Use of Ganoderma lucidum Grown on Agricultural Waste to Remove Antibiotics from Water

doi:10.21203/rs.3.rs-3210953/v1

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Use of Ganoderma lucidum Grown on Agricultural Waste to Remove Antibiotics from Water

https://doi.org/10.21203/rs.3.rs-3210953/v1

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Antibiotic-rich effluents from farming and medical applications into waterways pose a serious risk for antibiotic drug resistance, promoting a need for effective strategies of removal from the food chain and the environment. In this work, we show proof-of-concept laboratory-scale bioremediation experiments to remove antibiotics in synthetic wastewater. A white rot fungus, Ganoderma lucidum, was grown on biomass formed by agricultural waste from California (almond shells, cover crop stalks). Water containing or lacking Ganoderma lucidum was inoculated with twenty antibiotics from six different classes. The extent of antibiotic removal was measured at baseline and after 3 days with ultra-high pressure liquid chromatography coupled to tandem mass-spectrometry. The data were analyzed with a two-way repeated ANOVA for 17 antibiotics data sets meeting residuals' normality, and a mixed-effects model for 3 antibiotics sets that did not. Treatment with mycelial biomass caused a statistically significant reduction, compared to the baseline, in the concentration in 3 quinolones and 2 sulfonamides, after just 3 days. Our findings provide a first proof-of-concept to bioremediate certain antibiotics, (particularly quinolones), in synthetic wastewater and with repurposed agricultural waste.

Antibiotics

Ganoderma lucidum

bioremediation

LC-MS/MS

quinolones

Environmental pollution is an increasing global problem exacerbated by the growing use of active pharmaceutical ingredients such as antibiotics (Dąbrowska et. al. 2018). Antibiotics are used and misused in medical, veterinary, and farming (both aquaculture and agriculture) applications. They can enter the environment via wastewater effluents and agricultural runoffs, leading to the evolution and proliferation of antibiotic-resistant bacteria and genes (ARGs) in wastewater (e.g. Witte 1998; Batt et al. 2006; Kümmerer 2009a-b; Manzetti and Ghisi 2014; Jendrejewska and Karwowska 2018; Sanseverino et al. 2018; Pallares-Vega et al. 2019; Thiang et al. 2021). Antibiotic resistance in humans is estimated to have caused 4.95 million premature deaths worldwide in 2019, highlighting the need for remediation strategies to remove them from the environment (Murray et al. 2022).

Several methods are currently used to remove antibiotics from wastewater, including activated carbon adsorption, membrane filtration, coagulation and flocculation, advanced oxidation processes, bioadsorption and activated sludge systems (Copete-Pertuz et al. 2018). However, these methods are not highly effective in removing antibiotics, and vary in efficacy from plant to plant (reviews by Bouki et al. 2013, Fadário Frade et al. 2014, and Hazra and Durso 2022; also, work by Guerra et al. 2014, and report by Sanseverino et al. 2018). Additionally, there are several studies reporting that the conditions in wastewater treatment plants favor the proliferation of antibiotic-resistant bacteria and genes that can be discharged into the environment (Cacace et al. 2019). Wastewater and sewage sludge may be used as fertilizers, which means that they are constantly re-introduced to the food supply and natural ecosystems, despite containing antibiotic-resistant bacteria (Bouki et al. 2013, Fadário Frade et al. 2014; Guerra et al. 2014; Sanseverino et al. 2018; Hazra and Durso 2022).

