Chemicals, filters and flasks
Neomycin trisulfate hydrate (≥ 600 µg neomycin mg-1, dry basis) was purchased from Sigma Aldrich (Buchs, Switzerland). Ammonium tartrate dibasic (≥98%) and D-(+)-glucose (≥99.5%) from Sigma Aldrich and sulfuric acid (95–97%; Merck Millipore (Darmstadt, Germany) were used to prepare a nutrient solution at pH 4.0. Acetonitrile, Optima™, LC/MS grade from Fisher Scientific (Reinach, Switzerland), water for chromatography Lichrosolv® LC-MS grade (Merck Millipore) and formic acid (98-100%) from Merck Millipore were used in the UHPLC-Q-TOF analyses. Polyurethane foam (PUF) filter cartridges for aquariums (Pickup 200 EHEIM, Germany) were used for the immobilization of T. versicolor. The porosity of this open-cell filter type with a reticular structure is between 95-98%. Water, purified at Cytiva (Uppsala, Sweden) using a Milli-Q® water system (Milli-Q Millipore 0.22 µm serial no 1550, Massachusetts, USA) was used in the UHPLC-Q-TOF mobile phases, and tap water softened at Cytiva, Uppsala, Sweden was used in the experimental solutions that contained fungal cultures, thereby providing trace amounts of minerals. D-ribose with a purity of ≥ 99.0% from Merck, was used as UHPLC-Q-TOF standard.
Glass fibre prefilters, cat. no. AP 2504700 from Merck Millipore (Burlington, USA) were used to determine initial biomass weights of T. versicolor mycelia. Munktell, 1003, 9 cm-filters (Munktell Filter AB, Sweden) were used to determine the biomasses of R. ericae in experimental cultures. Furthermore, 300 µL fixed insert amber vials (Agilent Technologies, Santa Clara, USA) were used both for calibration standards and samples. For the biodegradation experiments, 500 mL Duran® Erlenmeyer flasks with bottom-baffles and air permeable lids from Sigma Aldrich were used
Fungi
T.versicolor (strain AG 1383) was donated from Culture Collection of Basidiomycetes (CCBAS, Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague). R. ericae (Hymenoscyphus ericae) was donated by Dr. Martin Vohnik from Institute of Botany (Academy of Science of the Czech Republic, Prague). This strain is derived directly from the type culture (Acc. Number UAMH6735) in the collection at the University of Alberta, GenBank number AJ319078, originally isolated from a Calluna Vulgaris hair root in the UK. Mycelia of both strains were grown on malt agar and transported to Sweden at refrigerated conditions (2-8 o C).
Instrumentation
An Edmund Bühler KS10 shaking table from Tübingen, Germany was used to stir the Erlenmeyer flasks (E-flasks). A Termaks, TS 9053 drying oven from Bergen, Norway was used to dry mycelia containing agar plugs.
An Agilent Technologies 6550 iFunnel Q-TOF LC/MS system (Agilent Technologies, Santa Clara, CA, USA), including an Agilent Technologies 1290 Infinity UHPLC system consisting of a 1260 iso pump (G1310B), 1290 binary pump (G4220A), thermostat (G1330B), 1290 sampler (G4226A), 1290 thermostated column compartment (G1316C), and electrospray ionization (ESI) system, was used for qualitative and quantitative analysis. Agilent MassHunter software (V. 06.00) was used for instrument control, data acquisition and processing. For separation purposes, a zwitterionic UHPLC column (SeQuant® ZIC®-cHILIC, 100 x 2.1 mm, 100 Å, 30 µm, Merck, Darmstadt, Germany) was used. Solid Phase Extraction (SPE) was performed with Isolute® C18 (EC) 100 mg 1 mL SPE columns, Biotage (Uppsala, Sweden). A pH meter, Mettler Toledo Steven Compact ID 12-137 (Greifensee, Switzerland) was used to adjust and measure the pH in all fungal experiment solutions.
