Alcohols as inhibitors of ammonia oxidizing archaea and bacteria

Abstract Ammonia oxidizers are key players in the global nitrogen cycle and are responsible for the oxidation of ammonia to nitrite, which is further oxidized to nitrate by other microorganisms. Their activity can lead to adverse effects on some human-impacted environments, including water pollution through leaching of nitrate and emissions of the greenhouse gas nitrous oxide (N2O). Ammonia monooxygenase (AMO) is the key enzyme in microbial ammonia oxidation and shared by all groups of aerobic ammonia oxidizers. The AMO has not been purified in an active form, and much of what is known about its potential structure and function comes from studies on its interactions with inhibitors. The archaeal AMO is less well studied as ammonia oxidizing archaea were discovered much more recently than their bacterial counterparts. The inhibition of ammonia oxidation by aliphatic alcohols (C1-C8) using the model terrestrial ammonia oxidizing archaeon ‘Candidatus Nitrosocosmicus franklandus’ C13 and the ammonia oxidizing bacterium Nitrosomonas europaea was examined in order to expand knowledge about the range of inhibitors of ammonia oxidizers. Methanol was the most potent specific inhibitor of the AMO in both ammonia oxidizers, with half-maximal inhibitory concentrations (IC50) of 0.19 and 0.31 mM, respectively. The inhibition was AMO-specific in ‘Ca. N. franklandus’ C13 in the presence of C1-C2 alcohols, and in N. europaea in the presence of C1-C3 alcohols. Higher chain-length alcohols caused non-specific inhibition and also inhibited hydroxylamine oxidation. Ethanol was tolerated by ‘Ca. N. franklandus’ C13 at a higher threshold concentration than other chain-length alcohols, with 80 mM ethanol being required for complete inhibition of ammonia oxidation.


Introduction
Ammonia oxidizing archaea (A O A) and bacteria (A OB) are major microbial drivers of the terrestrial nitrogen cycle, along with comammox bacteria (r e vie wed in Lehtovirta-Morley 2018 ).A O A and AOB oxidize ammonia to nitrite, which is further oxidized to nitrate by nitrite oxidizing bacteria.Nitrate is a water pollutant and can be leac hed fr om soils, causing eutrophication of aquatic ecosystems.Nitrous oxide is a po w erful greenhouse gas and the majority of its anthropogenic emissions are from agricultural soils (Reay et al. 2012 ).The activity of ammonia oxidizing micr oor ganisms is responsible for significant loss of nitrogen from soil and has broad ecological and economic consequences (Raun and Johnson 1999 ).
Ammonia monooxygenase (AMO) is a key enzyme in ammonia oxidation and belongs to the copper monooxygenase family (CuMMO).It catalyzes the first step in nitrification, the oxidation of ammonia to hydroxylamine .T his enzyme is found in all groups of aerobic ammonia oxidizers.It has never been purified in its activ e form, whic h makes this enzyme challenging to study.Current knowledge of the structure and function of this enzyme comes from the similarity of bacterial AMO with the particulate methane monooxygenase (pMMO) from methanotrophs (Lontoh et al. 2000 ;Semrau et al. 2010 ;Ross et al. 2019, Zhu et al. 2022 ), as well as studies based on its interaction with inhibitors (Taylor et al. 2013, 2015, Wright et al. 2020 ).The archaeal AMO is phylogenetically distinct from the bacterial AMO and pMMO, and its structure is likely to be different (Hodgskiss et al. 2023 ).Studies comparing the effects of inhibitors, particularly compounds that are substrate analogues, on the archaeal and bacterial AMO can provide insights into the substr ate r ange and function of the AMO.
