Antonie van Leeuwenhoek DOI 10.1007/s10482-014-0206-4
SHORT COMMUNICATION
Assessment of planctomycetes cell viability after pollutants exposure Carlos Flores • Jose´ A. M. Catita Olga Maria Lage
•
Received: 8 April 2014 / Accepted: 23 May 2014 Ó Springer International Publishing Switzerland 2014
Abstract In this study, the growth of six different planctomycetes, a particular ubiquitous bacterial phylum, was assessed after exposure to pollutants. In addition and for comparative purposes, Pseudomonas putida, Escherichia coli and Vibrio anguillarum were tested. Each microorganism was exposed to several concentrations of 21 different pollutants. After exposure, bacteria were cultivated using the drop plate method. In general, the strains exhibited a great variation of sensitivity to pollutants in the order: V. anguillarum [ planctomycetes [ P. putida [ E. coli. E. coli showed resistance to all pollutants tested, with the exception of phenol and sodium azide. Copper, RidomilÒ (fungicide), hydrazine and phenol were the most toxic pollutants. Planctomycetes were resistant C. Flores O. M. Lage (&) Departamento de Biologia, Faculdade de Cieˆncias, Universidade do Porto, FC4 Rua do Campo Alegre s/n8, 4169-007 Porto, Portugal e-mail: [email protected] C. Flores O. M. Lage CIMAR/CIIMAR – Centro Interdisciplinar de Investigac¸a˜o Marinha e Ambiental, Universidade do Porto, Rua dos Bragas, 289, 4050-123 Porto, Portugal J. A. M. Catita Paralab, SA, Travessa Calva´rio da Giesta 65, 4420-392 Valbom, Portugal J. A. M. Catita CEBIMED - Faculty of Health Sciences, University Fernando Pessoa, Porto, Portugal
to extremely high concentrations of nitrate, nitrite and ammonium but they were the only bacteria sensitive to Previcur NÒ (fungicide). Sodium azide affected the growth on plates of E. coli, P. putida and V. anguillarum, but not of planctomycetes. However, this compound affected planctomycetes cell respiration but with less impact than in the aforementioned bacteria. Our results provide evidence for a diverse response of bacteria towards pollutants, which may influence the structuring of microbial communities in ecosystems under stress, and provide new insights on the ecophysiology of planctomycetes. Keywords Cell viability Planctomycetes Pollutants Sodium azide
Introduction Extreme exploitation of water resources by anthropogenic activities, particularly intensive agriculture and industrial development, can lead to an increase of water pollution. Contaminated water treatment is a reality in many countries but, even so, many water bodies remain contaminated as some pollutants persist in it (Westerhoff et al. 2005). Furthermore, some treatments such as the chlorination process, can originate genotoxic compounds in the water (Park et al. 2000). The lists of ‘‘priority pollutants’’ created in order to ensure water quality vary according to the
123
Antonie van Leeuwenhoek
country and their regulatory entities (EPA 2007; NPi 2007; ATSDR 2011; Directive 2008/105/EC). These lists include hydrocarbons, heavy metals, phenols, active pharmaceutical ingredients (APIs) and detergents mainly found in urban/industrial effluents, but also phosphates, nitrates, nitrites, ammonia and pesticides commonly found in fertilizers and agricultural runoffs. The mechanisms of action of these pollutants are variable and can be: (i) enzyme inhibition (e.g. sodium azide, heavy metals); (ii) binding to ion channels and regulatory proteins, (iii) gene expression modification; (iv) reactive oxygen species generation; (v) cell growth limitation (e.g. ammonia, nitrites, nitrates); and (vi) unpredicted biological activity (e.g. APIs). Additionally, many of these pollutants are carcinogenic or potentially carcinogenic (e.g. hydrazine, heavy metals). Many methods have been developed for monitoring water bodies. Due to their easy manipulation and high biotechnological application, microbial biosensors have been widely used for water quality assessment (Eltzov and Marks 2011). The common bacteria used as biosensors are nitrifying bacteria (Woznica et al. 2013), sulphuroxidizing bacteria (Oh et al. 2011), bacterial communities from activated sludge (Jordan et al. 2010), as well as genetically modified Escherichia coli (Gao et al. 2011) and Pseudomonas sp. (Liu et al. 2010). Planctomycetes are a very distinct and diverse group of bacteria, with ecological and evolutionary relevance (Fuerst and Sagulenko 2011; Fuerst 2013; Devos 2013). Some of the most notable features of these organisms are (i) the absence of peptidoglycan in their cell walls, which are rich in proteins; (ii) a nucleoid with permanently condensed DNA; (iii) budding reproduction in many species; vi) an organelle, present in only some species, called the anammoxosome and responsible for the anaerobic ammonium oxidation–annamox, a fundamental step in the nitrogen cycle (van Niftrik 2013). Despite the importance of anammox planctomycetes in wastewater treatment (Kartal et al. 2010), a lack of knowledge still persists about the physiology of this phylum, as well as, its potential applications in biotechnology. Moreover, a recent comprehensive planctomycetal genome mining approach indicated planctomycetes as a source of novel bioactive molecules (Jeske et al. 2013). Despite their habitat ubiquity and particular features, toxicity has been scarcely studied in this group of bacteria (Lieber et al. 2009; Lage et al. 2012).
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The main objective of this work was the acute toxicity assessment of different pollutants on cell viability of selected planctomycetal strains, in comparison with the toxic effects observed in other bacteria commonly used in toxicity assays. A wide range of pollutants, including heavy metals, detergents, inorganic nitrogen compounds, phosphates, fungicides, pharmaceuticals, phenol, sodium azide and hydrazine were tested. Furthermore, by focusing on planctomycetes, this work will provide new insights on their ecophysiology, and may reveal their utility in aquatic toxicity assessment.
Materials and methods Bacterial strains and growth conditions Rhodopirellula rubra (strains LF2, UC9 and MsF5) and Rhodopirellula lusitana (strains UC17 and Sm4) were isolated from the biofilm community on the surface of marine macroalgae from the north coast of Portugal (Lage and Bondoso 2011). The reference strain Rhodopirellula baltica was isolated from the water column of the Baltic Sea (Schlesner 1994). E. coli was isolated from a River Febros water sample - Avintes, Portugal (Cabral and Marques 2006). Pseudomonas putida NB3L was isolated from a marine sponge (Pimenta 2010) and Vibrio anguillarum from a sea bass’s kidney. Planctomycetes strains were cultivated on modified M13 medium (Lage and Bondoso 2011). E. coli was cultivated in Luria Broth (LB)/Luria Agar (LA) (10 g/L Tryptone, 5 g/L BactoTM Yeast Extract, 1 g/L D-glucose, 5 g/L sodium chloride, ± 1.6 % agar-Bacteriological American Type). P. putida and V. anguillarum were cultivated in Nutrient Broth (NB)/Nutrient Agar (NA) (5 g/L BactoTM Peptone, 3 g/L Yeast extract, 1 g/L D-glucose, ± 1.6 % agar-Bacteriological American Type) made with natural sea water. Growth of the cultures was monitored by measuring the absorbance at 600 nm (OD600nm). All cultures were incubated at 26 °C with the exception of E. coli which was incubated at 37 °C. Toxicity assays Chemicals The tested pollutants were zinc (as ZnCl2, Merck), chromium (as KCr(SO4)212H2O, Merck), copper (as
Antonie van Leeuwenhoek
CuCl22H2O, Merck), arsenic (as KH2AsO4, Sigma), nickel (as NiCl26H2O, Sigma- Aldrich), cadmium (as CdCl221/2H2O, Sigma-Aldrich), nitrite (as NaNO2, Merck), nitrate (as NaNO3, Merck), ammonia (as NH4Cl, MandB), phosphate (NaH2PO4H2O, Merck), sodium azide (NaN3, Riedel-de-Haen), phenol (C6H6O, Sigma-Aldrich), hydrazine (as (NH2)2H2SO4, Sigma- Aldrich), Previcur NÒ (propamocarb hydrochloride, Bayer CropScience), Ridomil Gold SLÒ (metalaxil-M, Agro), IGEPAL CA-630 (SigmaAldrich), a common dish detergent (Super Pop lemon), a common hand cleanser (supermarket brand from Continente), Diclofenac (Sigma-Aldrich), Acetaminophen (Sigma-Aldrich) and Caffeine (SigmaAldrich). Cell viability assays Stock solutions of the pollutants were prepared in Milli-Q water for heavy metals (5,866 lM), ammonia (1.87 M), nitrate (3.53 M), nitrite (1.45 M), RidomilÒ (1 %), Previcur NÒ (10 %), phosphates (0.83 M), hydrazine (15.40 mM), sodium azide (1.54 M), phenol (1.06 M), Diclofenac (31.40 mM), Acetaminophen (66.2 mM) and Caffeine (51.50 mM). For the detergents (IGEPAL CA-630, dish detergent and hand cleanser), the original formulations were used as stock solutions. The exposure of the microorganisms to the pollutants was performed in liquid cultures at the exponential growth phase, which corresponds to an OD600nm of 0.5 AU for E. coli and 0.2 to 0.3 AU for the other bacteria. Absorbance measurements were performed in a spectrophotometer GenesysTM 10 Series (Thermo Spectronic). One millilitre of liquid culture was harvested by centrifugation at 13,400 rpm for 60 s (MiniSpinÒ, eppendorf) and the cells were resuspended in one millilitre of each pollutant concentration tested. The same concentration range for each pollutant was used for all the bacteria. The exposure times were 30 and 60 min. After pollutant exposure, the suspension was centrifuged and resuspended in 1 mL of Milli-Q water, which was afterwards cultivated by means of the drop plate method, i.e. three drops of 10 lL sample were placed on the appropriate medium for each strain. The microbial growth was checked after 24 h for E. coli, V. anguillarum and P. putida or 3 days for planctomycetes. Images of the cultures were recorded using the GenoPlex system (VWR). For results validation, each
experiment was done in triplicate from three different initial cultures. Different growth levels were scored in a range from 0 to 4 (Fig. 1), in which 4 represent the maximum growth level (same as control) and 0 represents the absence of growth. The level 0.5 was applied when only one or two colonies were formed after a longer incubation period. Controls were performed in Milli-Q water using the same procedure as for pollutant exposure and no impairment in bacterial growth was observed comparative to the bacteria kept in marine medium. Oxygen consumption measurements For respirometric assays, representative bacteria were selected: E. coli, P. putida, R. baltica and R. rubra strain LF2. The oxygen consumption was measured with a Clark-type oxygen electrode (Oxygraph System, Hansatech). Temperature was controlled with a water-bath at 37 °C for E. coli and 26 °C for P. putida, R. baltica and R. rubra strain LF2. Culture samples of E. coli, P. putida, R. baltica and R. rubra strain LF2 were harvested and resuspended in the appropriate growth medium up to a final OD600nm of 0.5 AU for E. coli, and 0.2 to 0.3 AU for the other bacteria. One millilitre of culture was placed in the oxygraphic chamber. When a constant slope of the oxygen plot was reached, the culture was exposed to 0.77 M sodium azide in order to determine the effect of this pollutant on respiratory activity. Each experiment was performed three times with independent cultures.