Experimental procedures
Preparation and composition of nutrient solutions
In the nutrient solution that was used for inoculation purposes, sulfuric acid was added to a 1000 mL measuring flask containing 6.0 g D-glucose, 3.3 g ammonium tartrate, and 900 mL softened tap water to obtain pH 4.0. The pH was selected with the aim of suppressing bacterial growth. The carbon-to-nitrogen ratio (C:N ratio) was 6.5. The carbon and nitrogen contents in a 300 mL volume of nutrient solution (experimental volume) were 971 and 150 mg respectively. The procedure was similar for nutrient solutions that contained alternative compositions of glucose and ammonium tartrate that were used in the biodegradation experiments (see section “Experimental design”).
Cultivation of T. versicolor and R. ericae
T. versicolor and R. ericae were grown on petri dishes with extracted malt agar from the Swedish National Veterinary Institute (Uppsala, Sweden). Growth was conducted in an aseptic environment where a malt agar plug was removed with a 10 mm (internal diameter) punch, previously sprayed with 70% EtOH and allowed to dry, from a prior culture of mycelia to a new dish. The petri dishes were kept under dark conditions by wrapping with aluminum foil for one week (T. versicolor) and two weeks (R. ericae) and then storing in a refrigerator at a temperature of +4 oC. This sub-culturing procedure was performed every second month.
Inoculation and PUF immobilization of T. versicolor
PUF pieces, cut in centimeter sized cubes, with a total weight of approximately 2.7 g were added to nineteen baffled E-flasks each. These flasks were filled with 300 mL nutrient solution each and then autoclaved at 125 oC for 30 minutes using a Certoclav CV-EL 12 L autoclave (Leonding, Austria). The flasks were then cooled to ambient temperature. In an aseptic environment, fifteen malt agar plugs containing cultivated T.versicolor were added to each flask. For twelve days prior to the experiments, the flasks were kept in the dark, covered with aluminum foil, and mounted on a shaking table operating at a low shaking speed (50 min-1) at room temperature. After 12 days of inoculation/immobilization, two of the flasks (heat killed controls (HKCs)), were autoclaved once more. The HKCs were aimed for monitoring biosorption to dead T.versicolor. Immobililization is motivated by increased suppression of bacterial growth, increased secretion of extracellular enzymes and less residual mycelia in the liquid broth (Libra et al. 2003; Lu et al. 2009; Ullah et al. 2000).
Inoculation of R. ericae
Previously non-published experiments, performed by the authors have shown that it is difficult to immobilize R. ericae on PUF. The mycelia did not colonize the PUF-pores, like the mycelia of T. versicolor. Therefore, the inoculation was performed in the absence of PUF. Twelve 300 mL baffled E-flasks were filled with 300 mL nutrient solution each, autoclaved and cooled according to section 2.4.3. Twenty agar plugs containing cultivated R. ericae mycelia, were added to each flask. The same inoculation conditions (time, darkness and shake speed) as for T.versicolor were maintained. Two of the flasks (HKCs) were after 12 days autoclaved once more.
Biomass calculations
Punched pieces of the cultivated biomasses of T.versicolor and R. ericae were weighed after storage in a refrigerator for two weeks (see section “Cultivation of T. versicolor and R. ericae”). The weights of the initial biomasses (BM) were later used in the calculation of initial carbon biomass (C:BM) ratios. These ratios thus give information on the relation between carbon content in nutrients and neomycin, and BM at the start of the inoculation step.
The used methodology by which agar plugs containing fungal mycelia are dried for 20 minutes at 105 oC to determine initial biomasses is previously described (Stenholm et al. 2022). In the present study, 15 agar plugs containing fungal mycelia from T. versicolor and 20 agar plugs containing R. ericae, were used for this purpose. The mean weight of the biomass in each T.versicolor and R.ericae plug was 2.6 mg ± 4.8% (n=10) and 4.5 mg ± 6.7% (n=10) respectively. Thus, the total biomass weight in 15 T.versicolor and 20 R.ericae agar plugs was 39 and 90 mg respectively.