Bacterial AMO, like other CuMMO superfamily enzymes, can oxidize a wide variety of substrates, including methane, methanol, linear 1-alkanes , alkenes , halogenated hydrocarbons , sulphides , and aromatic compounds (Hyman et al. 1988a ;Rasche et al. 1991, Juliette et al. 1993, Keener and Arp 1993, 1994 ), but oxidation of these substrates does not yield energy and a source of reductant is r equir ed for these co-oxidation reactions .T here are no known alternativ e substr ates for the arc haeal AMO, but se v er al inhibitors hav e been described.Pr e vious studies found that archaeal AMO fr om se v er al gener a of A O A w as inhibited b y C 2 to C 5 1-alkynes, and AMO from the bacterium Nitrosomonas europaea was inhibited b y C 2 -C 8 1-alk ynes (Taylor et al. 2013, 2015, Wright et al. 2020 ).This indicates that AOB has a broader substrate and inhibitor range than A O A, and 1-octyne is ther efor e r outinel y used for selectiv e inhibition of bacterial (but not archaeal) ammonia oxidizers (Lu et al. 2015, Hink et al. 2017, Lin et al. 2021 ).Inter estingl y, phen ylacetylene and octyne do not interact with the same site of the archaeal AMO as ammonia and acetylene, and A O A are inhibited by phenylacetylene and octyne at higher concentrations than AOB (Wright et al. 2020 ).Methane is a competitive inhibitor of the AMO in both archaea and bacteria (Suzuki et al. 1976, Oudova-Riv er a et al. 2023 ).Oxidation of alkanes and alkenes by CuMMO superfamily monooxygenases, including the bacterial AMO, generates alcohols as reaction products (Hyman et al. 1988b ).For example, N. europaea oxidizes alkanes of up to C 8 , and the reaction pr oducts ar e a mixtur e of differ ent isomers of alcohols (Hyman et al. 1988b ).Whilst these alcohols may be further oxidized by alcohol dehydrogenases in microbial cells, they can also interact with the CuMMO due to their structural similarity to the nativ e substr ates.Methanol is also oxidized by bacterial AMO, as was demonstrated by the inhibition of methanol oxidation in N. europaea by acetylene or all ylthiour ea (ATU), specific inhibitors of AMO (Voysey and Wood 1987 ).Although the production of alcohols by both bacterial AMO and pMMO has been studied pr e viousl y, v ery little is known about the inhibition of archaeal and bacterial AMO by alcohols.Recently, methanol was shown to be a specific competitive inhibitor of the archaeal AMO fr om ' Ca.N. fr anklandus' C13 (Oudov a-Riv er a et al. 2023 ).Furthermore, specific inhibition of the bacterial AMO from N. europaea by short-chain linear C 1 -C 4 alcohols, including methanol, ethanol, and both isomers of propanol and butanol, was pr e viousl y r eported (Hooper and Terry 1973 ).Soluble methane monooxygenase (sMMO) from Methylococcus capsulatus can oxidize methanol with K m(app) of 0.95 mM (Colby et al. 1977 ).Constitutiv e expr ession of the sMMO in a methanol dehydrogenase double mutant of the facultativ e methanotr oph Methylocella silvestris BL2 enables it to grow on methanol and ethanol, suggesting that sMMO can oxidize these alcohols (Crombie 2022 ).
To our knowledge, the effects of longer-chain alcohols ( > C 5 ) on either A O A or A OB have not been studied.AOB are sensitive to a wider range of alkynes than A O A, including chain lengths of up to C 9 (Ta ylor et al. 2013 ).Furthermore , AOB are inhibited by octyne and phenylacetylene at a lo w er concentration than A O A (Taylor et al. 2013, 2015, Wright et al. 2020 ).It seems plausible, although untested, that responses of AOB and AOA to alcohols may follow similar inhibition patterns.Ho w e v er, the c hemical pr operties of alcohols and alkynes ar e differ ent, as alcohols contain a polar, h ydrophilic h ydroxyl group (-OH).The active site of the AMO is thought to reside in a hydrophobic cavity, as it is in pMMO (Ng et al. 2008 ), and the incr easingl y amphiphilic nature of longer chainlength alcohols may cause them to interact with membranes differ entl y than alkynes would.
In addition to being potentially useful compounds to study the function of archaeal and bacterial AMO, alcohols naturally occur in soils as products of plant and microbial metabolism.The main sources of methanol, ethanol, and other alcohols in soils include the breakdown of pectin and other methoxylated compounds from plants (Kimmerer and MacDonald 1987 , Kirstine and Galball y 2012 ), r oot exudation (Sm uc ker and Eric kson 1987 ) and decay of organic matter (Warneke et al. 1999, Isidorov et al. 2003 ).In addition, methylotrophs are associated with plant roots, suggesting the presence of methanol in the rhizosphere (Macey et al. 2020 ).A O A and A OB ar e commonl y pr esent in the soil and rhizosphere, and could therefore be exposed to alcohols of various chain lengths.Although there is limited information on the envir onmental concentr ations of differ ent alcohols , their effect ma y be r ele v ant in specific envir onmental nic hes .T he aim of this study was to examine the effects of alcohols on model terrestrial ammonia oxidizing arc haeon ' Ca.N. fr anklandus' C13 and ammonia oxidizing bacterium N. europaea using a range of concentrations and chain lengths (C 1 -C 8 ).

