Using biobeds for the treatment of fungicide-contaminated effluents from various agro-food processing industries: Microbiome responses and mobile genetic element dynamics.

Agro-food processing industries generate large amounts of pesticide-contaminated effluents that pose a significant environmental threat if managed improperly. Biopurification systems like biobeds could be utilized for the depuration of these effluents although direct evidence for their efficiency are still lacking. We employed a column leaching experiment with pilot biobeds to (i) assess the depuration potential of biobeds against fungicide-contaminated effluents from seed-producing (carboxin, metalaxyl-M, fluxapyroxad), bulb-handling (thiabendazole, fludioxonil and chlorothalonil) and fruit-packaging (fludioxonil, imazalil) industries, (ii) to monitor microbial succession via amplicon sequencing and (iii) to determine the presence and dynamics of mobile genetic elements like intl1, IS1071, IncP-1 and IncP-1ε often associated with the transposition of pesticide-degrading genes. Biobeds could effectively retain (adsorbed but extractable with organic solvents) and dissipate (degraded and/or not extractable with organic solvents) the fungicides that were contained in the agro-industrial effluents with 93.1-99.98% removal efficiency in all cases. Lipophilic substances like fluxapyroxad were mostly retained in the biobed while more polar substances like metalaxyl-M and carboxin were mostly dissipated or showed higher leaching potential like metalaxyl-M. Biobeds supported a bacterial and fungal community that was not affected by fungicide application but showed clear temporal patterns in the different biobed horizons. This was most probably driven by the establishment of microaerophilic conditions upon water saturation of biobeds, as supported by the significant increase in the abundance of facultative or strict anaerobes like Chloroflexi/Anaerolinae, Acidibacter and Myxococcota. Wastewater application did not affect the dynamics of mobile genetic elements in biobeds whose abundance (intl1, IS1071, IncP-1ε) showed significant increases with time. Our findings suggest that biobeds could effectively decontaminate fungicide-contaminated effluents produced by agro-food industries and support a rather resilient microbial community.

The Effects of Quinone Imine, a New Potent Nitrification Inhibitor, Dicyandiamide, and Nitrapyrin on Target and Off-Target Soil Microbiota.

Dicyandiamide (DCD) and nitrapyrin (NP) are nitrification inhibitors (NIs) used in agriculture for over 40 years. Recently, ethoxyquin (EQ) was proposed as a novel potential NI, acting through its derivative quinone imine (QI). Still, the specific activity of these NIs on the different groups of ammonia-oxidizing microorganisms (AOM), and mostly their effects on other soil microbiota remain unknown. We determined the impact of QI, and comparatively of DCD and NP, applied at two doses (regular versus high), on the function, diversity, and dynamics of target (AOM), functionally associated (nitrite-oxidizing bacteria-NOB), and off-target prokaryotic and fungal communities in two soils mainly differing in pH (5.4 versus 7.9). QI was equally effective to DCD but more effective than NP in inhibiting nitrification in the acidic soil, while in the alkaline soil QI was less efficient than DCD and NP. This was attributed to the higher activity of QI toward AOA prevailing in the acidic soil. All NIs induced significant effects on the composition of the AOB community in both soils, unlike AOA, which were less responsive. Beyond on-target effects, we noted an inhibitory effect of all NIs on the abundance of NOB in the alkaline soil, with Nitrobacter being more sensitive than Nitrospira. QI, unlike the other NIs, induced significant changes in the composition of the bacterial and fungal communities in both soils. Our findings have serious implications for the efficiency and future use of NIs on agriculture and provide unprecedented evidence for the potential off-target effects of NIs on soil microbiota. IMPORTANCE NIs could improve N use efficiency and decelerate N cycling. Still, we know little about their activity on the distinct AOM groups and about their effects on off-target soil microorganisms. Here, we studied the behavior of a new potent NI, QI, compared to established NIs. We show that (i) the variable efficacy of NIs across soils with different pH reflects differences in the inherent specific activity of the NIs to AOA and AOB; (ii) beyond AOM, NIs exhibit negative effects on other nitrifiers, like NOB; (iii) QI was the sole NI that significantly affected prokaryotic and fungal diversity. Our findings (i) highlight the need for novel NI strategies that consider the variable sensitivity of AOM groups to the different NIs (ii) identify QI as a potent AOA inhibitor, and (iii) stress the need for monitoring NIs' impact on off-target soil microorganisms to ensure sustainable N fertilizers use and soil ecosystem functioning.

Comparison of Novel and Established Nitrification Inhibitors Relevant to Agriculture on Soil Ammonia- and Nitrite-Oxidizing Isolates.

Nitrification inhibitors (NIs) applied to soil reduce nitrogen fertilizer losses from agro-ecosystems. NIs that are currently registered for use in agriculture appear to selectively inhibit ammonia-oxidizing bacteria (AOB), while their impact on other nitrifiers is limited or unknown. Ethoxyquin (EQ), a fruit preservative shown to inhibit ammonia-oxidizers (AO) in soil, is rapidly transformed to 2,6-dihydro-2,2,4-trimethyl-6-quinone imine (QI), and 2,4-dimethyl-6-ethoxy-quinoline (EQNL). We compared the inhibitory potential of EQ and its derivatives with that of dicyandiamide (DCD), nitrapyrin (NP), and 3,4-dimethylpyrazole-phosphate (DMPP), NIs that have been used in agricultural settings. The effect of each compound on the growth of AOB (Nitrosomonas europaea, Nitrosospira multiformis), ammonia-oxidizing archaea (AOA; "Candidatus Nitrosocosmicus franklandus," "Candidatus Nitrosotalea sinensis"), and a nitrite-oxidizing bacterium (NOB; Nitrobacter sp. NHB1), all being soil isolates, were determined in liquid culture over a range of concentrations by measuring nitrite production or consumption and qPCR of amoA and nxrB genes, respectively. The degradation of NIs in the liquid cultures was also determined. In all cultures, EQ was transformed to the short-lived QI (major derivative) and the persistent EQNL (minor derivative). They all showed significantly higher inhibition activity of AOA compared to AOB and NOB isolates. QI was the most potent AOA inhibitor (EC50 = 0.3-0.7 µM) compared to EQ (EC50 = 1-1.4 µM) and EQNL (EC50 = 26.6-129.5 µM). The formation and concentration of QI in EQ-amended cultures correlated with the inhibition patterns for all isolates suggesting that it was primarily responsible for inhibition after application of EQ. DCD and DMPP showed greater inhibition of AOB compared to AOA or NOB, with DMPP being more potent (EC50 = 221.9-248.7 µM vs EC50 = 0.6-2.1 µM). NP was the only NI to which both AOA and AOB were equally sensitive with EC50s of 0.8-2.1 and 1.0-6.7 µM, respectively. Overall, EQ, QI, and NP were the most potent NIs against AOA, NP, and DMPP were the most effective against AOB, while NP, EQ and its derivatives showed the highest activity against the NOB isolate. Our findings benchmark the activity range of known and novel NIs with practical implications for their use in agriculture and the development of NIs with broad or complementary activity against all AO.