Results and discussion A semi-quantitative assay based on the drop plate method was used for the assessment of cell viability and consequently toxicity of pollutants. Since these bacteria can exist as isolated cells or as rosettes of different size (Bondoso et al. 2014), colonies can be formed by a variable number of cells which limits the accurate determination of colony forming units. As a consequence, a visual semi-quantitative scale based on the cell growth (Fig. 1) was used to evaluate toxicity. Although semi-quantitative methods lack formal statistical meaning, the results obtained give relevant information for future quantitative analysis. The growth level of the different bacteria after exposure to different pollutants concentrations is
123
Antonie van Leeuwenhoek
Fig. 1 Drops of planctomycetes cultures showing the different growth levels (0.5–4). The dashed lines correspond to the area where the drop was placed
summarised in Table 1. The results showed different growth levels for different bacteria, pollutants and concentrations suggesting different sensitivities. With the exception of phenol and sodium azide, no other pollutant affected the growth of E. coli. This strain, being isolated from a contaminated water source (Febros River), is probably well adapted to polluted environments. In contrast, V. anguillarum was, in general, the most sensitive bacterium, which may be due to the habitat it normally inhabits (as a pathogen of fishes), which can protect it from external stresses. Heavy metals In most cases, a dose–response effect was evident in the case of planctomycetes. The sensitivity order showed by planctomycetes to the different metals was: Ni \ As \ Zn \ Cr \ Cd \ Cu, with R. baltica being the most resistant strain. Nickel was the only metal that did not cause a complete loss of cell viability and a relatively low toxicity of arsenic, in the form of arsenate (As5?), was observed. Zinc showed a stronger toxic effect than arsenate, in particular for R. lusitana strains Sm4 and UC17. In fact, other studies have suggested that the effects of zinc in microbial communities, as well as in other aquatic living organisms, are underestimated (Paulsson et al. 2000). Copper, which is a widely known biocide (Borkow and Gabbay 2005), was the most toxic metal in this study. High toxicity of copper in other microorganisms has also been reported and several associated mechanisms were proposed (Boivin et al. 2005; Ore et al. 2010; Santo et al. 2011; Warnes et al. 2012). Kungolos et al. (2009) observed that copper induced a high toxic effect on different tested species (including V. fischeri) at concentration levels lower than 0.1 mg/L, which are lower than the ones tested in this study.
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In general, P. putida was more resistant to metals than the majority of the planctomycetes. The ability of P. putida to tolerate heavy metals observed in this work (with the exception of copper) agrees with previous genomic studies which have shown that the genome of this bacterium encodes several mechanisms for heavy metal tolerance and homeostasis. These include two systems for arsenic, one for chromate, four to six systems for divalent cations, two systems for monovalent cations, one metallothionein for metal(loid) binding and four ABC transporters for the uptake of essential Zn, Mn, Mo and Ni (Ca´novas et al. 2003; Hu and Zhao 2007). Mechanisms of resistance, tolerance and co-tolerance have also been reported as being a common phenomenon in E. coli, mainly due to its high genomic plasticity (Cohen et al. 1991; Abskharon et al. 2010; Su et al. 2011; Bouzat and Hoostal 2013). In general, the concentration 59 lM did not affect the growth of the bacteria tested. Inorganic nitrogen compounds These compounds (nitrate, nitrite and ammonium) did not cause the complete loss of cell viability in planctomycetes. Viability was slightly affected only by ammonium. On the other hand, P. putida and V. anguillarum were affected by the three compounds: P. putida growth was totally inhibited at 1.77 M nitrate (after 60 min of exposure) and V. anguillarum cells were not viable at 1.87 M ammonium. Ammonium toxicity at lower concentrations (10 mmol/L) affected cyanobacteria isolated from freshwater, reducing the growth by 65 % (Dai et al. 2008). Tolerance to the bacteriostatic action of sodium nitrite in different bacteria was reported early (Castellani and Niven 1955). Recently, Fu et al. (2013) induced resistance to
Antonie van Leeuwenhoek Table 1 Bacterial growth levels after acute exposure to pollutants (0 – Absence of growth; 0.5 to 4 – Different levels of growth) Pollutant
Arsenic
Cadmium
Chromium
Copper
Nickel
Zinc
time (min)
30
30
30
30
30
30
[]
R. rubra
R. baltica
LF2
UC9
MsF5
59 lM
4
4
4
293 lM
4
4
4
587 lM
4
4
1466 lM
3
4
2933 lM
2
4399 lM
R. lusitana
E. coli
P. putida
V. anguillarum
Sm4
UC17
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
1
4
4
3
4
4
3
0
1
1
3
4
2
3
4
1
0
5866 lM
0.5
0
2
2
0.5
0.5
4
0
0
59 lM
4
4
4
4
3
3
4
4
0.5
293 lM
3
3
3
4
0.5
0
4
4
0
587 lM
2
1
1
3
0
0
4
4
0
1466 lM
1
0.5
0
3
0
0
4
4
0
2933 lM
0.5
0
0
2
0
0
4
4
0
4399 lM
0
0
0
1
0
0
4
4
0
5866 lM
0
0
0
0
0
0
4
2
0
59 lM 293 lM
4 3
4 3
4 4
4 4
4 4
4 4
4 4
3 2
3 0
587 lM
2
2
4
4
3
3
4
2
0
1466 lM
1
1
1
2
0
0
4
1
0
2933 lM
0.5
0.5
0
1
0
0
4
0.5
0
4399 lM
0
0
0
1
0
0
4
0
0
5866 lM
0
0
0
0.5
0
0
4
0
0
59 lM
3
3
0.5
0.5
0
1
4
2
1
293 lM
0
1
0
0
0
0
4
0
0
587 lM
0
1
0
0
0
0
4
0
0
1466 lM
0
0.5
0
0
0
0
4
0
0
2933 lM
0
0.5
0
0
0
0
4
0
0
4399 lM
0
0
0
0
0
0
4
0
0
5866 lM
0
0
0
0
0
0
4
0
0
59 lM
4
4
4
4
4
4
4
4
4
293 lM 587 lM
4 4
4 4
4 4
4 4
4 4
4 4
4 4
4 4
3 3
1466 lM
3
4
4
4
4
4
4
4
0
2933 lM
3
4
4
4
4
4
4
4
0
4399 lM
3
4
4
4
4
4
4
4
0
5866 lM
2
3
3
4
2
3
4
4
0
59 lM
4
4
4
4
4
4
4
4
0
293 lM
3
4
4
4
4
2
4
4
0
587 lM
3
4
4
4
1
1
4
4
0
1466 lM
3
2
4
3
0
0.5
4
4
0
2933 lM
2
1
3
3
0
0.5
4
3
0
4399 lM
2
1
2
2
0
0
4
2
0
5866 lM
1
0.5
0.5
0.5
0
0
4
1
0
123
Antonie van Leeuwenhoek Table 1 continued Pollutant
Nitrate
Nitrite
time (min)
IGEPAL
MsF5
4 4
4 4
4 4
P. putida
V. anguillarum
Sm4
UC17
4 4
4 4
4 4
4 4
3 2
4 3
3.53 M
4
4
4
4
4
4
4
0.5
2
4
4
4
4
4
4
4
4
4
1.77 M
4
4
4
4
4
4
4
0
4
3.53 M
3
4
4
4
4
4
4
0
3
0.14 M
4
4
4
4
4
4
4
4
4
0.73 M
4
4
4
4
4
4
4
3
3
1.45 M
4
4
4
4
4
4
4
1
2
0.14 M
4
4
4
4
4
4
4
4
4
0.73 M
4
4
4
4
4
4
4
1
3
1.45 M
4
4
4
4
4
4
4
0.5
2
0.02 M
4
4
4
4
4
4
4
4
4
0.19 M
4
4
4
4
4
4
4
1
2
30
30
30
1.87 M
2
4
4
4
4
2
4
1
0
0.02 M 0.19 M
4 4
4 4
4 4
4 4
4 4
4 3
4 4
4 3
4 0.5
1.87 M
3
4
4
3
3
1
4
1
0
0.08 M
4
4
2
2
2
4
4
2
0
0.42 M
1
0.5
1
0
1
1
4
0.5
0
0.83 M
0.5
0.5
0.5
0
1
0.5
4
0
0
0.08 M
2
1
2
0
2
3
4
3
0
0.42 M
0
0.5
1
0
0.5
0.5
4
0.5
0
0.83 M
0
0
0.5
0
0.5
0
4
0
0
1%
3
4
4
4
4
4
4
4
2
5%
3
4
4
3
3
2
4
4
0
10 %
2
4
4
2
1
2
4
4
0
1%
3
4
4
4
3
3
4
4
0
5%
2
4
0.5
3
0.5
2
4
3
0
10 %
1
4
0
2
0
1
4
2
0
30
1% 5%
3 3
4 4
4 4
4 3
4 4
4 4
4 4
4 3
1 1
10 %
2
4
4
2
4
4
4
3
0.5
60
1%
4
4
4
4
4
4
4
3
0.5
5%
3
4
4
2
4
4
4
2
0.5
10 %
2
4
3
1
3
4
4
1
0
1%
3
4
2
4
4
4
4
4
0.5
5%
2
4
0.5
3
4
2
4
4
0.5
10 %
2
1
0
3
3
0.5
4
4
0
1%
3
4
1
4
4
4
4
4
0
5%
2
2
0
3
4
2
4
4
0
10 %
0.5
0.5
0
2
3
0.5
4
3
0
30
30
CA-630 60
123
UC9
E. coli
0.35 M
60
Dish detergent
LF2
R. lusitana
60
60
Hand cleanser
R. baltica
0.35 M 1.77 M
60
Phosphates
R. rubra
30
60
Ammonium
[]
Antonie van Leeuwenhoek Table 1 continued Pollutant
RidomilÒ
time (min)
30
60
Previcur NÒ
30
60
Phenol
30
60
Hydrazine
30
60
Sodium Azide
30
60
[]
R. rubra
R. baltica
R. lusitana UC17
4 2
4 1
P. putida
V. anguillarum
4 4
4 4
4 0
LF2
UC9
MsF5
0.1 % 0.5 %
4 2
4 2
4 1
1%
0
0
0
0
0
0
4
1