In a separate study, BM of R. ericae was determined after (i) the 12:th day of the inoculation step (BMRe), and (ii) after 168 h biodegradation in six experimental cultures which did not contain any neomycin but only nutrient solution (NS0Re), 34 mg neomycin L-1 in the presence of nutrient solution (NS4Re), 34 mg neomycin L-1 (Neo1Re), 1.0 g neomycin L-1 (Neo2Re), 6.0 g neomycin L-1 (Neo3Re) and 15 g neomycin L-1 (Neo4Re). The four last experiments contained solely neomycin solutions. The experiments were performed in duplicate. See Table 1 for experimental culture compositions of neomycin containing samples. The procedure was as follows: The filter that was going to be used to filtrate BM was initially dried in an oven for 20 minutes at 105 oC and allowed to cool to room temperature. Thereafter, BM was filtrated by gravity using the dried filter. BM was washed excessively with Milli-Q water to get rid of neomycin residues. Washed BM was dried for 20 minutes on the filter paper at 105 oC and allowed to cool to room temperature. Then the weight of the filterpaper and BM was determined, and finally, the previously determined weight of the filter paper was subtracted.
Experimental design
After the 12-day inoculation period, the broths were separated from mycelia immobilized PUF-pieces (T. versicolor) and the free mycelia (R. ericae) in most of the flasks, leaving solely the solids in the flasks. In the experiments with titles beginning with NS4, no decanting took place (see Table 1). In these experiments, neomycin was added to the inoculation solutions by gentle stirring. To the other flasks, differently composed solutions (300 mL each) were added to the flasks. The main purpose of the design was to evaluate to what extent neomycin could be biodegraded with and without the presence of other nutrients. The neomycin solutions that were added to the fungal cultures included softened tap water which was adjusted to pH 4.0 using 0.1 M sulfuric acid. These solutions were not autoclaved. In Table 1, the compositions are shown. Some of the experiments which were performed with T. versicolor and contained nutrient solution (NS) at different concentrations, were excluded for R. ericae. This was done mainly because this species had previously shown excellent capabilities to grow and survive on toxic nitrogen containing substances (Stenholm et al. 2021). The flasks were then subjected to biodegradation experiments that lasted for 168 h. The flasks were covered with aluminum foil and agitated (50 rpm). Samples (300 µL) were pipetted at 0, 1, 2, 4, 6, 8, 24, 30, 48, 72, 96, 120, 144 and 168 h. They were immediately frozen at -20 oC. Prior to UHPLC-Q-TOF MS analyses, the samples were thawed and worked-up with SPE. See section “Solid Phase Extraction”.
In Table 1, the experimental titles and conditions are shown. Each experiment was performed in duplicate except for NS4Tv that was done in triplicate and HKCs that were not replicated. The reason for the choice of the four HKC experimental conditions was that neomycin adsorption to dead mycelia may be affected (i) by the choice of species and (ii) by the neomycin and NS concentrations. The experiments that included neomycin concentrations > 34 mg neomycin L-1 excluding NS were motivated by the wish to understand whether NS can be used as sole nutrient. The NS composition in NS4TV and NS4Re approximately resembles the carbon and nitrogen concentration in the NS that is used in the recombinant protein production application.
Table 1
Neomycin containing compositions that were used in the biodegradation experiments. Tv and Re are denotations of T. versicolor and R. ericae. HKC is heat control. NS and Neo are abbreviations of nutrient solution and neomycin
The compositions in Table 1 were used to build a model matrix X aimed for Principle Component Analysis (PCA). In this matrix (see Appendix 1), a number of independent variables were used. These were:
CNS (amount of carbon in NS), NNS (amount of nitrogen in NS), Cneo (amount of carbon in neomycin), Nneo (amount of nitrogen in neomycin), Ctot (total amount of carbon), Ntot (total amount of nitrogen), Ctot:Ntot, Cneo:BM (initial biomass), Ctot:BM (initial biomass). Two dependent variables were included in the PCA matrix: These were “Removal degree” and “Removal amount”.
Solid Phase Extraction
Thawed collected fractions from the biodegradation experiments were purified from lipids originating from the immobilized mycelia (Stenholm et al. 2018).
