Dr. Sabine Sané

Lecturer and researcher at the Institute for Microsystem Technology - IMTEK at Albert-Ludwigs-Universität Freiburg

Expertise: Bio-electrochemical enzymatic systems to produce electricity and to degrade micropollutants in wastewater

Find them here: https://www.biooekonomie-bw.de/fachbeitrag/aktuell/pilzsuppe-als-klaeranlage-und-biobrennstoffzelle

Helped us with: Advice on laccase experimental conditions and laccase based system placement within a wastewater treatment plant, discussed her work with laccase that led to her winning the international Huber Technology Prize 2014: Future Water

Dr. Dietmar Schlosser

Group Leader Environmental Mycology at Helmholtz Center for Environmental Research - UFZ

Expertise: Laccases

Find them here: https://www.ufz.de/index.php?de=39080

Helped us with: Advice on different algae laccases, best growing conditions and lab advice

Prof. Dr. Antonio Pierik

Professor of Biochemistry at Technical University of Kaiserslautern

Expertise: Biochemistry and metallic proteins (such as laccase)

Find them here: https://www.chemie.uni-kl.de/en/pierik/members-of-the-group/prof-dr-antonio-pierik/

Helped us with: Proteins with metallic atoms such as laccase, gave recommendations for production in algae and how to avoid potential difficulties that could arise

PhD Yunrong Chai

Associate Professor of Biology at Northwestern University

Expertise: Microbial genomics and biofilm formation

Find them here: https://cos.northeastern.edu/people/yunrong-chai/

Helped us with: Improvement of B. subtilis for our needs, fusion proteins with TasA

Prof. Dr. Jörg Stülke

Professor of Microbiology at the Institute for Microbiology and Genetics in Göttingen Graduate Center for Neurosciences, Biophysics, and Molecular Biosciences

Expertise: Bacillus subtilis

Find them here: https://www.uni-goettingen.de/en/58040.html

Helped us with: Provided the tasA sinR knockout strain (GP1622), lab advice, advice on B. subtilis

Prof. Dr. Susanne Lackner

Professor in the department of wastewater management at the Technical University of Darmstadt

Expertise: Microbial communities for wastewater purification and degradation of harmful pharmaceutical residues

Find them here: https://www.iwar.tu-darmstadt.de/abwasserwirtschaft/fachgebiet_abwasserwirtschaft/mitarbeiter_8/Lackner.de.jsp

Helped us with: Identifying harmful substances in wastewater, carrier material for implementation

Prof. Dr. Jörg Oehlmann

Professor of Aquatic ecotoxicology at Goethe University Frankfurt am Main

Expertise: Micropollutants’ effects on aquatic environment

Find them here: https://www.bio.uni-frankfurt.de/45735703/Aquatische_%C3%96kotoxikologie

Helped us with: Identifying harmful substances in water, implementation in wastewater treatment plants (carrier material)

Dr. Patrick Schröder

Research assistant at Federal Environment Agency

Expertise: Biodegradation of pharmaceuticals and development of antimicrobial resistances

Find them here: https://www.umweltbundesamt.de/impressum

Helped us with: Provided understanding of dangers caused by pharmaceuticals and prevention of unnecessary water toxification through science communication

Dr. Bertram Kuch

Institute Sanitary Engineering, Water Quality and Solid Waste management University of Stuttgart

Expertise: Head of the department for the analysis of micropollutants

Find them here: https://www.iswa.uni-stuttgart.de/institut/team/Kuch-00001/

Helped us with: Decision on what substrates we should focus on degrading with our laccases as well as theoretical background for the establishment of an analytical method

Dr.-Ing. Henning Knerr

Manager of tectra and Division manager of wastewater treatment and water quality at Technical University of Kaiserslautern

Expertise: Engineering and wastewater management

Find them here: https://www.bauing.uni-kl.de/en/wir/team/leadership/dr-ing-henning-knerr/

Helped us with: Understanding major micropollutants in Germany (Diclofenac specific) and Rheinland-Pfalz wastewater treatment plant design and smaller treatment plant methods of cleaning micropollutants

Dr. Gerd Maack

Environmental Risk Assessment of Pharmaceuticals in German Environment Agency (UBA), EDA-EMERGE Advisory Board Member

Expertise: German micropollutants and environmental effects

Find them here: https://www.umweltbundesamt.de/en/topics/chemicals/pharmaceuticals https://www.ufz.de/eda-emerge/index.php?en=30284

