Enrichment experiment changes microbial interactions in an ultra-oligotrophic environment.

The increase of nutrients in water bodies, in particular nitrogen (N) and phosphorus (P) due to the recent expansion of agricultural and other human activities is accelerating environmental degradation of these water bodies, elevating the risk of eutrophication and reducing biodiversity. To evaluate the ecological effects of the influx of nutrients in an oligotrophic and stoichiometrically imbalanced environment, we performed a replicated in situ mesocosm experiment. We analyzed the effects of a N- and P-enrichment on the bacterial interspecific interactions in an experiment conducted in the Cuatro Cienegas Basin (CCB) in Mexico. This is a desert ecosystem comprised of several aquatic systems with a large number of microbial endemic species. The abundance of key nutrients in this basin exhibits strong stoichiometric imbalance (high N:P ratios), suggesting that species diversity is maintained mostly by competition for resources. We focused on the biofilm formation and antibiotic resistance of 960 strains of cultivated bacteria in two habitats, water and sediment, before and after 3 weeks of fertilization. The water habitat was dominated by Pseudomonas, while Halomonas dominated the sediment. Strong antibiotic resistance was found among the isolates at time zero in the nutrient-poor bacterial communities, but resistance declined in the bacteria isolated in the nutrient-rich environments, suggesting that in the nutrient-poor original environment, negative inter-specific interactions were important, while in the nutrient-rich environments, competitive interactions are not so important. In water, a significant increase in the percentage of biofilm-forming strains was observed for all treatments involving nutrient addition.

Introduction

A central goal of ecology is the understanding of the driving principles underpinning biodiversity (Gaston, 2000). Several lines of research have tried to explain the differences in diversity among local communities, focusing on approaches ranging from network interactions in food webs to random assemblages resuln class="Chemical">ting from the dispersion capacity of organpan>isms (Hutchinpan>son, 1959; Dykhuizenpan>, 1998; Kassenpan> et al., 2000; Benpan>nie et al., 2006; Marinpan>i et al., 2007). These theories, as well as other more complex explanpan>ations of biodiversity (such as chaotic inpan>teractions betweenpan> compepan> class="Chemical">ting strains; Huisman and Weissing, 1999) are partially successful, depending on the system studied and are frequently complementary. The patterns of biodiversity are determined by the combined impacts of interactions of several biotic and abiotic environmental factors, and cost-benefit strategies followed by each species can change according to the complexity of the community and its nutrient availability (Marini et al., 2007; Werner et al., 2014).

For instance, it is known that the abundances and proportions of n class="Chemical">nitrogen anpan>d pan> class="Chemical">phosphorus in the environment have a major influence in the composition of species at macro and microscopic scales (Makino and Cotner, 2005; Jansson et al., 2006; Marini et al., 2007; Christofoli et al., 2010; Nelson and Carlson, 2011). Classic trade-off competition theory places resource availability at the center of the processes that influence community structure (Tilman et al., 1981; Smith, 1993; Brauer et al., 2012). MacArthur and Wilson (1967) coined the terms “r-selection” and “K-selection.” This theory takes into account biotic and abiotic factors such as climate, mortality, survivorship, population size, intra and interspecific competition, relative abundance and length of life. Under this theory, r-strategists are adapted to abundant nutrients, which are rapidly exploited. On the other hand, K-strategists are adapted to a long-term survival on limited resources (Pianka, 1970; Fuchs et al., 2000; Singer et al., 2011). Experimental evidence from different microcosm experiments shows that nitrogen and phosphorus enrichments result in significant changes in community structure in terms of uniformity and species richness (Schäfer et al., 2001; Nelson and Carlson, 2011).

The changes in community structure are probably driven mainly by the effects of nutrients on the growth rates of individual strains (Smith, 1993), but other factors could also play a role such as seasonal changes (Rodríguez-Verdugo et al., 2012; Bevivino et al., 2014), as well as changes in mutualistic interactions due to fluctuations in the supply and demand of “public goods” by cause of the entrance of nutrients to the ecosystem (Morris et al., 2012; Sachs and Hollowell, 2012; Werner et al., 2014).