The SPE columns were first activated with 1.0 mL of acetonitrile and then equilibrated with 1.0 mL NS containing 6.0 and 3.3 glucose and ammonium tartrate L-1 respectively. The pH in NS was adjusted to 4.0 using sulfuric acid. Then, 300 µL collected samples were loaded onto the SPE-columns twice and the non-retarded neomycin was eluted with an applied pressure using a Pasteur pipette rubber bulb. The reason for the choice of NS as equilibration solution was that even if many of the experiments did not contain NS, it was considered to be important to have a consistency in the SPE procedure. The SPE recovery was investigated by preparing two QC samples, containing neomycin at concentrations of 10.0 and 34.0 mg L-1 (dissolved in NS (SPE equilibration solution)) followed by SPE and analyzing them by triplicate injections. The mean recovery was calculated to 94 and 92% respectively. See equation (1). The concentration was determined using external standards according to section “Quantitative analysis of neomycin”.
Quantitative analysis of neomycin
The analyses were performed using external standards. The sample recovery for the SPE procedure was > 90% (see section “Solid Phase Extraction”). No matrix effects in SPE worked up samples that affected the neomycin quantification could be seen according to the initial neomycin concentration levels in the undiluted samples. They were all between 30 and 34 mg L-1 (see Fig. 4 and 6). The external standards consisted of neomycin dissolved in solely NS to 3.0, 6.0, 14.0, 20.0, 27.0 and 34.0 mg L-1. The NS concentration was similar with the inoculation and SPE equilibration solution, that is 6.0 and 3.3 g glucose and ammonium tartrate L-1 respectively at pH 4.0. Using the selected zwitterionic SeQuant ZIC-cHILIC column and the chosen instrumental settings, it was noticed that the composition of the neomycin solvent (different concentration levels of NS) and also pure MilliQ water did not alter the neomycin ionization degree or the linearity of the calibration curves. For experiments in which exceeded concentration levels of neomycin were used, dilutions of the collected fractions with MilliQ water were made in such a manner that the results would fit to the used external standards. For these samples, the SPE procedure (see section “Solid Phase Extraction”) was performed before the dilution step.
The zwitterionic SeQuant ZIC-cHILIC column was thermostated at 60 oC to decrease peak width and asymmetry. The choice of this temperature was based on initial experiments that were performed at 30, 45 and 60 oC. It was observed that by increasing the temperature, the neomycin retention times were shortened and the peak widths and asymmetries were decreased. The mobile phases consisted of A; acetonitrile: water (5:95 (v/v)) with 0.1% formic acid and B; acetonitrile:water (95:5 (v/v)) with 0.1% formic acid. Initially, 100% B was maintained isocratically for one minute. Thereafter, mobile phase B decreased linearly to 5% while mobile phase A increased to 95% during a time period of 1.5 minutes. This composition was held for 4.5 minutes. Thereafter, the column was equilibrated to 100% B during a time period of 0.5 minutes. This composition was held to the end of the run (18 minutes). The flow rate was 0.3 mL min-1 and the injection volume was 15 µL. The MS settings were: capillary voltage 3.5 kV, gas temperature 200 oC, sheath gas temperature 350 oC, sheath gas flow 11 L min-1 and a nozzle voltage of 1.0 kV. MS data were acquired in the m/z range of 100 – 1700. The ESI ionization was performed in positive mode and the instrument was tuned and calibrated every day prior to use. The external calibration standards containing neomycin in the 3.0 to 34 mg L-1 concentration range were co-analyzed together with SPE-worked up samples and pure nutrition solution (blanks) in each sample sequence. The proton neomycin adduct at m/z 615.3201 (exact mass) was used in the quantification. The integrated peaks include both abundant neomycin isomeric forms (B and C) since they are not separable using the chosen SeQuant® ZIC®-cHILIC column and gradient elution The reference mass ions which were used for internal mass calibration (lock masses) were m/z 121.0509 and 922.0098. Three QC samples with the concentrations 10.0, 25.0 and 30.0 mg L-1 were used for determining the accuracy. It was 86.2, 99.3 and 99.7% (n=3), respectively. The linearity (r, i.e., the Pearson product-moment correlation coefficient) of 0.9975 was determined using triplicate injections of the calibration standards.