Fungi have been used as a natural form of remediation of environmental contaminants. This is because the enzymatic system of fungi is known to remove various chemicals, through sorption or degradation into less toxic/innocuous molecules, as observed in studies with hydrocarbons, polychlorinated biphenyls, PCBs (Tomasini and León-Santiesteban 2019) and dichlorodiphenyltrichloroethane, DDT, in water by Phanerochaete chrysosporium (Bumpus et al. 1985). White rot fungi were also shown to transform antibiotics in synthetic wastewater (aqueous solutions of antibiotics) or in bioreactors, although the fungi are typically in the form of cultures grown in malt-based medium (Prieto et al. 2011; Migliore et al. 2012; Yang et al. 2013; Copete-Pertuz et al. 2018; Čvančarová et al. 2015; Vasiliadou et al. 2016) or in colonized agar (Gao et al. 2018; Rodríguez-Rodríguez et al. 2019). Data on the remediation potential of fungi grown in natural environments such as agricultural waste are lacking. From a practical standpoint, it would be useful and more sustainable to simply utilize fungi grown on natural waste products (as they do anyway) rather than in controlled media.

In this paper, the mycelium (the root structure) of a white rot fungus, Ganoderma lucidum, was grown on agricultural waste to investigate its remediation potential in synthetic wastewater. Ganoderma lucidum is rarely found in nature, and it is mostly cultivated artificially because it is in high demand for its medicinal properties (Boh et al. 2007). It was chosen in this experiment because of prior published work on its use on bioremediation (Prieto et al. 2011; Vasiliadou et al. 2016; Chakraborty and Abraham, 2017; Dąbrowska, et al. 2018 and the review by Rodríguez-Rodríguez et al. 2019). Twenty antibiotics from the six classes (Amphenicols, Sulfonamides, B-lactams, Lincosamides, Quinolones, and Macrolides) were tested in this experiment, as they are commonly detected in wastewater and aquaculture farming (Thiang et al. 2019).

2.1 Materials

LC/MS grade methanol, optima grade methanol, and toluene were obtained from Fisher Chemical, LC/MS grade. Acetonitrile was obtained from Thermo Scientific. Formic acid, HCl 37% and Na₂EDTA were purchased from Sigma-Aldrich (St. Louis, MO). Dimethyldichlorosilane was obtained from Fisher Scientific (Hampton, NH, USA).

Antibiotic standards used in this study were from the following classes: Amphenicols chloramphenicol (CAP), thiamphenicol (TAP), florfenicol (FF), florfenicol amine (FFA); Sulfonamides sulfadimethoxine (SDM), sulfasalazine (SSZ), sulfamethoxazole (SMX), sulfadiazine (SDZ); B-lactams ampicillin anhydrous (AMP), penicillin G potassium salt (PEN-G), penicillin V (PEN-V) and amoxicillin (AMOX); Lincosamides lincomycin (LIN); Quinolones enrofloxacin (ENRO), flumequine (FLU), norfloxacin (NOR), enoxacin (ENO); Macrolides erythromycin (ERYTH), virginiamycin complex (VIRG-M1 and VIRG-S1).

CAP (98.5%) was purchased from Crescent Chemical (Islandia, NY). TAP (99.3%), FF (98%), SDZ (99%) and AMOX (98%) were purchased from Fisher Scientific (Hampton, NH, USA). ERYTH (94.8%), ENRO (99.8%), FFA (99.3%), SDM (98.5%), AMP (99.6%), and NOR (98%) were purchased from Sigma Aldrich (St. Louis, MO). SSZ (100%), SMX (100%), LIN (98%), PEN-V (98.8%), PEN-G, FLU, ENO, and VIRG were purchased from Cayman Chemicals (Ann Arbor, MI).

Isotopically labeled surrogate standards including FFA-D3 (chemical purity: 98%; isotopic purity: 98.7%), CAP-D5 (chemical purity: 98%; isotopic purity: 98.3%), LIN-D3 (chemical purity: 95%; isotopic purity: 99.6%), SMX-D4 (chemical purity: 98%; isotopic purity: 99.2%), SMZ-D4 (chemical purity: 98%; isotopic purity: 95.9%), ERYTH-D6 (chemical purity: 95%; isotopic purity: 98.1%) and ENRO-D5 (chemical purity: 99.61%; isotopic purity: 99.40%), AMP-D5 (chemical purity: 95%; isotopic purity: 99.00%), were purchased from Toronto Research Chemicals (Toronto, Ontario, Canada).