The release of chemical compounds into the environment, which are toxic or inhibitory to the competitors, is one of the commonly observed antagonistic interactions (Riley and Gordon, 1999; Lenski and Riley, 2002; Riley and Wertz, 2002; Kirkup and Riley, 2004; Hibbing et al., 2010; Kohanski et al., 2010; Majeed et al., 2011, 2013; Pérez-Gutiérrez et al., 2013; Aguirre-von-Wobeser et al., 2014). These secretions highly influence community structure and maintain cohesion of bacterial populations by leading to the exn class="Chemical">tinction of senpan>sitive strainpan>s inpan> liquid media, but to coexistenpan>ce inpan> a structured media (Validov et al., 2005; Greig anpan>d Travisanpan>o, 2008; Rypienpan> et al., 2010; Cordero et al., 2012). Inpan> the non-tranpan>sitive model rock-paper-scissors (RPS), one anpan>tagonist, one senpan>sitive anpan>d one resistanpan>t strainpan> coexist inpan> structured media (Czarán et al., 2002; Kerr et al., 2002; Kirkup anpan>d Riley, 2004). Inpan> a natural enpan>vironmenpan>t, this non-tranpan>sitive relation may occur if toxic production is costly, both senpan>sitive anpan>d resistanpan>t strainpan>s exist anpan>d costs associated with resistanpan>ce are less thanpan> those of toxinpan> production. However, the relative magnpan>itude of each of these features is critical for coexistenpan>ce (Kerr et al., 2002).

The effects of nutrient addition in microbial community structure can also depend on the extant biodiversity prior to the increase of nutrient availability, as shown by an experiment performed on the bacterial community of Owasso Lake (Minnesota, USA), which has been reported as one of the least diverse bacterial communities known (Makino and Cotner, 2005). When this lake was enriched with n class="Chemical">nitrogen, pan> class="Chemical">phosphorus and carbon, the community showed a response that would be expected from a single strain, rather than from a community (Jürgens and Güde, 1990), suggesting that if bacterial diversity in a given environment is low, this diversity will remain low, because of its low potential to respond at a community level to changes in its environment. In contrast, in more diverse environments, nutrient enrichment usually homogenizes the composition of the community assemblage (Donohue et al., 2009).

The Cuatro Cienegas Basin (n class="Chemical">CCB) located at the Chihuahuanpan> desert of north cenpan>tral Mexico is a good system to study the effects of the addition of nutrienpan>ts on bacterial communities. pan> class="Chemical">CCB harbors a number of highly oligotrophic aquatic ecosystems that have very low available phosphorus levels, as well as a stoichiometric disequilibrium with nitrogen and thus are strongly limited by phosphorus (Elser et al., 2005, 2006). We have suggested that the high microbial species diversity in CCB is strongly shaped by the stress of low nutrient supplies and the impacts of interspecific competition (Souza et al., 2008). Previous studies indicate that CCB bacterial communities display strategies to cope with this lack of nutrients, including a high representation of genes involved in phosphorus assimilation (e.g., pho and pst in Mesquites river; Breitbart et al., 2009) and the presence of a large number of genes related to the production and resistance to antibiotics (in Pozas Rojas; Bonilla-Rosso et al., 2012; Peimbert et al., 2012).

n class="Species">Pseudomonas anpan>d other cultivable proteobacteria are abundanpan>t inpan> differenpan>t aquatic systems inpan> pan> class="Chemical">CCB, including the Churince system (Escalante et al., 2009) and in the Los Hundidos region (Bonilla-Rosso et al., 2012; Peimbert et al., 2012). In a different study in the flow system Churince, it was found that the Pseudomonas genus exhibited a seasonal variation across summer and winter (Rodríguez-Verdugo et al., 2012).

In this paper, we analyze the seasonal response of bacteria cultivable in n class="Species">Pseudomonas Isolation Agar (pan> class="Chemical">PIA; Difco, Detroit, MI) in response to the nutrient amendment. Our study seeks to understand the effects of nitrogen and phosphorus fertilization on the interaction potential of the cultivable gamma-proteobacteria community in two different habitats, water and sediment, by determining how nutrient enrichment affected various features related to microbial species interactions, such as the tendency to form biofilms and resistance to antibiotic compounds. We are clearly aware of the limitations of culture media in order to capture the diversity of a site, as we have described elsewhere (see Souza et al., 2006, 2012), nevertheless, only in culture we can study the physiology and sociology of particular strains, which is the particular aim of this study. Our findings shed light on how the supplies of key nutrients, such as N and P modulate community structure, as well as the nature and intensity of interspecific interactions.