Whole-cell activity assays for examining inhibition by alcohols
Nitrosomonas europaea and ' Ca.N. franklandus' C13 cells were harvested at mid-exponential phase ( ∼800 μM NO 2 − accumulated) by filtration onto 0.22 μm pore-size membrane filter (PES, Merck Millipor e).Cells wer e then washed and resuspended in FWM salts (i.e.FWM without added NH 4 Cl and other medium components as described above) buffered with 10 mM HEPES buffer (pH 7).Cells were incubated at 28 • C for N. europaea and 37 • C for ' Ca.N. franklandus' C13 for ∼1 h until endogenous r espir ation ceased.A total volume of 5 ml of cell suspension was placed in 24 ml glass vials, alcohols (C 1 -C 8 ) were added to final concentrations in the range of 2-100 mM and immediately sealed with twiceautoclav ed gr ay butyl rubber stoppers and crimp sealed.Hexanol, heptanol, and octanol have relatively low solubility in water, r eac hing full saturation at 58, 17, and 2 mM, respectively.Although these alcohols were added at concentrations above their solubility for the consistency of the experimental design, the limit of solubility was considered when evaluating the results.Reactions were initiated by addition of 500 μM NH 4 Cl or 200 μM NH 2 OH.Vials containing N. europaea and ' Ca.Nitrosocosmicus franklandus' C13 cells were incubated at 28 • C and 37 • C, r espectiv el y.The activity of cultures was followed by monitoring nitrite accumulation over 1 h, measured in 15 min intervals using Griess reagent in a 96well plate format as pr e viousl y described (Lehtovirta-Morley et al. 2014 ).The final cell concentration in the activity assays was ∼10 7 cells ml −1 for both N. europaea and ' Ca .N. franklandus' C13, which equate to ∼1.2 and 5.55 μg pr otein ml −1 , r espectiv el y.Cell counts were performed as previously described (Oudova-Rivera et al. 2023 ).Pr otein concentr ations wer e determined using a Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Scientific) according to the manufacturer instructions .One-wa y ANOVA with LSD post-hoc tests were performed using SPSS statistics software version 27 (IBM).
The r eaction v elocity of ammonia and hydro xylamine o xidation was determined by measuring the rate of nitrite accumulation over time ( μM min −1 ).The logarithm of the reaction velocity, calculated as a percentage of the non-inhibited control, was plotted against the inhibitor concentration resulting in a linear relationship ( Figs.S1 and S2 ).The half-maximal inhibitory concentration (IC 50 ) was calculated from the slope and y-axis intercept over the linear part of the curve following the formula: where b is the estimated y-axis intercept and m is the estimated slope.
Inhibition by alcohols was tested at a fixed substrate concentration of 500 μM NH 4 Cl or 200 μM hydroxylamine for both N. europaea and ' Ca.N. franklandus' C13.Ammonia and hydroxylamine concentr ations wer e selected based on pr e viousl y published K m values and activity assays (Suzuki et al. 1974, Martens-Habbena et al. 2009, Oudov a-Riv er a et al. 2023 ).Full y inhibitory concentr ations of alcohols were determined experimentally with a series of activity assa ys , gr aduall y narr o wing do wn the range of alcohol concentr ations a pplied.Full inhibition was defined as a lac k of sustained nitrite accum ulation, wher e ther e was either no nitrite accumulation at all, or where a minor accumulation of nitrite was initially detected but ceased (potentially due to a delayed effect of the inhibitor).

Inhibition of the ammonia oxidizing archaeon ' Ca. N. franklandus' C13 by alcohols
Inhibition of ammonia oxidation was tested using whole-cell activity assays with the concentration of alcohols r anging fr om 2 to 100 mM and fixed substrate ammonia concentration of 500 μM NH 4 Cl.Specificity of the inhibition of AMO was tested by activity assays with 200 μM hydroxylamine and a concentration of each alcohol that resulted in complete inhibition of ammonia oxidation when using ammonia as substrate (Table 1 ).Because hydroxylamine, the product of ammonia oxidation, is a pathway intermediate downstream of AMO, any inhibitors specific to AMO should not interfere with hydroxylamine oxidation.