0
0.1 %
3
4
4
4
4
4
4
4
4 0
4 1
Sm4
E. coli
0.5 %
0.5
2
3
2
0
2
4
4
1%
0
0
0
0
0
0
4
1
0
1%
4
4
4
4
4
4
4
4
4
5%
3
4
3
4
3
4
4
4
4
10 %
0.5
4
1
0
1
3
4
4
4
1%
4
4
4
4
4
4
4
4
4
5%
0.5
4
2
3
2
4
4
4
4
10 %
0
3
0
0
1
0.5
4
4
4
0.01 M
4
4
4
4
4
4
4
4
0.5
0.11 M
0
0
0
0
0
0
0
0
0
1.06 M
0
0
0
0
0
0
0
0
0
0.01 M 0.11 M
4 0
4 0
4 0
4 0
4 0
4 0
4 0
4 0
0 0
1.06 M
0
0
0
0
0
0
0
0
0
0.15 mM
2
4
4
4
2
4
4
1
0
1.54 mM
0
0
0
0
0.5
0
4
0
0
15.4 mM
0
0
0
0
0
0
4
0
0
0.15 mM
1
0.5
0
0.5
0.5
0
4
0
0
1.54 mM
0
0
0
0
0
0
4
0
0
15.4 mM
0
0
0
0
0
0
4
0
0
0.15 M
4
4
4
4
4
4
4
4
3
0.77 M
4
4
4
4
4
4
3
0
2
1.54 M
4
4
4
4
4
4
2
0
1
0.15 M
4
4
4
4
4
4
4
4
3
0.77 M
4
4
4
4
4
4
2
0
2
1.54 M
4
4
4
4
4
4
2
0
0
Diclofenac
30
3.1 mM 31.4 mM
4 0
4 0
2 0
4 1
4 0
4 0
4 4
3 1
0 0
Acetaminophen
30
6.6 mM
4
4
4
4
4
4
4
4
4
66.2 mM
4
4
3
4
4
4
4
4
2
5.2 mM
4
4
4
4
4
4
4
4
4
51.5 mM
4
4
4
4
4
4
4
4
0
Caffeine
30
nitrite in E. coli and Shewanella oneidensis at concentration of 5 mM. However, to our knowledge, tolerance to nitrite has never been reported at the highest concentration tested in this study.
Furthermore, nitrate, nitrite and ammonium, when supplied as the only N-source were able to support the growth of these strains of planctomycetes (unpublished data). As these planctomycetes were isolated
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from coastal marine environments, they may also contribute to the inorganic part of the nitrogen cycle in these habitats. Phosphates Phosphate is a key nutrient for cells and for that reason, the majority of the microbial studies focus on phosphate depletion rather than on phosphate surplus. The main ecological problem related to phosphates is eutrophication due to high concentrations of this element in runoffs with agricultural fertilizers and detergents (Kleinman et al. 2011). However, some reports showed that even at high concentrations the microbial community is not affected and in some cases can increase their biomass (Lehman 1976; Jordaan and Bezuidenhout 2013). Other studies also suggested that phosphate doesn’t have a negative effect on the microbial growth (Appenzeller et al. 2001), microbial community structure, ecophysiological index and colony-development index (Sarathchandra et al. 2001). In the present work, phosphates had a strong effect on cell viability of some planctomycetes, P. putida and V. anguillarum (Table 1). Indeed, almost all bacteria studied were affected by the lowest phosphate concentration tested (0.08 M) and V. anguillarum growth was completely inhibited at this concentration. R. baltica lost completely cell viability at 0.42 M. These results are not in accordance with the ones mentioned above. The strong effect observed may be explained by the use of sodium phosphate as observed by others, particularly with Gram negative bacteria (Dickson et al. 1994; Kim and Marshall 1999). On the other hand, E. coli, R. lusitana strains MsF5 and Sm4 could tolerate 0.83 M of phosphates even after 60 min of exposure. In the case of E. coli, it was described early that when the inorganic phosphate is in excess, the PHO regulon (responsible for phosphorus assimilation) is inactivated and the necessary inorganic phosphate for the cell is taken up by low affinity with inorganic phosphate transporters (Wanner 1993). Detergents The detergents tested had a strong effect on the cell viability of V. anguillarum and when growth was observed it was minimal. In general IGEPAL-C630 was the most toxic detergent, followed by the hand
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cleanser. However, the complete loss of cell viability was only observed at a concentration of 10 % in hand cleanser for R. lusitana strains Sm4 and MsF5, and at 10 % in IGEPAL for R. rubra strain MsF5 (in all cases after 60 min of exposure). Being surfactants, detergents damage the phopholipid bilayer of the cell, particularly the inner membrane in Gram negative bacteria, and promote their self-uptake into the cells. According to Salton (1968), they can penetrate the cell, react with proteins (denaturation) and/or with lipids in order to induce membrane disorganization, causing the leakage of intracellular low molecular weight material. However, several resistance mechanisms to surfactants have been described in Gram negative bacteria, acquired either by chromosomal mutations or plasmid-mediation (Langsrud et al. 2003), and are strongly related to the outer membrane which may prevent their uptake. These resistance mechanisms may be present in the studied bacteria as they are Gram negative. This should also be the case of planctomycetes that recently have been proposed to possess an outer membrane comparable to the Gram negative bacteria (Speth et al. 2012; Santarella-Mellwig et al. 2013). Fungicides Among the fungicides tested, RidomilÒ had a stronger effect than Previcur NÒ on cell viability of all the bacteria. V. anguillarum and R. lusitana strain Sm4 lost their cell viability when exposed to 0.5 % RidomilÒ, and the other planctomycetes at 1 %. On the other hand, Previcur NÒ only affected planctomycetes and caused the complete loss of cell viability in R. baltica, and R. rubra strains LF2 and MsF5 at 10 % concentration. Previous studies showed that metalaxyl-M (RidomilÒ) was non-toxic for arthropods, vertebrate species (Sukul and Spiteller 2000) and earthworms (Mosleh et al. 2003). In contrast, tests performed with lower trophic level organisms such as V. fischeri, algae and Daphnia magna showed a considerable toxic effect of metalaxyl-M (Kungolos et al. 2009). Considering that concentrations of 1.3 mg/L were found by Graves et al. (2004) in storm water runoffs, our findings also suggest that metalaxyl-M can cause an environmental risk for non-target organisms. Whereas RidomilÒ inhibits fungal growth and reproduction by impairing rRNA synthesis (affecting
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the RNA polymerase activity and consequently the synthesis of nucleic acids), Previcur NÒ affects the permeability of fungi cells binding to sterols in the membranes and interfering with the biosynthesis of fatty acids and phospholipids within the cell. Planctomycetes contain lipids in their membranes like palmitic, oleic and palmitoleic lipids, which are typical of eukaryotes (Kerger et al. 1988). Additionally, Gemmata obscuriglobus, being a member of the phylum Planctomycetes, produces sterols through an abbreviated pathway which is not common in other bacteria (Pearson et al. 2003). These particular features of planctomycetes, may explain their higher sensitivity to Previcur NÒ. Fungicides like cycloheximide, amphotericin B and econazole nitrate are used to prevent fungal growth in planctomycetes isolation (Schlesner 1994; Lage and Bondoso 2011). Our results indicated that other fungicides could be used in isolation of planctomycetes, particularly Previcur N, but further studies are needed to assess their effectiveness. Phenol, hydrazine and sodium azide When exposed to phenol almost all the bacteria showed a similar sensitivity at the concentrations tested. The inhibition of growth was observed at 0.11 M after 30 min of exposure, with the exception of V. anguillarum, whose growth was inhibited also at 0.01 M after 60 min of exposure. Widely used as disinfectant, phenol acts on the cytoplasmic membrane of Gram-negative bacteria, can enter into the cells by a hydrophobic pathway and then it promotes a progressive leakage of the intracellular constituents (McDonnell and Russell 1999). Pulvertaft and Lumb (1948) showed that, at lower concentrations than the ones tested in this work (0.032 %), phenols agents lysed rapidly growing cultures of E. coli, staphylococci and streptococci without involvement of autolytic enzymes. Other studies revealed that phenol can act at the separation point of dividing cells, so that the offspring cells are more affected (Srivastava and Thompson 1965, 1966). Hydrazine, at 0.15 mM, strongly reduced the cell viability of R. rubra strain LF2, R. lusitana strain Sm4 and P. putida after 30 min of exposure, and also of R. baltica and R. rubra strain UC9 after 60 min. At this concentration V. anguillarum did not grow at all. Exposure to hydrazine at 1.54 mM for 60 min caused
