutrition, pp. 4449–4456. Academic press, New York (2003)

    Google Scholar 

  11. 11.

    Wang, D., Yeats, T.H., Uluisik, S., Rose, J.K.C., Seymour, G.B.: Fruit softening: revisiting the role of pectin. Trends Plant Sci. 23(4), 302–310 (2018)

    Article  Google Scholar 

  12. 12.

    Passos, F., Hom-Diaz, A., Blanquez, P., Vicent, T., Ferrer, I.: Improving biogas production from microalgae by enzymatic pretreatment. Bioresour. Technol. 199, 347–351 (2016)

    Article  Google Scholar 

  13. 13.

    Mahmoud, K.F., Abo-Elmagd, H.I., Housseiny, M.M.: Micro- and nano-capsulated fungal pectinase with outstanding capabilities of eliminating turbidity in freshly produced juice. Food Sci. Technol. Int. 24(4), 330–340 (2018)

    Article  Google Scholar 

  14. 14.

    Kuo, C.H., Huang, C.Y., Shieh, C.J., Wang, H.M.D., Tseng, C.Y.: Hydrolysis of orange peel with cellulase and pectinase to produce bacterial cellulose using Gluconacetobacter xylinus. Waste Biomass Valoriz. 10(1), 85–93 (2019)

    Article  Google Scholar 

  15. 15.

    Silva, D., Tokuioshi, K., Martins, E.D.S., Da Silva, R., Gomes, E.: Production of pectinase by solid-state fermentation with Penicillium viridicatum RFC3. Process Biochem. 40(8), 2885–2889 (2005)

    Article  Google Scholar 

  16. 16.

    Irshad, M., Anwar, Z., Mahmood, Z., Aqil, T., Mehmmod, S., Nawaz, H.: Bio-processing of agro-industrial waste orange peel for induced production of pectinase by Trichoderma viridi; its purification and characterization. Turk. J. Biochem. 39(1), 9–18 (2014)

    Article  Google Scholar 

  17. 17.

    Pili, J., Danielli, A., Zeni, J., Trentini, M.M.S., Cansian, R.L., Toniazzo, G., Valduga, E.: Utilization of orange peel, corn steep liquor, and parboiled rice water in the production of polygalacturonase from Aspergillus niger. Ind. Biotechnol. 11(5), 284–291 (2015)

    Article  Google Scholar 

  18. 18.

    Ortiz, G.E., Ponce-Mora, M.C., Noseda, D.G., Cazabat, G., Saravalli, C., López, M.C., Gil, G.P., Blasco, M., Albertó, E.O.: Pectinase production by Aspergillus giganteus in solid-state fermentation: optimization, scale-up, biochemical characterization and its application in olive-oil extraction. J. Ind. Microbiol. Biotechnol. 44(2), 197–211 (2017)

    Article  Google Scholar 

  19. 19.

    Lalitha Kumari, B., Sudhakar, P., Hemamalini, K., Satya Sree, N., Vijetha, P.: Studies on pectinase production by Bacillus subtilis using agro-industrial wastes. Res. J. Pharm. Biol. Chem. Sci. 5(6), 330–339 (2014)

    Google Scholar 

  20. 20.

    Rehman, H.U., Aman, A., Nawaz, M.A., Qader, S.A.U.: Characterization of pectin degrading polygalacturonase produced by Bacillus licheniformis KIBGE-IB21. Food Hydrocoll. 43, 819–824 (2015)

    Article  Google Scholar 

  21. 21.

    Bibi, N., Ali, S., Tabassum, R.: Statistical optimization of pectinase biosynthesis from orange peel by Bacillus licheniformis using submerged fermentation. Waste Biomass Valoriz. 7(3), 467–481 (2016)

    Article  Google Scholar 

  22. 22.

    Clesceri, L.S., Greenberg, A.E., Eaton, A.D.: Standard methods for the examination of water and wastewater. American Public Health Association (APHA), Washington DC (1998)

    Google Scholar 

  23. 23.

    Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F.: Colorimetric method for determination of sugars and related substances. Anal. Chem. 28(3), 350–356 (1956)

    Article  Google Scholar 

  24. 24.

    Bradford, M.M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72(1–2), 248–254 (1976)

    Article  Google Scholar 

  25. 25.

    Madrid, J., Martínez-Teruel, A., Hernández, F., Megías, M.D.: A comparative study on the determination of lactic acid in silage juice by colorimetric, high‐performance liquid chromatography and enzymatic methods. J. Sci. Food Agric. 79(12), 1722–1726 (1999)

    Article  Google Scholar 

  26. 26.

    Ntougias, S.: Phylogenetic identification and enzyme activities of indigenous bacteria from a landfill stabilization pond. Environ. Process. 3(2), 341–352 (2016)

    Article  Google Scholar 

  27. 27.