Materials and methods

Study site

The nutrient enrichment experiment was conducted in situ in a small shallow evaporitic pond, Lagunita (26.84810° n class="Chemical">N, 102.14160° W), lateral to the mainpan> Churinpan>ce flow system. The Churinpan>ce flow system, located at the westernpan> region of pan> class="Chemical">CCB is dominated by gypsum sediments and has a strong longitudinal gradient of salinity, temperature, pH and dissolved oxygen (Cerritos et al., 2011). Lagunita is characterized by low phosphorus concentrations (PO4 as low as 0.1 μM and often below detection) (Elser et al., 2005), but relatively high concentrations of inorganic N and thus high N:P ratios (>200:1 for total nutrients; Lee et al., in press). Lagunita is subjected to strong evaporation, the greatest water depth at the beginning of the experiment was 32 cm and decreased to as low as 12 cm by the end of the experiment.

Experimental design of mesocosm

The mesocosm experiment was conducted from May to June 2011. Each mesocosm consisted of a round clear plastic tube with a diameter of 40 cm. The tube had a depth of 20 cm into the sediment and approximately 20 cm above the n class="Chemical">water surface. The mesocosms were arranpan>ged inpan> a ranpan>domized complete block with a total of 5 blocks, separated about 2-4 m from each other. Each block consisted of four treatmenpan>ts. A non enpan>riched control treatmenpan>t, a pan> class="Chemical">phosphorus enrichment treatment (P), amended with KH2PO4 and maintained at 2 day intervals at a final concentration of 1 μM; a nitrogen and phosphorus enrichment (NP), amended as above with KH2PO4 to 1 μM but also with NH4NO3, to achieve N:P of 16:1; and a nitrogen and phosphorus enrichment with extra nitrogen (NNP), amended as above with 1 μM KH2PO4 but also with NH4NO3 at an N:P ratio = 75:1.

Methodology for chemical analyses

For the chemical analyses, n class="Chemical">water was collected in acid-washed 2 L cubitainers. pan> class="Chemical">Water samples were filtered through pre-combusted (24 h at 450°C) GF/F filters (Whatman, Piscataway, NJ) for seston elementary analysis and stored at −20°C. To measure total dissolved nutrients, water samples were filtered through 0.2 μm polyethersulfone membrane filters. Samples for dissolved organic carbon (DOC), and total dissolved nitrogen (TDN) were acidified with 12 N HCL to pH < 2 and stored in the dark at room temperature, while the remaining filtrate was frozen for total dissolved phosphorus (TDP) and soluble reactive phosphorus (SRP) analyses.

GF/F with seston were thawed, dried at 60°C and packed into n class="Chemical">tin disks (Elemenpan>tal Microanpan>alysis, U.K.) for pan> class="Chemical">N analyses with a Perkin Elmer™ PE 2400 CHN Analyzer at the Arizona State University Goldwater Environmental Laboratory (ASU GEL). Another set of dried GF/F filters from the same water samples was used in order to estimate seston P content. These filters were digested in persulfate followed by a colorimetric analysis to determine PO3−4 (APHA, 2005). TDP concentrations were determined using the colorimetric assay after persulfate digestion as previously described; SRP was measured without the persulfate digestion. DOC and TDN were analyzed using the Shimadzu TOC-VC/TN analyzer at the ASU GEL. Total Phosphorus (TP) concentration was calculated as the sum of the seston and total dissolved pools.

