Neomycin removal using the white rot fungus Trametes versicolor

Figures & data

Figure 1. Chemical structures of neomycin B and C, neamine (neomycin A), neobiosamine (divided by dotted line) and di-substituated 2-deoxy streptamine (2-DOS). NA means not applicable.

Figure 2. Full scan spectrum of neomycin calibration standard at a concentration of 10 mgL⁻¹.

Figure 3. Chemical structures of abundant neosamine B (a) and ribostamycin (b) ions.

Figure 4. Time course decline of neomycin. Neomycin added at 10 mg L⁻¹. Symbols: EC1 (■), HKC (◊), ECM1 (^), EC2 (▲), ECM2 (Х), CC (+).

Table 1. Summary of kinetic parameters of degradation of neomycin using different experimental conditions.

Experiment	Time (h)	Kinetics	Equation	Half-life (h)	k (mg L^−1 h^−1)*	k' (h^−1)**	Corr. coeff. (r)
EC1	0–8	Zero order	Ct = −0.3546t + 9.8170	13.8	0.3546	n/a	0.9798
EC1	24–168	Ps.-first order	ln Ct = −0.0102t + 1.9772	34.0	n/a	0.0102	0.9939
ECM1	0–168	Zero order	Ct = −0.0260t + 9.2503	177.9	0.0256	n/a	0.9635
ECM1	0–168	Ps.-first order	ln Ct = −0.0035t + 2.2305	99.0	n/a	0.0035	0.9766
ECM2	0–8	Zero order	Ct = −0.1840t + 9.3414	25.4	0.184	n/a	0.9130
ECM2	0–8	Ps.-first order	ln Ct = −0.0218t + 2.2370	229.4	n/a	0.0218	0.9129
EC1(1)	0–8	Zero order	Ct = −0.1073t + 9.8028	45.6	0.1073	n/a	0.9915
EC1(1)	24–240	Ps.-first order	ln Ct = −0.0062t + 1.9834	56.0	0.0062	0.9886
EC1(2)	0–8	Zero order	Ct = −0.1261t + 10.0490	39.8	0.1261	n/a	0.9870
EC1(2)	24–240	Ps.-first order	ln Ct = −0.0053t + 2.0338	65.4	n/a	0.0053	0.9910
EC1(3)	0–8	Zero order	Ct = −0.1147t + 10.0350	45.1	0.1147	n/a	0.9805
EC1(3)	24–240	Ps.-first order	ln Ct = −0.0069t + 2.0707	59.2	n/a	0.0069	0.9891

* k: rate constant for zero order kinetics.

** k': rate constant for pseudo-first order kinetics.

EC1(1–3) are replicate determinations.

Figure 5. Laccase enzymatic activities during the 168 h time course. Symbols: EC1 (■), ECM1 (^), EC2 (▲) and ECM2 (Х).

Figure 6. Time course decline of neomycin using EC1 experimental conditions 0–240 h (a) and initial part, 0–8 h (b). The experiment was performed in triplicate. Symbols: ■ shows mean results at each sampling occasion including standard deviation (SD) error bars. The symbols □, ^ and Δ show initial individual results.

Figure 7. Proposed biodegradation pattern of neomycin, where B-E show possible structural isomers of oxidation products of neosamine C (A). The tentative identities of the oxidation products (excluding isomer elucidation) could be verified by UHPLC-Q-TOF MS analyses. The *-sign indicates possible additional open-chain structures. Extracted ion chromatograms (EC1, 144 h) at m/z 157.0613 (C and D) and at m/z 175.0716 (E).

Figure 8. Proposed biodegradation pattern of neomycin, where G and H show possible structural isomers of oxidation products of 2-DOS (F). The tentative identities of the oxidation products (excluding isomer elucidation) could be verified by UHPLC-Q-TOF MS analyses.

Table 2. Summary of the accurate mass measurements of hydrolysis and oxidation products of neomycin, as determined for their abundant adduct ions using UHPLC-Q-TOF MS.

Compound	Elemental composition	Ion species	Accurate mass (m/z)	Exact mass (m/z)	Mass error		tR (min)
					mDa	ppm
Neomycin	C23H46N6O13	[M + H]^+	615.3206	615.3201	−0.5	−0.8	3.9
A	C6H14N2O4	[M + H-H2O]^+	161.0925	161.0926	0.1	0.6	3.9**
B	C6H12N2O4	[M + H-H2O]^+	159.0767	159.0770	0.3	−1.9	3.6
C + D	C6H10N2O4	[M + H-H2O]^+	157.0613	157.0610	−0.3	−1.9	3.4
E	C6H12N2O5	[M + H-H2O]^+	175.0716	175.0719	0.3	1.7	3.3
F	C6H14N2O3	[M + H]^+	163.1080	163.1083	0.3	1.8	3.9**
G	C6H12N2O3	[M + H]^+	161.0923	161.0926	0.3	1.9	3.8
H	C6H10N2O3	[M + H-H2O]^+	141.0664	141.0664	0.0	0.0	3.5

The elemental compositions are shown in their uncharged state. Data for detected ions correspond to acquisitions obtained in full scan mode. Ions that partly can be explained by in-source fragmentation are marked with (**). Most abundant peak t_R.

** hypothesized retention time since in-source fragmentation occurs.

Figure 9. Time course of oxidation product D at m/z 157.0613 (a) and oxidation product E at m/z 175.0716 (b). Symbols: EC1 (■) and ECM2 (Х).

Figure Caption ok

Availability of data and materials

The data that support the findings of this study are available from the corresponding author at reasonable request.