a complete loss of cell viability in all the bacteria tested, with the exception of E. coli. This compound affects gluconeogenesis in cells, blocking the conversion of oxaloacetic to phosphoenolpyruvic acid through inhibition of phosphoenolpyruvate carboxykinase (PEPCK) (Prajongtat et al. 2013). In silico analysis showed the presence of this enzyme in all the bacteria tested (data not shown). In addition, the high toxicity of this compound may be due to the potential induction of DNA damage and consequent inactivation, already well-described in Gram-negative Salmonella (McCann et al. 1975; Sugimura et al. 1976; Purchase et al. 1978) and mammalian cells (Damjanov et al. 1973; Petzold and Swenberg 1978; Bradley et al. 1979). Interestingly, the anammox group of planctomycetes produces hydrazine as an intermediate in the anammox process but keep it inside the anammoxosome which prevents the noxious effect of this compound (Kartal et al. 2011; van Niftrik 2013). Sodium azide affected E. coli, P. putida and V. anguillarum but not planctomycetes. The growth of P. putida was inhibited at 0.77 M, but the growth of V. anguillarum was inhibited at 1.54 M only after 60 min of exposure. Sodium azide is widely used as a biocide against Gram-negative bacteria. It is a respiration inhibitor, interfering with the electron transport chain of many different organisms. In bacterial cells, its main targets are the cytochromes of the electron transport chain (Li and Palmer 1993; Little et al. 1996) and ATPases (Daniel 1976; Noumi et al. 1987). Although planctomycetes are being considered as Gram-negative like bacteria (Devos 2013), their cell viability results to sodium azide were not as expected. These unexpected results led us to analyze the cell respiration in the studied bacteria. E. coli possessed the highest oxygen consumption (OC) (-62.8 ± 2.8 nmol/mL/min), followed by P. putida (-41.7 ± 7.2 nmol/mL/min). Planctomycetes showed much lower OC (-11.8 ± 2.4 and -19.1 ± 2.9 nmol/mL/ min of R. rubra strain LF2 and R. baltica, respectively). This data is in accordance with the growth rate of these bacteria: E. coli divides in approximately 58 min, P. putida in about 2.5 h and the planctomycetes in about 12.5 h. Even though cell viability of planctomycetes was not affected by sodium azide, their cell respiration was affected (Fig. 2) but to a lesser extent than in E. coli and P. putida. R. rubra strain LF2 was the least affected strain in the presence of sodium azide
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Fig. 2 Decrease in oxygen consumption induced by 0. 77 M sodium azide in liquid cultures of E. coli, P. putida, R. baltica and R. rubra strain LF2
(-4.3 ± 0.0 nmol/mL/min, 64 % OC decrease), followed by R. baltica (-5.1 ± 1.8 nmol/mL/min, 73 % OC decrease). E. coli showed a higher decrease in oxygen consumption (-10.9 ± 6.4 nmol/ml/min, 83 % OC decrease) and P. putida was the most affected bacterium (-1.4 ± 0.1 nmol/ml/min, 97 % OC decrease). Bacterial cell respiration is highly flexible and may involve various cytochromes (Richardson 2000). However, the respiratory mechanisms in planctomycetes are not well understood yet. An in silico analysis revealed the presence of cytochromes c and bd in these microorganisms but not of cytochromes bo or aa3 (data not shown). Further studies are needed to understand the planctomycetal behaviour. Active pharmaceutical ingredients The real impact of these ingredients in aquatic living organisms is still unclear and their emergence is increasing in natural waters (Sanderson et al. 2004). Among the APIs assayed, diclofenac was the only one that affected the growth of the planctomycetes, causing the complete loss of cell viability at 31.4 mM concentration, with the exception of R. baltica. Furthermore, it had also a strong effect on the cell viability of P. putida and V. anguillarum. The effect of diclofenac in all these bacteria is in agreement with effects reported on other microorganisms, such as V. fischeri (Yu et al. 2013). Caffeine only affected V. anguillarum at 51.5 mM concentration. These results disagree with previous ones that indicate the inhibitory effect of caffeine at lower concentrations, mainly in Pseudomonas sp., but also in E. coli (Ramanavicˇien_e et al. 2003; Dash and
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Gummadi 2008). However, our results support other studies that refer the ability to tolerate caffeine by Pseudomonas sp. and E. coli, mainly due to N-demethylase activity (Gibson et al. 2012; Summers et al. 2012). Up to now, only one demethylase is listed in genomic microbial databases for planctomycetes (G. obscuriglobus). Acetaminophen only slightly affected the cell viability of R. rubra strain MsF5 and V. anguillarum. The impact of APIs in ecosystems has only recently been assessed. The few studies available showed that they can have antibacterial activity as previously described and interfere with the bacteria biofilm communities (Lawrence et al. 2012). However, several bacteria may start to adapt to these conditions, along with genome evolution, including through horizontal gene transfer.
Conclusions The cell viability studies performed revealed diverse responses of the bacteria studied to different toxicants, which may help understanding of microbial community structure changes in habitats under pollution stress. Regarding the pollutants tested, stronger effects were observed in the presence of RidomilÒ, phenol, hydrazine and copper. In most cases, E. coli was the most resistant bacterium and V. anguillarum the most sensitive. The elevated resistance of this E. coli strain is consistent with the polluted habitat from where it was isolated. A potential role in the inorganic nitrogen cycle is envisaged for planctomycetes due to their resistance to high concentrations of inorganic nitrogen compounds. Although sodium azide did not reduce cell viability of planctomycetes, it affected their cell respiration, although in a reduced way in comparison to other bacterial species. Further studies are needed to clearly understand the mechanisms of cell respiration in planctomycetes, and the impact of sodium azide on the process. The findings obtained are a contribution to overcoming the gaps in knowledge that still remain about planctomycetes ecophysiology. These microorganisms, being widespread in aquatic environments and showing different dose–response behaviour to different pollutants, may be potential candidates for water quality assessment.
Antonie van Leeuwenhoek Acknowledgments We thank Paula Tamagnini for the oxygen consumption measurements. This research was supported by the European Regional Development Fund (ERDF) through the COMPETE Operational Competitiveness Programme and national funds through FCT – Foundation for Science and Technology, under the project PEst-C/MAR/LA0015/2013.