Both methanol and ethanol were specific inhibitors of the archaeal AMO in ' Ca .N. franklandus' C13 and did not interfere with hydro xylamine o xidation (Fig. 1 ).Ho w e v er, hydr o xylamine o xidation was incr easingl y inhibited by the incr easing c hain lengths of alcohols of > C 3 .Complete inhibition of ammonia oxidation was ac hie v ed at 8 mM methanol and the IC 50[methanol] was 0.19 mM (Table 1 ), making methanol the most potent inhibitor tested.Nitrite accumulation was not affected when the cells were supplied with hydroxylamine and 8 mM methanol, indicating that methanol is a specific inhibitor of the AMO (Fig. 1 ) as pr e viousl y r eported (Oudov a-Riv er a et al. 2023 ).In contrast to methanol, ' Ca.N. franklandus' C13 tolerated relatively high concentrations of ethanol.A total concentration of 80 mM ethanol was required for complete inhibition of ammonia oxidation in ' Ca.N. franklandus' C13 and the IC 50 for ethanol (7.2 mM) was more than an order of magnitude higher than the IC 50 for methanol.Although our study focussed on the effect of primary alcohols on ammonia oxidizers, a secondary alcohol (2-propanol) was also included.' Ca.N. franklandus' C13 toler ated 2-pr opanol better than 1-pr opanol, with IC 50 v alues 26.8 and 6.2 mM, r espectiv el y (Table 1 ).
In ' Ca.N. franklandus' C13, the inhibition by longer chain linear alcohols ( > C 4 ) was not specific to AMO, as they inhibited the oxidation of hydroxylamine as well as oxidation of ammonia (Fig. 1 ).At butanol and pentanol concentr ations full y inhibitory to ammonia oxidation, the rates of nitrite accumulation from hydro xylamine o xidation were 55% ±2.3% and 53% ± 2.8%, respectiv el y, compar ed to the non-inhibited control.A total volume of 15 mM hexanol completely inhibited ammonia oxidation with a IC 50[hexanol] of 2.4 mM (Table 1 ), and the nitrite accumulation rate in the presence of hydroxylamine was only 13% ± 0.9% compared to the non-inhibited contr ol.Similarl y, heptanol caused full inhibition at 17 mM concentration, IC 50[heptanol] was 3.6 mM and providing hydroxylamine as a substrate resulted in 10% ± 0.5% activity compared to non-inhibited control.The limit of solubility for octanol in water is 2 mM, a concentration which inhibited ammonia oxidation by 39%.Additional octanol caused further inhibition, possibly because of its general toxicity.Full inhibition was ac hie v ed when 10 mM octanol w as added.Ho w e v er, 33% ± 1.4% of o xidation acti vity was r estor ed in the pr esence of hydr oxylamine.

Inhibition of the ammonia oxidizing bacterium N . europaea b y alcohols
As observed for the A O A ' Ca .N. franklandus' C13, the most potent inhibitor of N. europaea among all alcohols tested was methanol (IC 50[methanol] = 0.31 mM) (Fig. 2 , Table 1 ).As with methanol, ethanol was a specific inhibitor of both archaeal and bacterial AMO.Consistent with the findings in ' Ca.N. franklandus' C13, N. europaea tolerated relatively high concentrations of ethanol with 90 mM ethanol r equir ed for complete inhibition of ammonia oxidation.The IC 50 for ethanol in both micr oor ganisms was one to two orders of ma gnitude gr eater than for methanol (7.2 and 20.9 mM for ' Ca.N. franklandus' C13 and N. europaea, r espectiv el y).The cause for the substantial difference between the inhibition profiles of methanol and ethanol is not clear but may be related to production of aldehydes, which are potential oxidation products of alcohols, or to free radical scavenging, as suggested by Hooper and Terry ( 1973 ).
Unlike with ' Ca .N. franklandus' C13, with N. europaea both isomers of propanol were specific inhibitors of AMO (Fig. 2 B).2propanol was better tolerated than 1-propanol, with the highest IC 50 (32.6mM) for 2-propanol.This differs from results obtained by Hooper and Terry ( 1973 ), who found that N. europaea was maximally inhibited by a lower concentration of 2-propanol (0.13 M) than 1-propanol (0.33 M).Observed differences between the isomers of propanol are unlikely to be due to steric hindrance alone, because hydrocarbons of < C 5 are thought to interact with the active site of the bacterial and archaeal AMOs (Taylor et al. 2013, Wright et al. 2020 ).