Helped us with: Understanding major micropollutants in Germany (Diclofenac specific) and worldwide environmental effects

Dipl. Ing. Udo Bäuerle

Construction Engineer, co-owner of “B&P Beratende Ingenieure”

Expertise: Water management and wastewater treatment

Find them here: http://www.bp-bi.de/ger/pages/personal01.htm

Helped us with: Explanation of wastewater purification, implementation of a biofilm in wastewater treatment plants

Prof. Dr. Karl-Heinrich Engesser

Institute of Sanitary Engineering, Water Quality and Solid Waste Management University of Stuttgart

Expertise: Wastewater management and biocatalysis

Find them here: https://www.iswa.uni-stuttgart.de/en/institute/team/Engesser-00004/

Helped us with: The implementation possibilities of our project in a wastewater plant and possible difficulties and what would be required to overcome them

Christian Kaiser

Member of the Initiative Progressive Agrarwende

Expertise: Biology student at the University of Bonn, focus in pharmaceutical biology on bacterial natural substances and their biosynthesis

Find them here: https://progressive-agrarwende.org/ueber-uns/

Helped us with: Our project’s design and realistic implementation, discussion on GMO regulations and micropollutants generally

Mike Short

Village of Archbold Wastewater Superintendent

Expertise: American Wastewater Treatment plant

Find them here: http://archbold.com/wastewater/

Helped us with: Comparisons between US wastewater treatment and German wastewater treatment methods

Dan Avers

Village of Archbold Water Distribution Chief

Expertise: American Water treatment plant

Find them here: http://archbold.com/water/

Helped us with: Comparisons between US water treatment and German water treatment methods

Florian Heyn

Management of wastewater treatment plant in Alzenau, Germany

Expertise: German wastewater treatment plant

Find them here: https://www.alzenau.de/B%C3%BCrger/Rathaus-Service/Verwaltung/Mitarbeiter-von-A-Z/index.php?ModID=9&object=tx%7C2413.11&FID=2413.148.1&NavID=2413.39&La=1

Helped us with: Understanding how a German wastewater treatment plant operates

Thomas Seeger

Management of wastewater treatment plant in Weiterstadt, Germany

Expertise: German wastewater treatment plant

Find them here: https://www.weiterstadt.de/verwaltung-service/rathaus/telefonverzeichnis/index.php?we_objectID=1403

Helped us with: Understanding how a German wastewater treatment plant operates

Dr. Ulrich Ehlers

Head of Unit 403 “Deliberate Release and Placing on the Market” of 'Genetic Engineering' department of the Federal Office of Consumer Protection and Food Safety (BVL)

Expertise: Genetic engineering regulations in Germany

Find them here: https://www.bvl.bund.de/EN/Tasks/06_Genetic_engineering/genetic_engineering_node.html

Helped us with: Understanding wastewater treatment plant restrictions for GMO in Germany and bioreactor specifications

Prof. Dr. Sibylle Gaisser

Professor of Industrial Biotechnology at Hochschule Ansbach

Expertise: Socio-economic analysis and technology assessment

Find them here: https://www.hs-ansbach.de/personen/gaisser-sibylle/

Helped us with: Assessment of risks, advice on complying with ethical standards and safety concerns.

Prof. Dr. Andreas Jürgens

Professor of Chemical Plant Ecology at the Technical University of Darmstadt

Expertise: Ethics

Find them here: https://www.bio.tu-darmstadt.de/biologie_1/mitarbeiter_1/mitarbeiterdetails_20096.en.jsp

Helped us with: Evaluation of the risks of our project, the cost-benefit evaluation of our implementation.

Prof. Dr. Alfred Nordmann

Professor of philosophy and history of the sciences and technosciences at the Technical University of Darmstadt

Expertise: Ethics

Find them here: https://www.philosophie.tu-darmstadt.de/institut_phil/mitarbeiter_innen_phil/professoren/a_nordmann/biography_2.de.jsp

Helped us with: Introduction into responsible research and innovation, importance of science communication

Arregui, L.; Ayala, M.; Gómez-Gil, X.; Gutiérrez-Soto, G. Laccases: Structure, Function, and Potential Application in Water Bioremediation. Microbial Cell Factories. BioMed Central Ltd. November 14, 2019, pp 1–33.DOI:10.1186/s12934-019-1248-0.
This review compares many fungal and bacterial laccases and their potential for use in wastewater treatment.