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SHORT COMMUNICATION
Assessment of planctomycetes cell viability after pollutants exposure Carlos Flores • Jose´ A. M. Catita Olga Maria Lage
•
Received: 8 April 2014 / Accepted: 23 May 2014 Ó Springer International Publishing Switzerland 2014
Abstract In this study, the growth of six different planctomycetes, a particular ubiquitous bacterial phylum, was assessed after exposure to pollutants. In addition and for comparative purposes, Pseudomonas putida, Escherichia coli and Vibrio anguillarum were tested. Each microorganism was exposed to several concentrations of 21 different pollutants. After exposure, bacteria were cultivated using the drop plate method. In general, the strains exhibited a great variation of sensitivity to pollutants in the order: V. anguillarum [ planctomycetes [ P. putida [ E. coli. E. coli showed resistance to all pollutants tested, with the exception of phenol and sodium azide. Copper, RidomilÒ (fungicide), hydrazine and phenol were the most toxic pollutants. Planctomycetes were resistant C. Flores O. M. Lage (&) Departamento de Biologia, Faculdade de Cieˆncias, Universidade do Porto, FC4 Rua do Campo Alegre s/n8, 4169-007 Porto, Portugal e-mail: [email protected] C. Flores O. M. Lage CIMAR/CIIMAR – Centro Interdisciplinar de Investigac¸a˜o Marinha e Ambiental, Universidade do Porto, Rua dos Bragas, 289, 4050-123 Porto, Portugal J. A. M. Catita Paralab, SA, Travessa Calva´rio da Giesta 65, 4420-392 Valbom, Portugal J. A. M. Catita CEBIMED - Faculty of Health Sciences, University Fernando Pessoa, Porto, Portugal
to extremely high concentrations of nitrate, nitrite and ammonium but they were the only bacteria sensitive to Previcur NÒ (fungicide). Sodium azide affected the growth on plates of E. coli, P. putida and V. anguillarum, but not of planctomycetes. However, this compound affected planctomycetes cell respiration but with less impact than in the aforementioned bacteria. Our results provide evidence for a diverse response of bacteria towards pollutants, which may influence the structuring of microbial communities in ecosystems under stress, and provide new insights on the ecophysiology of planctomycetes. Keywords Cell viability Planctomycetes Pollutants Sodium azide
Introduction Extreme exploitation of water resources by anthropogenic activities, particularly intensive agriculture and industrial development, can lead to an increase of water pollution. Contaminated water treatment is a reality in many countries but, even so, many water bodies remain contaminated as some pollutants persist in it (Westerhoff et al. 2005). Furthermore, some treatments such as the chlorination process, can originate genotoxic compounds in the water (Park et al. 2000). The lists of ‘‘priority pollutants’’ created in order to ensure water quality vary according to the
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Antonie van Leeuwenhoek
country and their regulatory entities (EPA 2007; NPi 2007; ATSDR 2011; Directive 2008/105/EC). These lists include hydrocarbons, heavy metals, phenols, active pharmaceutical ingredients (APIs) and detergents mainly found in urban/industrial effluents, but also phosphates, nitrates, nitrites, ammonia and pesticides commonly found in fertilizers and agricultural runoffs. The mechanisms of action of these pollutants are variable and can be: (i) enzyme inhibition (e.g. sodium azide, heavy metals); (ii) binding to ion channels and regulatory proteins, (iii) gene expression modification; (iv) reactive oxygen species generation; (v) cell growth limitation (e.g. ammonia, nitrites, nitrates); and (vi) unpredicted biological activity (e.g. APIs). Additionally, many of these pollutants are carcinogenic or potentially carcinogenic (e.g. hydrazine, heavy metals). Many methods have been developed for monitoring water bodies. Due to their easy manipulation and high biotechnological application, microbial biosensors have been widely used for water quality assessment (Eltzov and Marks 2011). The common bacteria used as biosensors are nitrifying bacteria (Woznica et al. 2013), sulphuroxidizing bacteria (Oh et al. 2011), bacterial communities from activated sludge (Jordan et al. 2010), as well as genetically modified Escherichia coli (Gao et al. 2011) and Pseudomonas sp. (Liu et al. 2010). Planctomycetes are a very distinct and diverse group of bacteria, with ecological and evolutionary relevance (Fuerst and Sagulenko 2011; Fuerst 2013; Devos 2013). Some of the most notable features of these organisms are (i) the absence of peptidoglycan in their cell walls, which are rich in proteins; (ii) a nucleoid with permanently condensed DNA; (iii) budding reproduction in many species; vi) an organelle, present in only some species, called the anammoxosome and responsible for the anaerobic ammonium oxidation–annamox, a fundamental step in the nitrogen cycle (van Niftrik 2013). Despite the importance of anammox planctomycetes in wastewater treatment (Kartal et al. 2010), a lack of knowledge still persists about the physiology of this phylum, as well as, its potential applications in biotechnology. Moreover, a recent comprehensive planctomycetal genome mining approach indicated planctomycetes as a source of novel bioactive molecules (Jeske et al. 2013). Despite their habitat ubiquity and particular features, toxicity has been scarcely studied in this group of bacteria (Lieber et al. 2009; Lage et al. 2012).
123
The main objective of this work was the acute toxicity assessment of different pollutants on cell viability of selected planctomycetal strains, in comparison with the toxic effects observed in other bacteria commonly used in toxicity assays. A wide range of pollutants, including heavy metals, detergents, inorganic nitrogen compounds, phosphates, fungicides, pharmaceuticals, phenol, sodium azide and hydrazine were tested. Furthermore, by focusing on planctomycetes, this work will provide new insights on their ecophysiology, and may reveal their utility in aquatic toxicity assessment.
Materials and methods Bacterial strains and growth conditions Rhodopirellula rubra (strains LF2, UC9 and MsF5) and Rhodopirellula lusitana (strains UC17 and Sm4) were isolated from the biofilm community on the surface of marine macroalgae from the north coast of Portugal (Lage and Bondoso 2011). The reference strain Rhodopirellula baltica was isolated from the water column of the Baltic Sea (Schlesner 1994). E. coli was isolated from a River Febros water sample - Avintes, Portugal (Cabral and Marques 2006). Pseudomonas putida NB3L was isolated from a marine sponge (Pimenta 2010) and Vibrio anguillarum from a sea bass’s kidney. Planctomycetes strains were cultivated on modified M13 medium (Lage and Bondoso 2011). E. coli was cultivated in Luria Broth (LB)/Luria Agar (LA) (10 g/L Tryptone, 5 g/L BactoTM Yeast Extract, 1 g/L D-glucose, 5 g/L sodium chloride, ± 1.6 % agar-Bacteriological American Type). P. putida and V. anguillarum were cultivated in Nutrient Broth (NB)/Nutrient Agar (NA) (5 g/L BactoTM Peptone, 3 g/L Yeast extract, 1 g/L D-glucose, ± 1.6 % agar-Bacteriological American Type) made with natural sea water. Growth of the cultures was monitored by measuring the absorbance at 600 nm (OD600nm). All cultures were incubated at 26 °C with the exception of E. coli which was incubated at 37 °C. Toxicity assays Chemicals The tested pollutants were zinc (as ZnCl2, Merck), chromium (as KCr(SO4)212H2O, Merck), copper (as
Antonie van Leeuwenhoek
CuCl22H2O, Merck), arsenic (as KH2AsO4, Sigma), nickel (as NiCl26H2O, Sigma- Aldrich), cadmium (as CdCl221/2H2O, Sigma-Aldrich), nitrite (as NaNO2, Merck), nitrate (as NaNO3, Merck), ammonia (as NH4Cl, MandB), phosphate (NaH2PO4H2O, Merck), sodium azide (NaN3, Riedel-de-Haen), phenol (C6H6O, Sigma-Aldrich), hydrazine (as (NH2)2H2SO4, Sigma- Aldrich), Previcur NÒ (propamocarb hydrochloride, Bayer CropScience), Ridomil Gold SLÒ (metalaxil-M, Agro), IGEPAL CA-630 (SigmaAldrich), a common dish detergent (Super Pop lemon), a common hand cleanser (supermarket brand from Continente), Diclofenac (Sigma-Aldrich), Acetaminophen (Sigma-Aldrich) and Caffeine (SigmaAldrich). Cell viability assays Stock solutions of the pollutants were prepared in Milli-Q water for heavy metals (5,866 lM), ammonia (1.87 M), nitrate (3.53 M), nitrite (1.45 M), RidomilÒ (1 %), Previcur NÒ (10 %), phosphates (0.83 M), hydrazine (15.40 mM), sodium azide (1.54 M), phenol (1.06 M), Diclofenac (31.40 mM), Acetaminophen (66.2 mM) and Caffeine (51.50 mM). For the detergents (IGEPAL CA-630, dish detergent and hand cleanser), the original formulations were used as stock solutions. The exposure of the microorganisms to the pollutants was performed in liquid cultures at the exponential growth phase, which corresponds to an OD600nm of 0.5 AU for E. coli and 0.2 to 0.3 AU for the other bacteria. Absorbance measurements were performed in a spectrophotometer GenesysTM 10 Series (Thermo Spectronic). One millilitre of liquid culture was harvested by centrifugation at 13,400 rpm for 60 s (MiniSpinÒ, eppendorf) and the cells were resuspended in one millilitre of each pollutant concentration tested. The same concentration range for each pollutant was used for all the bacteria. The exposure times were 30 and 60 min. After pollutant exposure, the suspension was centrifuged and resuspended in 1 mL of Milli-Q water, which was afterwards cultivated by means of the drop plate method, i.e. three drops of 10 lL sample were placed on the appropriate medium for each strain. The microbial growth was checked after 24 h for E. coli, V. anguillarum and P. putida or 3 days for planctomycetes. Images of the cultures were recorded using the GenoPlex system (VWR). For results validation, each
experiment was done in triplicate from three different initial cultures. Different growth levels were scored in a range from 0 to 4 (Fig. 1), in which 4 represent the maximum growth level (same as control) and 0 represents the absence of growth. The level 0.5 was applied when only one or two colonies were formed after a longer incubation period. Controls were performed in Milli-Q water using the same procedure as for pollutant exposure and no impairment in bacterial growth was observed comparative to the bacteria kept in marine medium. Oxygen consumption measurements For respirometric assays, representative bacteria were selected: E. coli, P. putida, R. baltica and R. rubra strain LF2. The oxygen consumption was measured with a Clark-type oxygen electrode (Oxygraph System, Hansatech). Temperature was controlled with a water-bath at 37 °C for E. coli and 26 °C for P. putida, R. baltica and R. rubra strain LF2. Culture samples of E. coli, P. putida, R. baltica and R. rubra strain LF2 were harvested and resuspended in the appropriate growth medium up to a final OD600nm of 0.5 AU for E. coli, and 0.2 to 0.3 AU for the other bacteria. One millilitre of culture was placed in the oxygraphic chamber. When a constant slope of the oxygen plot was reached, the culture was exposed to 0.77 M sodium azide in order to determine the effect of this pollutant on respiratory activity. Each experiment was performed three times with independent cultures.