Sample collection and processing

n class="Chemical">Water anpan>d sedimenpan>t samples were takenpan> for each one of the four treatmenpan>ts for each experimenpan>tal block (20 samples total). Inpan>itial samples were obtainpan>ed from surface pan> class="Chemical">water and top of sediment prior to the application of the treatments (T0; 14 May 2011) and after 21 days of enrichment (4 June 2011) using sterile BD Falcon vials (BD Biosciences, San Jose, CA). Cultivable bacteria were obtained by plating 100 μl of each water sample or 100 μl of a 1:10 sediment dilution, prepared with 0.9% NaCl solution. Strains were isolated from water and sediment using PIA medium. Strains were incubated on agar plates at room temperature for 2 days at the field, and then were kept at 4°C until isolation in the laboratory. Individual colonies were transferred to new PIA plates and then were grown at 30°C. A total of 960 isolates were obtained.

DNA extraction and PCR amplification

Phylogenetic identification of the strains was performed using the 16S rRNA genpan>e. For the isolates, Dpan> class="Chemical">NA extraction was very complicated, and thus several methods were tested (Chen and Kuo, 1993; Aljanabi and Martinez, 1997; Reischl et al., 2000, and DNeasy Blood and Tissue kits, Qiagen, Hilden, Germany). Ultimately, the DNeasy Blood and Tissue kit was the method used, and from the 960 isolates, good quality DNA was obtained for 152 strains. 16S rRNA genes were amplified using universal primers 27F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492R (5′-GGT TAC CTT GTT ACG ACT T-3′) (Lane, 1991) and high fidelity Phusion hot start DNA polymerase (Finnzymes, Espoo, Finland). All reactions were carried out in a Techne TC-3000 thermal cycler (Barloworld Scientific, Staffordshire, UK) with the following program: 94°C for 5 min, followed by 30 cycles consisting of 94°C for 1 min, 50°C for 30 s, 72°C for 1 min and 72°C for 5 min. Polymerase chain reaction (PCR) amplification products were electrophoresed on 1% agarose gels. Sanger sequencing was performed at the University of Washington High Throughput Genomics Center. The sequences have been uploaded to GenBank with accession numbers (KF317734-KF317770, KM352505-KM352636).

Phylogenetic analysis

The 16S rRNA sequenpan>ces were alignpan>ed with CLUSTALW (Larkinpan> et al., 2007) anpan>d pan> class="Disease">MUSCLE (Edgar, 2004), and the alignments were manually revised. For the reconstruction of the phylogenetic tree, a maximum likelihood analysis was done with PhyML version 3.0 (Guindon et al., 2010) with the TrN+G model. The substitution model was calculated with jModelTest 2.1.3 (Darriba et al., 2012). The degree of statistical support for the branches was determined with 1000 bootstrap replicates. Genera level identification of the strains was made using the classifier tool (Wang et al., 2007) from the Ribosomal Database Project (RDP) Release 10, update 30 (Cole et al., 2009; Table 1). We performed a local BLAST search (Altschul et al., 1990) to find the nearest neighbors using the 16S ribosomal RNA. These analyses were performed with 700 bp from the 5′ end of all the sequences.

Table 1

Summary of nutrient-induced shifts in relative abundance of dominant bacterial lineages.

Class	Consensus clade	Total Count	T0		Control		NP		NNP		P
			H20	Sed	H20	Sed	H20	Sed	H20	Sed	H20	Sed
α-Proteobacteria	Rhizobium	8	0	6	0	0	0	0	0	0	0	2
γ-Proteobacteria	Stenotrophomonas	9	0	9	0	0	0	0	0	0	0	0
γ-Proteobacteria	Pseudomonas	54	20	0	9	0	0	0	4	0	21	0
γ-Proteobacteria	Shewanella	1	1	0	0	0	0	0	0	0	0	0
γ-Proteobacteria	Aeromonas	20	5	0	0	3	10	0	2	0	0	0
γ-Proteobacteria	Citrobacter	1	1	0	0	0	0	0	0	0	0	0
γ-Proteobacteria	Rheinheimera	5	5	0	0	0	0	0	0	0	0	0
γ-Proteobacteria	Halomonas	47	7	0	0	0	0	18	0	22	0	0
Actinobacteria	Kocuria	4	0	0	0	0	0	1	0	0	0	3
Actinobacteria	Brachybacterium	3	0	3	0	0	0	0	0	0	0	0

Antibiotic resistance assays

The 960 isolates were grown on LB plates supplemented with the following antibiotic concentrations: n class="Chemical">Carbenicillin 500 μg/ml, pan> class="Chemical">Kanamycin, 200 μg/ml, Tetracycline, 150 μg/ml, Streptomycin, 200 μg/ml and Gentamicin 150 μg/ml. Isolates were labeled as resistant to an antibiotic if we observed bacterial growth; otherwise, they were considered as nonresistant.