Mate, D. M.; Alcalde, M. Research Review Paper Laccase Engineering: From Rational Design to Directed Evolution. Biotechnol. Adv. 2014, 33, 25–40. DOI:10.1016/j.biotechadv.2014.12.007.
Many different laccases, both fungal and bacterial, are compared and it discusses how these could be modified to improve effectiveness in reactions.

Margot, J.; Bennati-Granier, C.; Maillard, J. Bacterial versus Fungal Laccase: Potential for Micropollutant Degradation; 2013; Vol. 3. DOI:/10.1186/2191-0855-3-63.
Evaluation and comparison of laccase-producing organisms, particularly useful for the comparison of T. versicolor and S. cyaneus.

Kittl R, Mueangtoom K, Gonaus C, A chloride tolerant laccase from the plant pathogen ascomycete Botrytis aclada expressed at high levels in Pichia pastoris. J Biotechnol. 2012 Jan 20;157(2):304-14. doi: 10.1016/j.jbiotec.2011.11.021. Epub 2011 Dec 9. PMID: 22178779. DOI:10.1016/j.jbiotec.2011.11.02.
Botrytis aclada laccase (BaLac) is discussed at length in this paper as a viable option for a laccase with a low pI and chloride tolerant construct, factors which normally limit laccase effectiveness.

Scheiblbrandner, S., Breslmayr, E., Csarman, F. Evolving stability and pH-dependent activity of the high redox potential Botrytis aclada laccase for enzymatic fuel cells. Sci Rep 7, 13688 (2017). DOI:10.1038/s41598-017-13734-0.
Experiments that delve into increasing the pH optimum of Botrytis aclada laccase (BaLac), a laccase with a low pI and chloride tolerant construct, factors which normally limit laccase effectiveness.

Yang Q, Zhang M, Zhang M Characterization of a Novel, Cold-Adapted, and Thermostable Laccase-Like Enzyme With High Tolerance for Organic Solvents and Salt and Potent Dye Decolorization Ability, Derived From a Marine Metagenomic Library. Front Microbiol. 2018;9:2998. Published 2018 Dec 5. DOI:10.3389/fmicb.2018.02998
Investigation over the marine laccase know as Lac1326, an extremely thermostable laccase construct.

Zeng, J.; Zhu, Q.; Wu, Y. Oxidation of Polycyclic Aromatic Hydrocarbons Using Bacillus Subtilis CotA with High Laccase Activity and Copper Independence. Chemosphere 2016, 148, 1–7. DOI:10.1016/j.chemosphere.2016.01.019.
The bacterial laccases CueO and CotA are compared regarding the oxidation rates of 15 different polycyclic aromatic hydrocarbons.

German literature:
Ricklefs, E. Charakterisierung Einer Neuen Bakteriellen Laccase Und Deren Anwendung in Einer Multi-Enzymatischen Kaskade Zur Synthese von Lignanen. https://docserv.uni-duesseldorf.de/servlets/DerivateServlet/Derivate-41000/Ricklefs%2C%20Esther_Doktorarbeit_pdf_a1.pdf (accessed on Oct 13, 2020)
A new bacterial laccase from Corynebacterium glutamicum has been characterized and analysed. Furthermore bacterial laccases were used for multi-enzymatic cascades for fine chemical production.

Alharbi, S. K.; Nghiem, L. D.; van de Merwe, J. P. Degradation of Diclofenac, Trimethoprim, Carbamazepine, and Sulfamethoxazole by Laccase from Trametes Versicolor: Transformation Products and Toxicity of Treated Effluent. Biocatal. Biotransformation 2019, 37 (6), 399–408. DOI:10.1080/10242422.2019.1580268 .
Degradation products of diclofenac and carbamazepine with the laccase of Trametes versicolor discussed, as well as HPLC method.

Jones, S. M.; Solomon, E. I. Electron Transfer and Reaction Mechanism of Laccases. Cellular and Molecular Life Sciences. Birkhauser Verlag AG March 1, 2015, pp 869–883. DOI:10.1007/s00018-014-1826-6.
The electron transfer process and the O₂ reduction in laccases is outlined.

Naghdi, M.; Taheran, M.; Brar, S. K. Removal of Pharmaceutical Compounds in Water and Wastewater Using Fungal Oxidoreductase Enzymes. Environmental Pollution. Elsevier Ltd March 1, 2018, pp 190–213. DOI:10.1016/j.envpol.2017.11.060.
This review summarizes and analyzes the studies performed on pharmaceutically active compounds in wastewater using oxidoreductase enzymes for their degradation. The review also offers an insight into enzymes immobilization, fungal reactors, mediators, degradation mechanisms and transformation products.