2.2 Biomass Preparation

The biomass was prepared by mixing 140 g of oak pellets (MushroomMediaOnline, IA, USA), 98 g of almond shells from California (leftovers from a research project completed before Fall 2020) and 400 ml of filtered tap water. Used coffee grounds (3 tablespoons) from a local household and 28 g of fava stalks from a local vegetable garden (a Spring 2021 cover crop harvested, dried in the sun and then pulverized) were added as nitrogen-rich nutrient source. The biomass mix was sterilized in a conventional autoclave and in an autoclaveable bag with a filter patch (grow.bio, USA), at 121 deg. C and at 0.103 MPa (15 psi) for 20 minutes. It was then cooled for 1 hour in a fume hood. The autoclaved biomass mix was then inoculated with 5 ml Ganoderma lucidum liquid culture (Root Mushroom Farm, WA, USA). After 13 days in ambient conditions (21 deg. C in an air-conditioned room, October 2021 in Davis, CA), sustained growth of the mycelium into the biomass was observed (Fig. 1), and the bag containing mycelium and biomass was relocated to a -80 deg. C freezer for storage.

2.3 Antibiotic Mixture Preparation

Antibiotics were first prepared individually in methanol (amphenicols, sulfonamides, quinolones, macrolides) or water (B-lactams). Individual stock solutions of CAP, TAP, FF, FFA, SDM, SMX, ENRO, ERYTH, VIRG, LIN, CAP-D5, SMX-D4, SMZ-D4, ERYTH-D6, ENRO-D5 and TRIM-D3 were prepared in methanol at 1 mg/mL concentration. SSZ, SDZ, and LIN- D3 were prepared in methanol at a concentration of 0.5 mg/mL. ENO, NOR and FLU were prepared in methanol at concentration of 0.2 mg/mL. Β-lactams (AMP, PEN-G, PEN-V, AMOX and PEN-V-D5) were prepared in Milli-Q water at 1 mg/mL. AMP-D5 was prepared in Milli-Q water at 0.5 mg/mL. The stock solutions were diluted from 0.2 -1 mg/mL to individual ‘intermediate’ solutions of 10 µg/mL using the same solvent as the stock solution.

The individual intermediate solutions of unlabeled and labeled standards were used to prepare antibiotic mixture solutions of methanol-soluble and water-soluble antibiotic standards. These mixes were prepared separately for unlabeled and labeled antibiotics. For unlabeled antibiotics, both methanol-soluble and water-soluble mixes were prepared at concentration of 400 ng/mL. For labeled antibiotics, both methanol-soluble and water-soluble mixes were prepared at concentration of 1000 ng/mL. The water-soluble and methanol-soluble antibiotics were mixed at a 1:1 ratio, before use. The unlabeled and labeled standards were mixed separately, as they were needed for different purposes: the unlabeled mixes were used for spiking water samples, and the labeled mixes were spiked to each sample right before extraction, for the purpose of quantification.

Briefly, for methanol-soluble unlabeled antibiotic standards (n = 15; Table A.1 Appendix), 30 µL of each antibiotic from their individual intermediate solutions (10 µg/mL) were added to a 2 ml amber glass LC vial. The mixture was evaporated under nitrogen and reconstituted in 750 µL LC-MS methanol. For water-soluble antibiotic standards (n = 4; Table A.1 in the Appendix), 630 µL Milli-Q water was added to a 2 ml amber glass LC vial followed by adding, 30 µL of each of the four unlabeled water-soluble antibiotic standards (from their individual intermediate solutions (10 µg/mL).