Results and discussion A semi-quantitative assay based on the drop plate method was used for the assessment of cell viability and consequently toxicity of pollutants. Since these bacteria can exist as isolated cells or as rosettes of different size (Bondoso et al. 2014), colonies can be formed by a variable number of cells which limits the accurate determination of colony forming units. As a consequence, a visual semi-quantitative scale based on the cell growth (Fig. 1) was used to evaluate toxicity. Although semi-quantitative methods lack formal statistical meaning, the results obtained give relevant information for future quantitative analysis. The growth level of the different bacteria after exposure to different pollutants concentrations is
123
Antonie van Leeuwenhoek
Fig. 1 Drops of planctomycetes cultures showing the different growth levels (0.5–4). The dashed lines correspond to the area where the drop was placed
summarised in Table 1. The results showed different growth levels for different bacteria, pollutants and concentrations suggesting different sensitivities. With the exception of phenol and sodium azide, no other pollutant affected the growth of E. coli. This strain, being isolated from a contaminated water source (Febros River), is probably well adapted to polluted environments. In contrast, V. anguillarum was, in general, the most sensitive bacterium, which may be due to the habitat it normally inhabits (as a pathogen of fishes), which can protect it from external stresses. Heavy metals In most cases, a dose–response effect was evident in the case of planctomycetes. The sensitivity order showed by planctomycetes to the different metals was: Ni \ As \ Zn \ Cr \ Cd \ Cu, with R. baltica being the most resistant strain. Nickel was the only metal that did not cause a complete loss of cell viability and a relatively low toxicity of arsenic, in the form of arsenate (As5?), was observed. Zinc showed a stronger toxic effect than arsenate, in particular for R. lusitana strains Sm4 and UC17. In fact, other studies have suggested that the effects of zinc in microbial communities, as well as in other aquatic living organisms, are underestimated (Paulsson et al. 2000). Copper, which is a widely known biocide (Borkow and Gabbay 2005), was the most toxic metal in this study. High toxicity of copper in other microorganisms has also been reported and several associated mechanisms were proposed (Boivin et al. 2005; Ore et al. 2010; Santo et al. 2011; Warnes et al. 2012). Kungolos et al. (2009) observed that copper induced a high toxic effect on different tested species (including V. fischeri) at concentration levels lower than 0.1 mg/L, which are lower than the ones tested in this study.
123
In general, P. putida was more resistant to metals than the majority of the planctomycetes. The ability of P. putida to tolerate heavy metals observed in this work (with the exception of copper) agrees with previous genomic studies which have shown that the genome of this bacterium encodes several mechanisms for heavy metal tolerance and homeostasis. These include two systems for arsenic, one for chromate, four to six systems for divalent cations, two systems for monovalent cations, one metallothionein for metal(loid) binding and four ABC transporters for the uptake of essential Zn, Mn, Mo and Ni (Ca´novas et al. 2003; Hu and Zhao 2007). Mechanisms of resistance, tolerance and co-tolerance have also been reported as being a common phenomenon in E. coli, mainly due to its high genomic plasticity (Cohen et al. 1991; Abskharon et al. 2010; Su et al. 2011; Bouzat and Hoostal 2013). In general, the concentration 59 lM did not affect the growth of the bacteria tested. Inorganic nitrogen compounds These compounds (nitrate, nitrite and ammonium) did not cause the complete loss of cell viability in planctomycetes. Viability was slightly affected only by ammonium. On the other hand, P. putida and V. anguillarum were affected by the three compounds: P. putida growth was totally inhibited at 1.77 M nitrate (after 60 min of exposure) and V. anguillarum cells were not viable at 1.87 M ammonium. Ammonium toxicity at lower concentrations (10 mmol/L) affected cyanobacteria isolated from freshwater, reducing the growth by 65 % (Dai et al. 2008). Tolerance to the bacteriostatic action of sodium nitrite in different bacteria was reported early (Castellani and Niven 1955). Recently, Fu et al. (2013) induced resistance to
Antonie van Leeuwenhoek Table 1 Bacterial growth levels after acute exposure to pollutants (0 – Absence of growth; 0.5 to 4 – Different levels of growth) Pollutant
Arsenic
Cadmium
Chromium
Copper
Nickel
Zinc
time (min)
30
30
30
30
30
30
[]
R. rubra
R. baltica
LF2
UC9
MsF5
59 lM
4
4
4
293 lM
4
4
4
587 lM
4
4
1466 lM
3
4
2933 lM
2
4399 lM
R. lusitana
E. coli
P. putida
V. anguillarum
Sm4
UC17
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
1
4
4
3
4
4
3
0
1
1
3
4
2
3
4
1
0
5866 lM
0.5
0
2
2
0.5
0.5
4
0
0
59 lM
4
4
4
4
3
3
4
4
0.5
293 lM
3
3
3
4
0.5
0
4
4
0
587 lM
2
1
1
3
0
0
4
4
0
1466 lM
1
0.5
0
3
0
0
4
4
0
2933 lM
0.5
0
0
2
0
0
4
4
0
4399 lM
0
0
0
1
0
0
4
4
0
5866 lM
0
0
0
0
0
0
4
2
0
59 lM 293 lM
4 3
4 3
4 4
4 4
4 4
4 4
4 4
3 2
3 0
587 lM
2
2
4
4
3
3
4
2
0
1466 lM
1
1
1
2
0
0
4
1
0
2933 lM
0.5
0.5
0
1
0
0
4
0.5
0
4399 lM
0
0
0
1
0
0
4
0
0
5866 lM
0
0
0
0.5
0
0
4
0
0
59 lM
3
3
0.5
0.5
0
1
4
2
1
293 lM
0
1
0
0
0
0
4
0
0
587 lM
0
1
0
0
0
0
4
0
0
1466 lM
0
0.5
0
0
0
0
4
0
0
2933 lM
0
0.5
0
0
0
0
4
0
0
4399 lM
0
0
0
0
0
0
4
0
0
5866 lM
0
0
0
0
0
0
4
0
0
59 lM
4
4
4
4
4
4
4
4
4
293 lM 587 lM
4 4
4 4
4 4
4 4
4 4
4 4
4 4
4 4
3 3
1466 lM
3
4
4
4
4
4
4
4
0
2933 lM
3
4
4
4
4
4
4
4
0
4399 lM
3
4
4
4
4
4
4
4
0
5866 lM
2
3
3
4
2
3
4
4
0
59 lM
4
4
4
4
4
4
4
4
0
293 lM
3
4
4
4
4
2
4
4
0
587 lM
3
4
4
4
1
1
4
4
0
1466 lM
3
2
4
3
0
0.5
4
4
0
2933 lM
2
1
3
3
0
0.5
4
3
0
4399 lM
2
1
2
2
0
0
4
2
0
5866 lM
1
0.5
0.5
0.5
0
0
4
1
0
123
Antonie van Leeuwenhoek Table 1 continued Pollutant
Nitrate
Nitrite
time (min)
IGEPAL
MsF5
4 4
4 4
4 4
P. putida
V. anguillarum
Sm4
UC17
4 4
4 4
4 4
4 4
3 2
4 3
3.53 M
4
4
4
4
4
4
4
0.5
2
4
4
4
4
4
4
4
4
4
1.77 M
4
4
4
4
4
4
4
0
4
3.53 M
3
4
4
4
4
4
4
0
3
0.14 M
4
4
4
4
4
4
4
4
4
0.73 M
4
4
4
4
4
4
4
3
3
1.45 M
4
4
4
4
4
4
4
1
2
0.14 M
4
4
4
4
4
4
4
4
4
0.73 M
4
4
4
4
4
4
4
1
3
1.45 M
4
4
4
4
4
4
4
0.5
2
0.02 M
4
4
4
4
4
4
4
4
4
0.19 M
4
4
4
4
4
4
4
1
2
30
30
30
1.87 M
2
4
4
4
4
2
4
1
0
0.02 M 0.19 M
4 4
4 4
4 4
4 4
4 4
4 3
4 4
4 3
4 0.5
1.87 M
3
4
4
3
3
1
4
1
0
0.08 M
4
4
2
2
2
4
4
2
0
0.42 M
1
0.5
1
0
1
1
4
0.5
0
0.83 M
0.5
0.5
0.5
0
1
0.5
4
0
0
0.08 M
2
1
2
0
2
3
4
3
0
0.42 M
0
0.5
1
0
0.5
0.5
4
0.5
0
0.83 M
0
0
0.5
0
0.5
0
4
0
0
1%
3
4
4
4
4
4
4
4
2
5%
3
4
4
3
3
2
4
4
0
10 %
2
4
4
2
1
2
4
4
0
1%
3
4
4
4
3
3
4
4
0
5%
2
4
0.5
3
0.5
2
4
3
0
10 %
1
4
0
2
0
1
4
2
0
30
1% 5%
3 3
4 4
4 4
4 3
4 4
4 4
4 4
4 3
1 1
10 %
2
4
4
2
4
4
4
3
0.5
60
1%
4
4
4
4
4
4
4
3
0.5
5%
3
4
4
2
4
4
4
2
0.5
10 %
2
4
3
1
3
4
4
1
0
1%
3
4
2
4
4
4
4
4
0.5
5%
2
4
0.5
3
4
2
4
4
0.5
10 %
2
1
0
3
3
0.5
4
4
0
1%
3
4
1
4
4
4
4
4
0
5%
2
2
0
3
4
2
4
4
0
10 %
0.5
0.5
0
2
3
0.5
4
3
0
30
30
CA-630 60
123
UC9
E. coli
0.35 M
60
Dish detergent
LF2
R. lusitana
60
60
Hand cleanser
R. baltica
0.35 M 1.77 M
60
Phosphates
R. rubra
30
60
Ammonium
[]
Antonie van Leeuwenhoek Table 1 continued Pollutant
RidomilÒ
time (min)
30
60
Previcur NÒ
30
60
Phenol
30
60
Hydrazine
30
60
Sodium Azide
30
60
[]
R. rubra
R. baltica
R. lusitana UC17
4 2
4 1
P. putida
V. anguillarum
4 4
4 4
4 0
LF2
UC9
MsF5
0.1 % 0.5 %
4 2
4 2
4 1
1%
0
0
0
0
0
0
4
1
0
0.1 %
3
4
4
4
4
4
4
4
4 0
4 1
Sm4
E. coli