Some experiments contained neomycin at elevated concentrations (1.0, 6.0 and 15 g neomycin L-1). The highest external calibration concentration was 34 mg neomycin L-1. To be able to quantify neomycin in these experiments, dilutions of SPE-cleaned collected fractions (200 µL) with MilliQ water were made. These dilutions were made in such a manner that the maximum end concentration would be 34 mg L-1. The initial concentration: theoretical final volume relations were as follows; 1.0 g L-1: 5882 µL, 6.0 g L-1: 35294 µL, 15 g L-1: 88235 µL. The SPE recoveries for the elevated neomycin concentrations were proven to be close to 100% by dividing the initial concentrations in Fig. 4f, 4g, 4h, 6c, 6d and 6e with the final volumes (5882, 35294 and 88235 µL) and then multiplying with 200 µL. The majority of the back calculated concentrations were 33 mg neomycin L-1.
Qualitative analysis of biodegradation products
Molecular formulas of possible oxidation products of neosamine, 2-DOS, D-ribose, neobiosamine and neamine (see Fig. 1) including ketones, aldehydes and acids were added to a PCDL-database which was integrated in the MassHunter software. Also, a number of compounds, previously identified as fungal metabolites and secondary metabolites (small organic molecules that are not directly involved in primary metabolism processes) were added to the database. These were glucuronic acid, fumaric acid, gluconic acid and citric acid (metabolites) and itaconic acid and ribitol (secondary metabolites). After a finalized run, the database was searched and compounds were preliminarily identified, based on mass errors, isotopic abundances, and spacings. Ions with mass errors and scores of < 2.5 ppm and > 80% respectively, were considered. An alternative approach was to search in the TIC-chromatograms for ions that were missing from the blanks. The “Generate Formula From Peak Spectra” algorithm was used and the ions with mass errors and scores of < 2.5ppm and > 80%, respectively, were further evaluated using the software ChemSpider (Chemspider 2020).
Statistical analysis
In Table 4, the calculations of the accurate and exact masses in MS were facilitated by the use of Agilent MassHunter software (v. 06.00). Furthermore, using this software, elemental compositions were suggested for detected ions. Excel, Microsoft Office Version 2110, was used for regression analysis purposes to construct least square fitted (i) calibration curves and (ii) kinetic models. The Pearson product-moment correlation coefficient (r) was used to determine linearity. Excel was also used to calculate confidence intervals to confirm whether any biodegradation occurred or not.
Kinetics
Free radical processes which are present in the extracellular degradation of chemicals using white rot fungi can at co-metabolic conditions follow pseudo-first order kinetics (Barr and Aust 1994; Dhiman et al. 2022). See Equation (2).
where Ct is the concentration at time t. k´ is the degradation rate constant and C0 is the initial concentration.
When the target substance is present at a high concentration level, the reaction can obey zero order kinetics (Huang et al. 2019). See equation (3). The reaction rate is thus independent of the target compound concentration level. Zero-order reactions are only applicable for a narrow region of time. After this time period, other kinetic models are better applicable.
Evaluation of neomycin biodegradation curves
The neomycin biodegradation curves (excluding the four HKCs) were plotted and compared with each other. The results from the last collected fractions at 168 h, were used to determine the final removal degrees (%). Furthermore, these results were also used to calculate the total amount of neomycin that was biodegraded in each experiment. See the general equations (4) and (5).
where Ct is the neomycin concentration at time t and C01 is the initial neomycin concentration in the analyzed sample.
where 0.3 is the liquid volume (L) in the E-flask. C02 is the initial neomycin concentration (mg L-1) in the biodegradation experiments (four different concentrations).
A matrix which consisted of these two measures of the biodegradation and the carbon and nitrogen contents in the experiments were analyzed using Principal Component Analysis (PCA) to (i) determine whether the biodegradation could be classified as co-metabolism or nutrition mediated and (ii) possibly determine the most favourable conditions for the biodegradation. The raw data that were used in PCA were centered and univariately scaled (SIMCA, 2021).