For methanol-soluble labeled antibiotic standards, 50 µL of each labeled standard (from their individual intermediate solutions (10 µg/mL) were added to a 2 ml amber glass LC vial. Samples were vortexed, dried under nitrogen, and reconstituted in 500 µL LC-MS methanol. For water-soluble labeled antibiotic standard, 400 µL Milli-Q water was added to a 2 ml amber glass LC vial followed by adding 50 µL of the two water soluble standards from their individual intermediate solutions (10 µg/mL). Water-soluble and methanol-soluble antibiotic mixes were mixed at a 1:1 ratio before the experiment, resulting in working mix of unlabeled antibiotic standards at concentration of 200 ng/mL and working mix labeled standards at concentration of 500 ng/mL.

2.4 Glass Silanization Prior to Antibiotic Treatment

Some antibiotics including quinolones were shown to adsorb onto the glass surface, likely by interactions with the glass silanol groups (Emami et al. 2022). In order to avoid these interactions, glassware was silanized using dimethyldichlorosilane (DMDCS) prior to the experiment, in order to cover the silanol groups on the glass surface, using the method described by Ye et al. (2007). Briefly, the clean and dry glass vials (50 ml), were pre-washed with detergent-free soap and water and rinsed 5 times with distilled water, were treated with 5 ml 5% DMDCS in toluene solution. Silanization was completed by rinsing, vortexing, and dumping three more times with 5 ml of 5% DMDCS in toluene, 5 ml toluene, 5 ml methanol, and 5 ml Milli-Q water; respectively. Vials were left overnight to dry.

2.5 Experimental Design and Mycelium Treatment

Mycelial biomass (Ganoderma lucidum grown on the substrate described in Section 2.2) was thawed on ice for approximately 90 minutes. Approximately, 1 g of biomass was weighed and placed into 50 ml silanized Pyrex glass tubes. Milli-Q water (50 ml) was then added. Mycelium-water samples were spiked with 20 ng per 25 ml water of unlabeled antibiotics (Table A.1, Appendix) and incubated for 0 (baseline) or 3 days (n = 4 samples per incubation period). A parallel set of samples (n = 4 samples per timepoint) contained 20 ng antibiotics in 25 ml Milli-Q water without mycelium, as a negative control. Each sample was set in its own independent 50 ml of silanized Pyrex tube. Samples corresponding to Day 3 were incubated for 3 days, then on Day 3, the Day 0 (baseline) samples were completed (in separate Pyrex tubes). Additionally, one water method blank containing 25 ml of Milli-Q water and no antibiotics (n = 1 on Day 0 and Day 3), as well as one matrix blank containing mycelium and water only (no antibiotics; n = 1 on Day 0 and n = 1 on Day 3) were incorporated into the design to control for any background antibiotics potentially coming from the water itself or the mycelium, respectively. Antibiotics were extracted from both Day 0 and Day 3 samples as described below. Vials representing “Day 3” sample were covered in foil and shaken at room temperature using an Excella E24 shaker at 100 rpm for 3 days.

The final design and sample size for each incubation day (i.e. Day 0 and Day 3) were as follows:

n = 1, Water method blank (Water only) = 25 ml Milli-Q water

n = 1, Matrix method blank (Water + Mycelium) = 0.5 g mycelial biomass in 25 ml Milli-Q water

n = 4, Control sample (Water + Antibiotics), named "Control" in the statistical analysis = 20 ng antibiotics in 25 ml Milli-Q water

n = 4, Treatment sample (Water + mycelial biomass + Antibiotics), named "Treated" in the statistical analysis = 0.5 g mycelial biomass + 20 ng antibiotics in 25 ml Milli-Q water

After incubation, the samples were centrifuged using SORVALL RT 6000D at 3000 rpm for 10 minutes at room temperature. One water-mycelial biomass Day 3 sample and one water-mycelial biomass antibiotics Day 3 sample broke in the centrifuge. To protect the rest of the samples, including all those from Day 0, samples were just vortexed and allowed to sit for a few minutes. Water samples were then transferred to 40 ml non-silanized glass vials for the extraction of antibiotics.