Longer chain-length alcohols ( > C 4 ) were non-specific inhibitors of ammonia oxidation by N. europaea .Butanol inhibited nitrite accum ulation fr om ammonia with a IC 50 of 7.6 mM.In the pr esence of hydroxylamine at fully inhibitory butanol concentration, the reaction velocity was significantly reduced to 83% ± 1.8% compared to the control, suggesting that AMO was not the sole target of inhibition.Pentanol (IC 50 = 5.3 mM) and hexanol (IC 50 = 2.2 mM) applied at a concentration which was fully inhibitory in the presence of ammonia, only allo w ed for minor accumulation of nitrite when cells were supplied with hydroxylamine, at rates of 27% ± 2.6% and 18% ± 5.6%, r espectiv el y, compar ed to the control.The inhibition of ammonia oxidation by heptanol (IC 50 = 4.5 mM) as with octanol was non-specific, with no activity detected in the presence of hydroxylamine.

Significance of findings in the context of ecology and physiology of ammonia oxidation
This study compared the inhibition by aliphatic alcohols of two widespread model nitrifiers, the archaeon ' Ca .N. franklandus' C13 and the bacterium N. europaea .Methanol and ethanol were specific inhibitors of the AMO, and longer-chain alcohols (C 4 -C 8 ) were nonspecific inhibitors of both nitrifiers.With the exception of ethanol, N. europaea was more tolerant than ' Ca.N. franklandus' C13 to all alcohols tested.Although there is currently a lack of knowledge about environmental sources, sinks and concentrations of alcohols, especiall y longer-c hain alcohols ( > C 5 ), a fe w studies hav e measur ed concentr ations of methanol, ethanol, and pr opanol pr edominantly in aquatic habitats.Methanol concentrations in the N/A * * 20 * * * Full inhibition was defined as a lack of sustained nitrite accum ulation, wher e ther e was either no nitrite accumulation at all, or where a minor accumulation of nitrite was initially detected but ceased (potentially due to a delayed effect of the inhibitor).* * The limit of solubility for octanol in water is 2 mM.seaw ater range betw een 61 and 97 nM (Dixon et al. 2011, Bates et al. 2021 ).In the Atlantic, the concentration of ethanol was reported to be 2-33 nM, and concentrations of 1-propanol and 2propanol 2-22 nM and 1-19 nM, r espectiv el y (Beale et al. 2010 ).
In an ice core from Greenland, methanol concentrations were 83-1666 nM andethanol 16-424 nM (Felix et al. 2019 ).In soil ecosystems, methanol and ethanol are present in the rhizosphere as a component of root exudates or product of decomposition and − mg protein −1 min −1 and 0.54 μmol NO 2 − mg protein −1 min −1 for ammonia and hydroxylamine as substrates, respectively.Significant differences between cells inhibited with the alcohols shown and non-inhibited contr ols ar e shown as * P < 0.05; * * * P < 0.001.Err or bars r epr esent standard err or ( n = 3).fermentation (Young et al. 1977, Haldar and Sengupta 2015, Macey et al. 2020 ).Concentr ations of ethanol in the rhizospher e of Lupinus angustifolius L. were found to be between 1 and 5 mM (Young et al. 1977 ), which is significantly higher than in e .g. sea water and could potentially have minor inhibitory effect on ammonia oxidizers.Although methanol concentration in soil is unknown, it has been suggested that there may be 'hotspots' with ele v ated methanol concentration in the vicinity of plant material and soil methylotrophs have been shown to oxidize methanol at low ( < 10 μM) concentr ations (Stac heter et al. 2013 ).In addition, it is estimated that 28 ×10 12 mol yr −1 of methanol ar e pr oduced fr om decaying and living plant material, but only 4.9 ×10 12 mol yr −1 of methanol are emitted from soils, potentially suggesting that the ga p between pr oduction and emission of methanol in soils may be due to active methanol oxidizing microbial communities (Kolb 2009 ).Both A O A and A OB are ubiquitous microorganisms, and A O A can sometimes dominate the arc haeal comm unity in the rhizosphere (Prudence et al. 2021 ).Intriguingly, a previous DNA stable isotope probing study using 13 C-labelled methanol in soil micr ocosms r etrie v ed a meta genome-assembled genome affiliated to the genus Candidatus Nitrosocosmicus in the 13 C-DNA fraction, implying that methanol-derived carbon was either dir ectl y or in-dir ectl y assimilated by soil A O A (Macey et al. 2020 ).Ne v ertheless, the question as to whether aliphatic alcohols could affect nitrification in soil ecosystems remains largely unexplored.