    Adeleke, A.J., Odunfa, S.A., Olanbiwonninu, A., Owoseni, M.C.: Production of cellulase and pectinase from orange peels by fungi. Nat. Sci. 10(5), 107–112 (2012)

    Google Scholar 

  28. 28.

    Miller, G.L.: Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 32, 426–428 (1959)

    Article  Google Scholar 

  29. 29.

    Tadakittisarn, S., Songpim, M., Vaithanomsat, P.: Polygalacturonase and pectate lyase activity during ripening of Kluay Hom Thong fruit. Kasetsart J. (Nat. Sci.) 43, 267–274 (2009)

    Google Scholar 

  30. 30.

    Huang, X., Madan, A.: CAP3: a DNA sequence assembly program. Genome Res. 9(9), 868–877 (1999)

    Article  Google Scholar 

  31. 31.

    Sievers, F., Wilm, A., Dineen, D., Gibson, T.J., Karplus, K., Li, W., Lopez, R., McWilliam, H., Remmert, M., Söding, J., Thompson, J.D., Higgins, D.G.: Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 (2011)

    Article  Google Scholar 

  32. 32.

    Kumar, S., Stecher, G., Li, M., Knyaz, C., Tamura, K.: MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 35(6), 1547–1549 (2018)

    Article  Google Scholar 

  33. 33.

    Kimura, M.: A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16, 111–120 (1980)

    Article  Google Scholar 

  34. 34.

    Saitou, N., Nei, M.: The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4(4), 406–425 (1987)

    Google Scholar 

  35. 35.

    Navrozidou, E., Remmas, N., Melidis, P., Karpouzas, D.G., Tsiamis, G., Ntougias, S.: Biodegradation potential and diversity of diclofenac-degrading microbiota in an immobilized cell biofilter. Processes 7(9), 554 (2019)

    Article  Google Scholar 

  36. 36.

    Remmas, N., Melidis, P., Zerva, I., Kristoffersen, J.B., Nikolaki, S., Tsiamis, G., Ntougias, S.: Dominance of candidate Saccharibacteria in a membrane bioreactor treating medium age landfill leachate: effects of organic load on microbial communities, hydrolytic potential and extracellular polymeric substances. Bioresour. Technol. 238, 48–56 (2017)

    Article  Google Scholar 

  37. 37.

    Cole, J.R., Wang, Q., Fish, J.A., Chai, B., McGarrell, D.M., Sun, Y., Brown, C.T., Porras-Alfaro, A., Kuske, C.R., Tiedje, J.M.: Ribosomal database project: data and tools for high throughput rRNA analysis. Nucleic Acids Res. 42(D1), D633–D642 (2014)

    Article  Google Scholar 

  38. 38.

    Masella, A.P., Bartram, A.K., Truszkowski, J.M., Brown, D.G., Neufeld, J.D.: PANDAseq: paired-end assembler for Illumina sequences. BMC Bioinf. 13(1), 31 (2012)

    Article  Google Scholar 

  39. 39.

    Edgar, R.C.: UNOISE2: improved error-correction for Illumina 16S and ITS amplicon sequencing. bioRxiv (2016). https://doi.org/10.1101/081257

    Article  Google Scholar 

  40. 40.

    Sebastian, A., Herdegen, M., Migalska, M., Radwan, J.: AmpliSAS: a web server for multilocus genotyping using next-generation amplicon sequencing data. Mol. Ecol. Resour. 16, 498–510 (2016)

    Article  Google Scholar 

  41. 41.

    Dhariwal, A., Chong, J., Habib, S., King, I., Agellon, L.B., Xia, J.: MicrobiomeAnalyst - a web-based tool for comprehensive statistical, visual and meta-analysis of microbiome data. Nucleic Acids Res. 45, W180–W188 (2017)

    Article  Google Scholar 

  42. 42.

    Rodrigues, P.H.M., Borgatti, L.M.O., Gomes, R.W., Passini, R., Meyer, P.M.: Effect of increasing levels of citrus pulp on the fermentation quality and nutritive value of elephantgrass silage. Rev. Bras. de Zootec. 34(4), 1138–1145 (2005)

    Article  Google Scholar 

  43. 43.

    Liu, R., Zhang, Q., Chen, F., Zhang, X.: Analysis of culturable yeast diversity in spontaneously fermented orange wine, orange peel and orangery soil of a Ponkan plantation in China. Ann. Microbiol. 65(4), 2387–2391 (2015)

    Article  Google Scholar 

  44. 44.

    Hu, L., Wang, J., Ji, X., Liu, R., Chen, F., Zhang, X.: Selection of non-Saccharomyces yeasts for orange wine fermentation based on their enological traits and volatile compounds formation. Int. J. Food Sci. Technol. 55(10), 4001–4012 (2018)

    Article  Google Scholar 

  45. 45.