2.6 Antibiotics Extraction

25 ml water samples placed in non-silanized glass vials were spiked with 20 ng of surrogate standard mixture containing CAP-D5, FFA-D3, TRIM-D3, SMZ-D4, SMX-D4, LIN-D3, ENRO-D5, ERYTH-D6, AMP-D5, and PEN-V-D5 (500 ng/mL per surrogate standard). Then, 1340 µL of 0.05 M Na₂EDTA in water (corresponding to 0.1% Na₂EDTA) was added to the glass vials. Aliquots were let to stay for 1 hour covered with aluminum foil with occasional shaking before solid phase extraction (SPE).

2.7 Solid Phase Extraction (SPE)

Antibiotics were extracted from the water samples using Waters OASIS HLB cartridges (60 mg, 3 cm). The cartridges were pre-conditioned with methanol (5ml), Milli-Q water (5ml), and pH = 2.5 water (5ml) made by adding 90 µL HCL (37%) to 150 ml Milli-Q water in a flask and verifying the pH with litmus paper. Water samples were loaded onto the conditioned HLB cartridges and allowed to elute. The SPE cartridges were then washed with 6 ml Milli-Q water and the SPE cartridges were dried under the vacuum manifold (Supelco Visiprep 24 SPE) for five minutes at 0.117 MPa (17 psi). Antibiotics were eluted into 8 ml glass vials using 5 ml Optima grade methanol. The vials were stored overnight in -20 deg. C freezer. The following day, the samples were evaporated under nitrogen for approximately two hours. Samples were reconstituted in 1 ml LC-MS methanol: water (1:1). All samples (n = 17) from the SPE step were vortexed for 3 minutes, transferred to 2 ml centrifuge tubes, and centrifuged for 2 min at 12000 rpm at 0 deg. C (Eppendorf, 5424 R, 13523 ×g). The samples were then transferred into filter centrifuge tubes (two tubes per sample each containing approx. <500 µL of sample per tube). The actual sample amount pipetted was 480 µL per tube. The filter tubes were centrifuged for 10 minutes at 12000 rpm (Eppendorf, 5424 R, 13523 ×g). after which the filters were discarded, and the extract was transferred to LC vials with slit caps to be analyzed by UPLC-MS/MS (total volume of 960 µL in LCMS vial).

2.8 LC-MS/MS Instrumentation

Antibiotic analysis was performed on an Agilent ultra-high pressure liquid chromatography system coupled to a 6460 Agilent triple quad (LC-MS/MS). Chromatographic separation of the antibiotics mixture was performed on AQUITY BEH C18 column (100 × 2.1 mm, 1.8 µm), using 0.1% formic acid in water (mobile phase A, (MPA)) and 0.1% formic acid in acetonitrile (mobile phase B, MPB) running at a flow rate of 0.300 mL/min and column temperature of 30 deg. C. MS/MS analysis was performed using Agilent Jetstream electrospray ionization (ESI) operating at both positive and negative mode. MS source parameters were as follows: sheath gas temperature of 375 deg. C, sheath gas flow of 8 L/min, drying gas temperature of 250 deg. C, nozzle voltage of 0 V, nebulizer gas pressure of 40 psi and capillary voltage of 3500 V. Collision-induced dissociation was carried out using nitrogen at the collision cell.

2.9 Antibiotics Quantification

Antibiotic concentrations in water samples were calculated by the internal standard calibration method where isotopically labeled surrogates were used to correct for recoveries and unlabeled standards were used to correct for the detector response factor. An 11-point standard calibration curve (0.01–100 ng/mL) containing a fixed amount of surrogate standard (20 ng/mL) was made to derive the response factor. Calibration curves were generated by quadratic regression and \(\frac{1}{{x}^{2}}\) weighting factor was applied. Peaks were integrated and analyzed using MassHunter Workstation Software, QQQ Quantitative Analysis (Agilent Technologies, Inc.).