Erythromycin Esterase Type II (EreB):
Morar, M.; Pengelly, K.; Koteva, K. Mechanism and Diversity of the Erythromycin Esterase Family of Enzymes. 2012, 51, 1740–1751. DOI:10.1021/bi201790u.
The mechanisms of the Erythromycin and Azithromycin inactivation by the enzyme EreB are described.This takes place by the enzymatic hydrolysis of the macrolactone ring.

Patel, S.; Kalia, V. C.; Choi, J.-H. Immobilization of Laccase on SiO₂ Nanocarriers Improves Its Stability and Reusability Biological Hydrogen Production, Polyhydroxyalkanoates, Quorum Sensing Inhibitors, Bacterial Identification View Project INJM Special ISSUE View Project. Artic. J. Microbiol. Biotechnol. 2014. DOI:10.4014/jmb.1401.01025.
Immobilization of laccase on SiO₂ nanoparticles to overcome problems associated with stability and reusability of the free enzyme is presented.

Huang, J.; Liu, S.; Zhang, C. Programmable and Printable Bacillus Subtilis Biofilms as Engineered Living Materials. Nat. Chem. Biol. 2019. DOI:10.1038/s41589-018-0169-2.
Immobilization of proteins by producing a fusion protein with the major protein component (TasA) of Bacillus subtilis biofilm is shown.

German literature:
Dreifke, M.; Fröba, M. Immobilisierung von Enzymen: Spielerei Oder Biotechnologischer Fortschritt? Springer 2017, 23 (1), 95–97. DOI:10.1007/s12268-017-0769-5.
Different enzyme immobilization strategies especially about the enzyme immobilization on silica foam are explained and compared.

Following the Flow An Inside Look at Wastewater Treatment https://www.wef.org/globalassets/assets-wef/3---resources/for-the-public/public-information/following-the-flow-book-an-inside-look-at-wastewater-treatment.pdf (accessed Oct 1, 2020).
There is general information about Wastewater Treatment Plant structures as an initial overview described by the Water Environment Federation (WEF).

Pistocchi, A.; Dorati, C.; Grizzetti, B.; Udias, A.; Vigiak, O.; Zanni, M. Water Quality in Europe : Effects of the Urban Wastewater Treatment Directive; 2019. DOI:10.2760/303163.
This report shows what micropollutants can be found in wastewater and whether they are transformed in wastewater treatment.

German literature:
Arzneimittelwirkstoffe | Umweltbundesamt https://www.umweltbundesamt.de/themen/wasser/fluesse/zustand/arzneimittelwirkstoffe#undefined (accessed Sep 3, 2020).
Problematic pollutants in German wastewater are described in this article.

Teams that used laccase for inactivation of micro pollutants:
Team TU Munich in 2013:
They used a laccase from Bacillus pumilus and EreB from Escherichia coli for biodegradation of Xenobiotics.

Team Bielefeld-Germany in 2012:
They used five different laccases and even compared them to each other to clean wastewater from synthetic estrogens and aromatic compounds.

Team Stockholm in 2018:
They used the laccase from Trametes versicolor, immobilized on magnetic beads, to inactivate sulfamethoxazole.

Team Western Canada in 2019:
They also used the laccase from Trametes versicolor and immobilized it via the spy-tag/spy-catcher-system on the major curlin subunit (CsgA) of E. coli to clean wastewater from emerging compounds.

Teams that used laccase for other targets:
Team Saint Joseph in 2019:
They used the laccase from Trametes versicolor for dye-decolorization.

Team Edinburgh OG in 2019:
They used the laccase from Bacillus pumilus for dye-decolorization.

Team Hong Kong HKUST in 2018:
They used the laccase from Escherichia coli for poly-ethylen degradation.

Team British Columbia in 2016:
They displayed small laccases on the cell surface of C. crescentus for degradation of lignin cellulose.

Team SHSBNU China in 2018:
They used the CotA laccase from Bacillus subtilis and immobilized it via spy-tag/spy-catcher-system on the major curlin subunit (CsgA) of E. coli for dye-decolorization.

Team Paris Bettencourt in 2016:
They used six enzymes, among which the laccase from Bacillus pumilus to remove stains made by Anthocyanins.