    Zerva, I., Remmas, N., Ntougias, S.: Diversity and biotechnological potential of xylan-degrading microorganisms from orange juice processing waste. Water 11(2), 274 (2019)

    Article  Google Scholar 

  46. 46.

    Renard, A., Gómez di Marco, P., Egea-Cortines, M., Weiss, J.: Application of whole genome amplification and quantitative PCR for detection and quantification of spoilage yeasts in orange juice. Int. J. Food Microbiol. 126(1–2), 195–201 (2008)

    Article  Google Scholar 

  47. 47.

    Albertin, W., Miot-Sertier, C., Bely, M., Marullo, P., Coulon, J., Moine, V., Colonna-Ceccaldi, B., Masneuf-Pomarede, I.: Oenological prefermentation practices strongly impact yeast population dynamics and alcoholic fermentation kinetics in Chardonnay grape must. Int. J. Food Microbiol. 178, 87–97 (2014)

    Article  Google Scholar 

  48. 48.

    Luan, Y., Zhang, B.Q., Duan, C.Q., Yan, G.L.: Effects of different pre-fermentation cold maceration time on aroma compounds of Saccharomyces cerevisiae co-fermentation with Hanseniaspora opuntiae or Pichia kudriavzevii. LWT-Food Sci. Technol. 92, 177–186 (2018)

    Article  Google Scholar 

  49. 49.

    Cavello, I., Albanesi, A., Fratebianchi, D., Garmedia, G., Vero, S., Cavalitto, S.: Pectinolytic yeasts from cold environments: novel findings of Guehomyces pullulans, Cystofilobasidium infirmominiatum and Cryptococcus adeliensis producing pectinases. Extremophiles 21(2), 319–329 (2017)

    Article  Google Scholar 

  50. 50.

    Ling, L., Li, Z., Jiao, Z., Zhang, X., Ma, W., Feng, J., Zhang, J., Lu, L.: Identification of novel endophytic yeast strains from tangerine peel. Curr. Microbiol. 76(9), 1066–1072 (2019)

    Article  Google Scholar 

  51. 51.

    Arias, C.R., Burns, J.K., Friedrich, L.M., Goodrich, R.M., Parish, M.E.: Yeast species associated with orange juice: evaluation of different identification methods. Appl. Environ. Microbiol. 68(4), 1955–1961 (2002)

    Article  Google Scholar 

  52. 52.

    Udota, H.I.J., Urua, E.E.: Comparative analysis of fungal growth in commercially and laboratory prepared fruit juices - Using orange and pineapple as a case study. J. Ind. Pollut. Control 26(2), 125–130 (2010)

    Google Scholar 

  53. 53.

    Samagaci, L., Ouattara, H., Niamké, S., Lemaire, M.: Pichia kudrazevii and Candida nitrativorans are the most well-adapted and relevant yeast species fermenting cocoa in Agneby-Tiassa, a local Ivorian cocoa producing region. Food Res. Int. 89, 773–780 (2016)

    Article  Google Scholar 

  54. 54.

    Huang, Z.R., Guo, W.L., Zhou, W.B., Li, L., Xu, J.X., Hong, J.L., Liu, H.P., Zeng, F., Bai, W.D., Liu, B., Ni, L., Rao, P.F., Lv, X.C.: Microbial communities and volatile metabolites in different traditional fermentation starters used for Hong Qu glutinous rice wine. Food Res. Int. 121, 593–603 (2019)

    Article  Google Scholar 

  55. 55.

    Camu, N., De Winter, T., Verbrugghe, K., Cleenwerck, I., Vandamme, P., Takrama, J.S., Vancanneyt, M., De Vuyst, L.: Dynamics and biodiversity of populations of lactic acid bacteria and acetic acid bacteria involved in spontaneous heap fermentation of cocoa beans in Ghana. Appl. Environ. Microbiol. 73(6), 1809–1824 (2007)

    Article  Google Scholar 

  56. 56.

    Zhong, W., Chen, T., Yang, H., Li, E.: Isolation and selection of non-Saccharomyces yeasts being capable of degrading citric acid and evaluation its effect on kiwifruit wine fermentation. Fermentation 6(1), 25 (2020)

    Article  Google Scholar 

  57. 57.

    Xia, X., Li, G., Ye, X., Kan, J., Zheng, J.: Dynamic changes of bacteria community diversity during the fermentation of pickled Ma bamboo shoots. Zhongguo Shipin Xuebao 15(11), 206–211 (2015)

    Google Scholar 

  58. 58.

    de MedinaFigueroa, R., Oliver, G., Benito de Cárdenas, I.L.: Influence of temperature on flavour compound production from citrate by Lactobacillus rhamnosus ATCC 7469. Microbiol. Res. 155(4), 257–262 (2001)

    Article  Google Scholar 

  59. 59.