2.11 Statistical Analysis

MATLAB (Mathworks) was used to perform a \({\chi }^{2}\)goodness-of-fit tests on the data and its log10 transformed value. The rest of the statistical analysis was conducted with Graphpad Prism9 (GraphPad) using for most cases a two-way repeated ANOVA (time as repeated measure and treatment as a main factor) with a 5% significance level applied to the log10 values (for reduced data skewness) of control and treated data. For 3 out of the 20 sets of log10 data (namely, SSZ, SDM and SDZ), the residuals were found not to be normally distributed (a key requirement for ANOVA). Therefore, a mixed-effects model (with Šidák's multiple comparison) was employed in place of ANOVA, also with Prism9 software.

As shown in Fig. 2, Tables A.6-A.8 and Figure A.3 in the Appendix, there was a significant time effect for SSZ and PEN-G antibiotics on Day 3 compared to Day 0 (baseline). There were significant treatment and time effects for ERYTH and VIRG-S1 antibiotics, with the control samples exhibiting a larger decrease in concentration over time compared to antibiotic-spiked samples (Figure A.6 in the Appendix). For SDM, SSZ, ENO, ENRO, NOR antibiotics, the mycelium + biomass treatment reduced antibiotic concentrations more than the control samples on Day 3, compared to Day 0 (Fig. 2). No significant reductions were seen in the parallel samples of water containing the antibiotics but lacking the mycelium after 3 days. As seen in Fig. 2, the mycelium + biomass treatment appeared to be most useful to remediate quinolones (particularly ENRO), a notoriously recalcitrant class of antibiotics, followed by those two sulfonamides, SDM and SSZ. The analysis of matrix effects (ME) from the water-mycelium matrix is reported in the Appendix: matrix effects were comparable between the groups.

The main finding of this study is that a 3-day treatment of antibiotic-containing water with mycelium grown on agricultural waste reduced antibiotic concentrations in water by up to 82.4%. Our findings provide proof-of-concept demonstration of the use of naturally grown fungus for antibiotic remediation in contaminated water.

The effects of mycelium incubation on antibiotic reduction in water were mostly greater than previously reported with other fungal strains or similar strains grown on different media, highlighting the importance of utilizing the right substrate (i.e. agricultural waste) for maximizing fungal growth and potency. With respect to published results in mycelium cultures without agricultural waste, Vasiliadou et al. (2016) reported a less than 20% change after an exposure of 7 days to sulfamethoxazole (SMX) by Ganoderma lucidum grown with malt extract. Chakraborty and Abraham (2017) reported 100% removal after 4 days of exposure to enrofloxacin (ENRO) by Ganoderma lucidum, findings which are comparable to our results (-82.4% after 3 days of treatment). Martens et al. (1996) showed considerably less success when enrofloxacin (ENRO) was degraded by other mycelial species (varying between 0.19% for white rot fungus, and 25.6% for brow rot fungus, and after 56 days), likely due to differences in the species used to degrade antibiotics. Čvančarová et al. (2015) showed that norfloxacin (NOR) degraded by up to 100% with Irpex lacteus after 10 days, and by 10% with Dichomitus squalens after 10 days. Again, with all these studies, a longer duration was needed to achieve similar antibiotic reduction magnitude as in this study, where it was achieved within 3 days.

The reduction in antibiotic concentrations after 3-day mycelium incubation could be due to increased adsorption of antibiotics to the mycelium surface, or internalization and degradation by mycelium enzymes. Adsorption is an unlikely mechanism as it is non-specific, meaning that all 20 antibiotics applied would have adhered to the mycelium non-selectively. Enzymatic degradation is more plausible, given that 5 antibiotics belonging to the quinolone and sulfonamide families decreased in concentration after 3 days. Studies have demonstrated the ability of fungi to oxidize antibiotics in vitro at various success levels, via peroxidases, ligninase, xylanases, cellulases (Dąbrowska et al. 2018).