0.5 %
0.5
2
3
2
0
2
4
4
1%
0
0
0
0
0
0
4
1
0
1%
4
4
4
4
4
4
4
4
4
5%
3
4
3
4
3
4
4
4
4
10 %
0.5
4
1
0
1
3
4
4
4
1%
4
4
4
4
4
4
4
4
4
5%
0.5
4
2
3
2
4
4
4
4
10 %
0
3
0
0
1
0.5
4
4
4
0.01 M
4
4
4
4
4
4
4
4
0.5
0.11 M
0
0
0
0
0
0
0
0
0
1.06 M
0
0
0
0
0
0
0
0
0
0.01 M 0.11 M
4 0
4 0
4 0
4 0
4 0
4 0
4 0
4 0
0 0
1.06 M
0
0
0
0
0
0
0
0
0
0.15 mM
2
4
4
4
2
4
4
1
0
1.54 mM
0
0
0
0
0.5
0
4
0
0
15.4 mM
0
0
0
0
0
0
4
0
0
0.15 mM
1
0.5
0
0.5
0.5
0
4
0
0
1.54 mM
0
0
0
0
0
0
4
0
0
15.4 mM
0
0
0
0
0
0
4
0
0
0.15 M
4
4
4
4
4
4
4
4
3
0.77 M
4
4
4
4
4
4
3
0
2
1.54 M
4
4
4
4
4
4
2
0
1
0.15 M
4
4
4
4
4
4
4
4
3
0.77 M
4
4
4
4
4
4
2
0
2
1.54 M
4
4
4
4
4
4
2
0
0
Diclofenac
30
3.1 mM 31.4 mM
4 0
4 0
2 0
4 1
4 0
4 0
4 4
3 1
0 0
Acetaminophen
30
6.6 mM
4
4
4
4
4
4
4
4
4
66.2 mM
4
4
3
4
4
4
4
4
2
5.2 mM
4
4
4
4
4
4
4
4
4
51.5 mM
4
4
4
4
4
4
4
4
0
Caffeine
30
nitrite in E. coli and Shewanella oneidensis at concentration of 5 mM. However, to our knowledge, tolerance to nitrite has never been reported at the highest concentration tested in this study.
Furthermore, nitrate, nitrite and ammonium, when supplied as the only N-source were able to support the growth of these strains of planctomycetes (unpublished data). As these planctomycetes were isolated
123
Antonie van Leeuwenhoek
from coastal marine environments, they may also contribute to the inorganic part of the nitrogen cycle in these habitats. Phosphates Phosphate is a key nutrient for cells and for that reason, the majority of the microbial studies focus on phosphate depletion rather than on phosphate surplus. The main ecological problem related to phosphates is eutrophication due to high concentrations of this element in runoffs with agricultural fertilizers and detergents (Kleinman et al. 2011). However, some reports showed that even at high concentrations the microbial community is not affected and in some cases can increase their biomass (Lehman 1976; Jordaan and Bezuidenhout 2013). Other studies also suggested that phosphate doesn’t have a negative effect on the microbial growth (Appenzeller et al. 2001), microbial community structure, ecophysiological index and colony-development index (Sarathchandra et al. 2001). In the present work, phosphates had a strong effect on cell viability of some planctomycetes, P. putida and V. anguillarum (Table 1). Indeed, almost all bacteria studied were affected by the lowest phosphate concentration tested (0.08 M) and V. anguillarum growth was completely inhibited at this concentration. R. baltica lost completely cell viability at 0.42 M. These results are not in accordance with the ones mentioned above. The strong effect observed may be explained by the use of sodium phosphate as observed by others, particularly with Gram negative bacteria (Dickson et al. 1994; Kim and Marshall 1999). On the other hand, E. coli, R. lusitana strains MsF5 and Sm4 could tolerate 0.83 M of phosphates even after 60 min of exposure. In the case of E. coli, it was described early that when the inorganic phosphate is in excess, the PHO regulon (responsible for phosphorus assimilation) is inactivated and the necessary inorganic phosphate for the cell is taken up by low affinity with inorganic phosphate transporters (Wanner 1993). Detergents The detergents tested had a strong effect on the cell viability of V. anguillarum and when growth was observed it was minimal. In general IGEPAL-C630 was the most toxic detergent, followed by the hand
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cleanser. However, the complete loss of cell viability was only observed at a concentration of 10 % in hand cleanser for R. lusitana strains Sm4 and MsF5, and at 10 % in IGEPAL for R. rubra strain MsF5 (in all cases after 60 min of exposure). Being surfactants, detergents damage the phopholipid bilayer of the cell, particularly the inner membrane in Gram negative bacteria, and promote their self-uptake into the cells. According to Salton (1968), they can penetrate the cell, react with proteins (denaturation) and/or with lipids in order to induce membrane disorganization, causing the leakage of intracellular low molecular weight material. However, several resistance mechanisms to surfactants have been described in Gram negative bacteria, acquired either by chromosomal mutations or plasmid-mediation (Langsrud et al. 2003), and are strongly related to the outer membrane which may prevent their uptake. These resistance mechanisms may be present in the studied bacteria as they are Gram negative. This should also be the case of planctomycetes that recently have been proposed to possess an outer membrane comparable to the Gram negative bacteria (Speth et al. 2012; Santarella-Mellwig et al. 2013). Fungicides Among the fungicides tested, RidomilÒ had a stronger effect than Previcur NÒ on cell viability of all the bacteria. V. anguillarum and R. lusitana strain Sm4 lost their cell viability when exposed to 0.5 % RidomilÒ, and the other planctomycetes at 1 %. On the other hand, Previcur NÒ only affected planctomycetes and caused the complete loss of cell viability in R. baltica, and R. rubra strains LF2 and MsF5 at 10 % concentration. Previous studies showed that metalaxyl-M (RidomilÒ) was non-toxic for arthropods, vertebrate species (Sukul and Spiteller 2000) and earthworms (Mosleh et al. 2003). In contrast, tests performed with lower trophic level organisms such as V. fischeri, algae and Daphnia magna showed a considerable toxic effect of metalaxyl-M (Kungolos et al. 2009). Considering that concentrations of 1.3 mg/L were found by Graves et al. (2004) in storm water runoffs, our findings also suggest that metalaxyl-M can cause an environmental risk for non-target organisms. Whereas RidomilÒ inhibits fungal growth and reproduction by impairing rRNA synthesis (affecting
Antonie van Leeuwenhoek
the RNA polymerase activity and consequently the synthesis of nucleic acids), Previcur NÒ affects the permeability of fungi cells binding to sterols in the membranes and interfering with the biosynthesis of fatty acids and phospholipids within the cell. Planctomycetes contain lipids in their membranes like palmitic, oleic and palmitoleic lipids, which are typical of eukaryotes (Kerger et al. 1988). Additionally, Gemmata obscuriglobus, being a member of the phylum Planctomycetes, produces sterols through an abbreviated pathway which is not common in other bacteria (Pearson et al. 2003). These particular features of planctomycetes, may explain their higher sensitivity to Previcur NÒ. Fungicides like cycloheximide, amphotericin B and econazole nitrate are used to prevent fungal growth in planctomycetes isolation (Schlesner 1994; Lage and Bondoso 2011). Our results indicated that other fungicides could be used in isolation of planctomycetes, particularly Previcur N, but further studies are needed to assess their effectiveness. Phenol, hydrazine and sodium azide When exposed to phenol almost all the bacteria showed a similar sensitivity at the concentrations tested. The inhibition of growth was observed at 0.11 M after 30 min of exposure, with the exception of V. anguillarum, whose growth was inhibited also at 0.01 M after 60 min of exposure. Widely used as disinfectant, phenol acts on the cytoplasmic membrane of Gram-negative bacteria, can enter into the cells by a hydrophobic pathway and then it promotes a progressive leakage of the intracellular constituents (McDonnell and Russell 1999). Pulvertaft and Lumb (1948) showed that, at lower concentrations than the ones tested in this work (0.032 %), phenols agents lysed rapidly growing cultures of E. coli, staphylococci and streptococci without involvement of autolytic enzymes. Other studies revealed that phenol can act at the separation point of dividing cells, so that the offspring cells are more affected (Srivastava and Thompson 1965, 1966). Hydrazine, at 0.15 mM, strongly reduced the cell viability of R. rubra strain LF2, R. lusitana strain Sm4 and P. putida after 30 min of exposure, and also of R. baltica and R. rubra strain UC9 after 60 min. At this concentration V. anguillarum did not grow at all. Exposure to hydrazine at 1.54 mM for 60 min caused