    Ramsey, M., Hartke, A., Huycke, M.: The physiology and metabolism of Enterococci. In: Gilmore, M.S., Clewell, D.B., Ike, Y., Shankar, Ν (eds.) Enterococci: From Commensals to Leading Causes of Drug Resistant Infection, pp. 581–635. Massachusetts Eye and Ear Infirmary, Boston (2014)

    Google Scholar 

  60. 60.

    Hanchi, H., Mottawea, W., Sebei, K., Hammami, R.: The genus Enterococcus: between probiotic potential and safety concerns-an update. Front. Microbiol. 9, 1791 (2018)

    Article  Google Scholar 

  61. 61.

    Hutzler, M., Riedl, R., Koob, J., Jacob, F.: Fermentation and spoilage yeasts and their relevance for the beverage industry - a review. Brew. Sci. 65(3–4), 33–52 (2012)

    Google Scholar 

  62. 62.

    Ordoñez, R.G., Morlon-Guyot, J., Gasparian, S., Guyot, J.P.: Occurrence of a thermoacidophilic cell-bound exo-pectinase in Alicyclobacillus acidocaldarius. Folia Microbiol. 43(6), 657–660 (1998)

    Article  Google Scholar 

  63. 63.

    de Melo Pereira, G.V., Soccol, V.T., Pandey, A., Medeiros, A.B.P., Andrade Lara, J.M.R., Gollo, A.L., Soccol, C.R.: Isolation, selection and evaluation of yeasts for use in fermentation of coffee beans by the wet process. Int. J. Food Microbiol. 188, 60–66 (2014)

    Article  Google Scholar 

  64. 64.

    Feng, X., Dong, H., Yang, P., Yang, R., Lu, J., Lv, J., Sheng, J.: Culture-dependent and -independent methods to investigate the predominant microorganisms associated with wet processed coffee. Curr. Microbiol. 73(2), 190–195 (2016)

    Article  Google Scholar 

  65. 65.

    Bai, J., Baldwin, E.A., McCollum, G., Plotto, A., Manthey, J.A., Widmer, W.W., Luzio, G., Cameron, R.: Changes in volatile and non-volatile flavor chemicals of “Valencia” orange juice over the harvest seasons. Foods 5, 4 (2016)

    Article  Google Scholar 

  66. 66.

    Guan, Y., Wang, D., Lv, C., Zhang, Y., Gelbic, I., Ye, X.: Archives of microbiology: screening of pectinase-producing bacteria from citrus peel and characterization of a recombinant pectate lyase with applied potential. Arch. Microbiol. 202(5), 1005–1013 (2020)

    Article  Google Scholar 

  67. 67.

    Vilanova, L., López-Pérez, M., Ballester, A.-R., Teixidó, N., Usall, J., Lara, I., Viñas, I., Torres, R., González-Candelas, L.: Differential contribution of the two major polygalacturonases from Penicillium digitatum to virulence towards citrus fruit. Int. J. Food Microbiol. 282, 16–23 (2018)

    Article  Google Scholar 

  68. 68.

    Kumar, G.P., Suneetha, V.: Characterization of pectin lyase and polygalacturonase from novel Bacillus cereus GS-2 isolated from Chittoor and Vellore fruit industrial dump sites by SEM, 16S rRNA sequencing, ion-exchange, SDS and HPLC analysis. J. Pure Appl. Microbiol. 10(1), 741–749 (2016)

    Google Scholar 

  69. 69.

    Naga Padma, P., Anuradha, K., Reddy, G.: Pectinolytic yeast isolates for cold-active polygalacturonase production. Innov. Food Sci. Emerg. Technol. 12(2), 178–181 (2011)

    Article  Google Scholar 

  70. 70.

    Lu, X., Lin, J., Wang, C., Du, X., Cai, J.: Purification and characterization of exo-polygalacturonase from Zygoascus hellenicus V25 and its potential application in fruit juice clarification. Food Sci. Biotechnol. 25(5), 1379–1385 (2016)

    Article  Google Scholar 

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Ioanna Zerva would like to acknowledge the Eugenides Foundation for its financial support through a Ph.D. scholarship.

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Zerva, I., Remmas, N., Melidis, P. et al. Microbial Succession and Identification of Effective Indigenous Pectinolytic Yeasts From Orange Juice Processing Wastewater. Waste Biomass Valor (2021). https://doi.org/10.1007/s12649-021-01364-7

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  • Citrus waste
  • Microbial succession
  • Polygalacturonase activity
  • Spontaneous fermentation
  • Pichia fermentans
  • Heterofermentative leuconostocs