The inhibition of both archaeal and bacterial AMO by linear alcohols was v ery differ ent than the r esults pr e viousl y r eported on inhibition by alkynes .T he inhibitory effect of alkynes is weaker with incr easing c hain length in A O A, whereas N .europaea is also inhibited by long-chain alkynes (Taylor et al. 2013, Wright et al. 2020 ).In contrast to alkynes, there was no substantial difference between the inhibition profiles of A O A and A OB with increasing chain lengths of alcohols.In addition, the inhibition of ammonia oxidizers by alkynes is specific to the AMO, whereas alcohols > C 3 also inhibited hydroxylamine oxidation in both A O A and A OB. Alcohols contain a h ydrophilic h ydr oxyl gr oup absent in alkynes.Although the active site of the AMO is thought to be hydrophobic (Welton and Reichardt 2011 ), polarity alone is unlikely to explain the differ ences observ ed.Alcohols ar e solv ents and may inter act with cellular structure, induce solvent stress and influence fluidity and permeability of membranes .T haumarchaeal membranes consist of gl ycer ol dibiphytan yl gl ycer ol tetr aether (GDGT) lipids, in contrast to the phospholipid bilayer found in bacteria (Bale et al. 2019 ).Inter estingl y, a pr e vious study found that archaea from phylum Eury ar chaeota w ere more sensitive to alcohol stress than bacteria (Huffer et al. 2011 ).Although the membrane composition of members of the phylum Eury ar chaeota is different to that of Thaumarchaeota (Villanueva et al. 2014 ), the greater sensitivity to alcohols compared to bacteria appears to be in line with these findings.
This study provides insights into the inhibition of ammonia oxidation by alcohols in the model nitrifiers archaeon ' Ca .N. franklandus' C13 and bacterium N. europaea .This work contributes to understanding of the function of AMO, but also raises questions about whether alcohols could affect nitrification under envir onmentall y r ele v ant conditions and whether other strains of A O A, A OB, and comammox Nitrospira would respond in a manner similar to the model organisms used in our study.Future studies should explore these questions and investigate the roles of cell env elope and membr anes of ammonia oxidizers in withstanding str essors suc h as alcohols.

Figure 1 .
Figure 1.Inhibition of nitrite production by 'Ca.N. franklandus' (A O A) in r esponse to linear alcohols in concentr ation r anging fr om 2 to 100 mM and supplied with 500 μM NH 4 Cl (panel A ), and in response to the lo w est fully inhibitory concentration of each alcohol and supplied with 200 μM hydroxylamine (panel B ).The reaction velocities of the non-inhibited controls were 177 nmol NO 2 − mg protein −1 min −1 and 52 nmol NO 2 − mg protein −1 min −1 for ammonia and hydroxylamine as substrates, respectively.Significant differences between cells inhibited with the alcohols shown and non-inhibited controls are shown as * P < 0.05; * * * 10 P < 0.001.Error bars represent the standard error ( n = 3).

Figure 2 .
Figure 2. Inhibition of nitrite production by N. europaea (AOB) in response to linear alcohols in concentration ranging from 2 to 100 mM and supplied with 500 μM NH 4 Cl (panel A ), and in response to the lo w est fully inhibitory concentration of each alcohol and supplied with 200 μM hydroxylamine (panel B ).The reaction velocities of the non-inhibited controls were 1.26 μmol NO 2− mg protein −1 min −1 and 0.54 μmol NO 2 − mg protein −1 min −1 for ammonia and hydroxylamine as substrates, respectively.Significant differences between cells inhibited with the alcohols shown and non-inhibited contr ols ar e shown as * P < 0.05; * * * P < 0.001.Err or bars r epr esent standard err or ( n = 3).

Table 1 .
Half-maximal inhibitory concentrations (IC 50 ) and lo w est fully inhibitory concentrations * of alcohols (I) in ' Ca.N. franklandus'  and N. europaea