a complete loss of cell viability in all the bacteria tested, with the exception of E. coli. This compound affects gluconeogenesis in cells, blocking the conversion of oxaloacetic to phosphoenolpyruvic acid through inhibition of phosphoenolpyruvate carboxykinase (PEPCK) (Prajongtat et al. 2013). In silico analysis showed the presence of this enzyme in all the bacteria tested (data not shown). In addition, the high toxicity of this compound may be due to the potential induction of DNA damage and consequent inactivation, already well-described in Gram-negative Salmonella (McCann et al. 1975; Sugimura et al. 1976; Purchase et al. 1978) and mammalian cells (Damjanov et al. 1973; Petzold and Swenberg 1978; Bradley et al. 1979). Interestingly, the anammox group of planctomycetes produces hydrazine as an intermediate in the anammox process but keep it inside the anammoxosome which prevents the noxious effect of this compound (Kartal et al. 2011; van Niftrik 2013). Sodium azide affected E. coli, P. putida and V. anguillarum but not planctomycetes. The growth of P. putida was inhibited at 0.77 M, but the growth of V. anguillarum was inhibited at 1.54 M only after 60 min of exposure. Sodium azide is widely used as a biocide against Gram-negative bacteria. It is a respiration inhibitor, interfering with the electron transport chain of many different organisms. In bacterial cells, its main targets are the cytochromes of the electron transport chain (Li and Palmer 1993; Little et al. 1996) and ATPases (Daniel 1976; Noumi et al. 1987). Although planctomycetes are being considered as Gram-negative like bacteria (Devos 2013), their cell viability results to sodium azide were not as expected. These unexpected results led us to analyze the cell respiration in the studied bacteria. E. coli possessed the highest oxygen consumption (OC) (-62.8 ± 2.8 nmol/mL/min), followed by P. putida (-41.7 ± 7.2 nmol/mL/min). Planctomycetes showed much lower OC (-11.8 ± 2.4 and -19.1 ± 2.9 nmol/mL/ min of R. rubra strain LF2 and R. baltica, respectively). This data is in accordance with the growth rate of these bacteria: E. coli divides in approximately 58 min, P. putida in about 2.5 h and the planctomycetes in about 12.5 h. Even though cell viability of planctomycetes was not affected by sodium azide, their cell respiration was affected (Fig. 2) but to a lesser extent than in E. coli and P. putida. R. rubra strain LF2 was the least affected strain in the presence of sodium azide
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Antonie van Leeuwenhoek
Fig. 2 Decrease in oxygen consumption induced by 0. 77 M sodium azide in liquid cultures of E. coli, P. putida, R. baltica and R. rubra strain LF2
(-4.3 ± 0.0 nmol/mL/min, 64 % OC decrease), followed by R. baltica (-5.1 ± 1.8 nmol/mL/min, 73 % OC decrease). E. coli showed a higher decrease in oxygen consumption (-10.9 ± 6.4 nmol/ml/min, 83 % OC decrease) and P. putida was the most affected bacterium (-1.4 ± 0.1 nmol/ml/min, 97 % OC decrease). Bacterial cell respiration is highly flexible and may involve various cytochromes (Richardson 2000). However, the respiratory mechanisms in planctomycetes are not well understood yet. An in silico analysis revealed the presence of cytochromes c and bd in these microorganisms but not of cytochromes bo or aa3 (data not shown). Further studies are needed to understand the planctomycetal behaviour. Active pharmaceutical ingredients The real impact of these ingredients in aquatic living organisms is still unclear and their emergence is increasing in natural waters (Sanderson et al. 2004). Among the APIs assayed, diclofenac was the only one that affected the growth of the planctomycetes, causing the complete loss of cell viability at 31.4 mM concentration, with the exception of R. baltica. Furthermore, it had also a strong effect on the cell viability of P. putida and V. anguillarum. The effect of diclofenac in all these bacteria is in agreement with effects reported on other microorganisms, such as V. fischeri (Yu et al. 2013). Caffeine only affected V. anguillarum at 51.5 mM concentration. These results disagree with previous ones that indicate the inhibitory effect of caffeine at lower concentrations, mainly in Pseudomonas sp., but also in E. coli (Ramanavicˇien_e et al. 2003; Dash and
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Gummadi 2008). However, our results support other studies that refer the ability to tolerate caffeine by Pseudomonas sp. and E. coli, mainly due to N-demethylase activity (Gibson et al. 2012; Summers et al. 2012). Up to now, only one demethylase is listed in genomic microbial databases for planctomycetes (G. obscuriglobus). Acetaminophen only slightly affected the cell viability of R. rubra strain MsF5 and V. anguillarum. The impact of APIs in ecosystems has only recently been assessed. The few studies available showed that they can have antibacterial activity as previously described and interfere with the bacteria biofilm communities (Lawrence et al. 2012). However, several bacteria may start to adapt to these conditions, along with genome evolution, including through horizontal gene transfer.
Conclusions The cell viability studies performed revealed diverse responses of the bacteria studied to different toxicants, which may help understanding of microbial community structure changes in habitats under pollution stress. Regarding the pollutants tested, stronger effects were observed in the presence of RidomilÒ, phenol, hydrazine and copper. In most cases, E. coli was the most resistant bacterium and V. anguillarum the most sensitive. The elevated resistance of this E. coli strain is consistent with the polluted habitat from where it was isolated. A potential role in the inorganic nitrogen cycle is envisaged for planctomycetes due to their resistance to high concentrations of inorganic nitrogen compounds. Although sodium azide did not reduce cell viability of planctomycetes, it affected their cell respiration, although in a reduced way in comparison to other bacterial species. Further studies are needed to clearly understand the mechanisms of cell respiration in planctomycetes, and the impact of sodium azide on the process. The findings obtained are a contribution to overcoming the gaps in knowledge that still remain about planctomycetes ecophysiology. These microorganisms, being widespread in aquatic environments and showing different dose–response behaviour to different pollutants, may be potential candidates for water quality assessment.
Antonie van Leeuwenhoek Acknowledgments We thank Paula Tamagnini for the oxygen consumption measurements. This research was supported by the European Regional Development Fund (ERDF) through the COMPETE Operational Competitiveness Programme and national funds through FCT – Foundation for Science and Technology, under the project PEst-C/MAR/LA0015/2013.
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