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Selective microbial resolution of lupanine racemate: Bioprocess development and the impact of carbon catabolite repression on industrial wastewater valorisation

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Abstract

An environmentally friendly bioprocess for the valorisation of the lupanine enantiomeric mixture existing in lupin bean processing wastewater was developed. Pseudomonas putida LPK411, which is capable of enantioselectively biodegrading lupanine enantiomers, was employed for the resolution of the lupanine racemate content of unrefined and pretreated industrial effluents. The optimal culture conditions for racemic lupanine biodegradation by LPK411 were determined as 31 °C, pH 6–7, and 1.5 g L−1 initial lupanine concentration. The results obtained for enantioselective resolution of the effluents by P. putida LPK411, grown in shake-flasks, and a lab-scale bioreactor under batch operation, demonstrated that lupanine resolution was substantially improved in the bioreactor, exhibiting L-(–)-lupanine enantiomeric excess > 93% for all feedstocks used. Moreover, a fed-batch bioprocess was conducted using racemic lupanine resulting in 53% and 49% enhanced D-( +)-lupanine biodegradation and biomass production compared to the corresponding batch experiment respectively, while L-(–)-lupanine concentration increased by 49%. Monitoring the transcriptional kinetics of luh and crc genes employing industrial wastewater and the alkaloid racemate in synthetic media demonstrated that although expression from the lupanine catabolic route was rapidly induced upon supply of lupanine as a single substrate, LPK411 preferably utilised other carbon molecules of the real effluent over lupanine, suppressing the alkaloid’s catabolic pathway via the carbon catabolite repression regulatory system. The study exemplified the impact of a preferred compound on the main metabolic route of a bioprocess, demonstrating the importance of molecular interactions in biorefineries developed based on the mixture of substrates contained in renewable bioresources. Future research should aim at optimising and up-scaling the enantioselective biodegradation process proposed.

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Funding

This work was supported by the M-ERA.NET project Biorg4WasteWaterVal + co-funded by the Research Promotion Foundation (RPF, Cyprus) [grant number ΚΟΙΝΑ/ΠΚΠ-WATER/0315/14]; the Fundação para a Ciência e a Tecnologia (FCT, Portugal) [grant numbers WaterJPI/0001/2014, WaterJPI/0002/2014, WaterJPI/0003/2014]; and the IBB-Institute for Bioengineering and Biosciences (UIDB/04565/2020), from Programa Operacional Regional de Lisboa 2020 (Lisboa-01–0145-FEDER-007317).

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Authors and Affiliations

  1. Department of Chemical Engineering, Cyprus University of Technology, 30 Archbishop Kyprianou Str, 3036, Limassol, Cyprus

    Stella Parmaki & Michalis Koutinas

  2. Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001, Lisbon, Portugal

    Teresa Esteves & Frederico C. Ferreira

  3. Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, 1049-001, Lisbon, Portugal

    Teresa Esteves & Frederico C. Ferreira

  4. Research Institute for Medicines (iMed. ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003, Lisbon, Portugal

    João M. J. Gonçalves & Carlos A. M. Afonso

  5. Department of Civil and Environmental Engineering - DICA, Environmental Section, Politecnico Di Milano, Piazza L. da Vinci 32, 20133, Milan, Italy

    Arianna Catenacci & Franscesca Malpei

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  1. Stella Parmaki
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  2. Teresa Esteves
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  6. Frederico C. Ferreira
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  7. Carlos A. M. Afonso
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  8. Michalis Koutinas
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Contributions

SP and MK designed the work. SP performed the main experiments and analysis, including fermentation experiments, q-PCR analysis, GC-FID, and HPLC analysis and statistical analysis. JMJG and CAMA produced purified lupanine racemate. TE and FCF produced the nanofiltrated wastewater. GB and FM produced the anaerobically digested wastewater. SP analysed the literature and experimental data and prepared the manuscript with the help of MK. MK supervised the work. All authors read and approved the final manuscript.

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Correspondence to Michalis Koutinas.

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Parmaki, S., Esteves, T., Gonçalves, J.M.J. et al. Selective microbial resolution of lupanine racemate: Bioprocess development and the impact of carbon catabolite repression on industrial wastewater valorisation. Biomass Conv. Bioref. 13, 14949–14961 (2023). https://doi.org/10.1007/s13399-022-03383-3

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