There are several limitations worth noting. Since our exposure lasted only 3 days, we do not know whether the results would be further enhanced with prolonged incubation with the mycelium. Additionally, different agri-waste substrates may have different priming effects on mycelium potency (in terms of antibiotic reduction capability) and growth potential. Studies involving prolonged incubation of mycelium grown on different agri-waste substrates (e.g. grape pomace) with contaminated water, are needed to optimize the remediation potential of mycelium. A final limitation relates to the risk of biomass contamination with antibiotic-degrading bacteria. Although the biomass was sterilized before inoculation, it would have been useful to test a separate control group consisting of water-uninoculated biomass plus antibiotics to test for symbiotic effects involving bacteria.

In our investigation, we showed that a mycelium (Ganoderma lucidum) grown on agricultural waste reduces the concentration of five antibiotics in synthetic wastewater. In just 3 days, the concentrations of 3 quinolones and 2 sulfonamides were reduced compared to baseline, and with respect to spiked synthetic water without mycelium/biomass. The results on quinolone antibiotics are particularly promising because of their extensive consumption worldwide (e.g. Thiang et al. 2019; Yang et al. 2021), and the inability of conventional wastewater treatment plants to remove them, thus fostering antibiotics resistance and harming the environment (Collignon et al. 2016). In summary, our work indicates that mycelium grown on re-purposed agricultural waste is a promising, novel method to remove certain antibiotics from water, and warrants further validation with contaminated water samples.

Amoxicillin

AMOX

Ampicillin

AMP

Chloramphenicol

CAP

Enrofloxacin

ENRO

Enoxacin

ENO

Erythromycin

ERYTH

Florfenicol

Florfenicol amine

FFA

Flumequine

FLU

Lincomycin

LIN

Norfloxacin

NOR

Penicillin G

PEN-G

Penicillin V

PEN-V

Solid phase extraction

SPE

Sulfadimethoxine

SDM

Sulfadiazine

SDZ

Sulfamethoxazole

SMX

Sulfasalazine

SSZ

Thiamphenicol

TAP

Tilmicosin

Virginiamycin M1:VIRG-M1

Virginiamycin S1

VIRG-S1.

Compliance with Ethical Standards

Declaration on funding sources

This work was partially supported by a grant by UC Davis The Green Initiative Fund (S21-84, to V.L.S.), and partially by the National Institute of Food and Agriculture, United States Department of Agriculture, USDA-NIFA [Grant no. 2018-67017-28116/Project Accession no. 1015597, to A.Y.T.]. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. The authors thank Dr. Nitin Nitin (UC Davis Food Science and Technology) and his Ph.D. student Yixing Lu for access to an autoclave.

Declaration of interest statement

The authors declare that they have no relevant financial or non-financial interests to disclose.

Declaration of author consent and contributions

All authors whose names appear on the submission

1) made substantial contributions to the conception or design of the work: V.S., V.L.S., S.E. and A.Y.T designed the experiments. V.S. and S.E. performed the experiments and analyzed the data. V.S. wrote the original draft. V.S. and S.E. edited the manuscript. V.L.S. and A.Y.T. reviewed and edited the manuscript and carried out the statistical analysis. V.L.S. and A.Y.T. share equally the intellectual property of this work.

2) approved the version to be published, and

3) agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Declaration of consent to participate

This research did not use human and/or animal subjects.

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Use of Ganoderma lucidum Grown on Agricultural Waste to Remove Antibiotics from Water

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1 Materials

2.2 Biomass Preparation

2.3 Antibiotic Mixture Preparation

2.4 Glass Silanization Prior to Antibiotic Treatment

2.5 Experimental Design and Mycelium Treatment

2.6 Antibiotics Extraction

2.7 Solid Phase Extraction (SPE)

2.8 LC-MS/MS Instrumentation

2.9 Antibiotics Quantification

2.11 Statistical Analysis

3. Results

4. Discussion

5. Conclusions

Abbreviations

Declarations

References

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