Abstract
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.
Graphic Abstract

This is a preview of subscription content, access via your institution.
We’re sorry, something doesn't seem to be working properly.
Please try refreshing the page. If that doesn't work, please contact support so we can address the problem.








References
- 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)
- 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)
- 3.
FAO: Citrus Fruit - Fresh and Processed, Statistical Bulletin 2016. Food and Agriculture Organization of the United Nations, Rome (2017)
- 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)
- 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)
- 6.
Mamma, D., Christakopoulos, P.: Biotransformation of citrus by-products into value added products. Waste Biomass Valoriz. 5(4), 529–549 (2014)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 26.
Ntougias, S.: Phylogenetic identification and enzyme activities of indigenous bacteria from a landfill stabilization pond. Environ. Process. 3(2), 341–352 (2016)
- 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)
- 28.
Miller, G.L.: Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 32, 426–428 (1959)
- 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)
- 30.
Huang, X., Madan, A.: CAP3: a DNA sequence assembly program. Genome Res. 9(9), 868–877 (1999)
- 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)
- 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)
- 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)
- 34.
Saitou, N., Nei, M.: The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4(4), 406–425 (1987)
- 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)
- 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)
- 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)
- 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)
- 39.
Edgar, R.C.: UNOISE2: improved error-correction for Illumina 16S and ITS amplicon sequencing. bioRxiv (2016). https://doi.org/10.1101/081257
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
- 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)
Acknowledgements
Ioanna Zerva would like to acknowledge the Eugenides Foundation for its financial support through a Ph.D. scholarship.
Author information
Affiliations
Corresponding author
Ethics declarations
Conflict of Interest
All authors declare no conflict of interest.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Keywords
- Citrus waste
- Microbial succession
- Polygalacturonase activity
- Spontaneous fermentation
- Pichia fermentans
- Heterofermentative leuconostocs