For statistical inference, the antibiotics data were organized as conn class="Chemical">tingenpan>cy tables, anpan>d Barnpan>ard's exact test (Barnpan>ard, 1945) was performed. Inpan>depenpan>denpan>t tests were conducted for each treatmenpan>t againpan>st the inpan>itial time, for pan> class="Chemical">water and sediment data. Since several treatments were compared to the initial condition, a Bonferroni correction was applied for multiple tests.

Biofilm formation assay

A microtiter dish assay was performed for the same 960 isolates as described by O'Toole (2011) with the following modifications. The overnight culture was grown in n class="Chemical">LB medium. The microtiter plates were inpan>cubated for 18 h at 30°C. Each strainpan> was anpan>alyzed by triplicate anpan>d we used the strainpan>s pan> class="Species">P. aeruginosa PAO1 and E. coli MC4100 as positive and negative controls, respectively. After incubation, plates were gently washed with water, and subsequently 125 μl of a 0.1% solution of crystal violet in water was added to each well. The plates were incubated at room temperature for 15 min and then washed with water. 125 μl of 30% acetic acid in water was added to each well to solubilize the crystal violet and it was incubated for 15 min. The volume was transferred to a new flat-bottomed microtiter dish. Absorbance was read with a Synergy HT plate reader (BioTek, Winooski, VT) at 550 nm.

To determine the statistical significance of differences in biofilm formation between the different treatments and the initial condition, we organized the data as conn class="Chemical">tingenpan>cy tables including the number of positive anpan>d negative strains for biofilm formation for the initial time anpan>d each treatmenpan>t, anpan>d applied Barnpan>ard's exact tests. To correct multiple tespan> class="Chemical">ting, a Bonferroni correction was applied.

Results

To characterize the changes in diversity of cultivated Proteobacteria related to pseudomonads associated with nutrient enrichment in a n class="Chemical">water system, we performed anpan> inpan> situ mesocosm experimenpan>t with three experimenpan>tal manpan>ipulations, addinpan>g pan> class="Chemical">phosphate (P), phosphate and nitrogen (NP), and phosphate and excess nitrogen (NNP), as well as an un-enriched control. The experiment is part of a bigger project that is described in detail elsewhere (Lee et al., in press). Cultures were obtained from surface water and sediment in two sampling events, prior to the experimental manipulations, and after 21 days, and a total of 960 isolates were analyzed for interaction phenotypes such as biofilm formation, and antibiotic resistance. A subsample of the isolates was characterized by 16S rRNA sequence.

Nutrient concentrations

n class="Chemical">Nutrienpan>t conditions durinpan>g the experimenpan>t were characterized by low concenpan>trations of P but relatively high concenpan>trations of pan> class="Chemical">N that increased in NP and NNP treatments. Despite nutrient enrichment, N:P ratios remained quite stoichiometrically imbalanced. Except for the NNP treatment, total dissolved phosphorus and soluble reactive phosphorus were significantly different respect to control. Total phosphorus, total dissolved phosphorus and the N:P ratio were significantly different in all enriched treatments. Total dissolved nitrogen was significantly different only in the nitrogen amended treatments (Table 2). Details of dynamics and fate of N and P in the mesocosms are given elsewhere (Lee et al., in press).

Table 2

Nutrient concentrations.

Treatment	DOC	TDN	Seston P	TP	TDP	SRP	N:P
T0	2177 ± 51.84	130 ± 5.98	1.21 ± 0.08	1.79 ± 0.20	0.58 ± 0.17	0.06 ± 0.02	47.26 ± 1.95
Control	2799.17 ± 212.81	182.77 ± 9.51	1.09 ± 0.11	2.47 ± 0.24	1.39 ± 0.25	0.31 ± 0.09	49.12 ± 6.04
NP	3005 ± 227.63	202.48 ± 12.11*	3.03 ± 0.26*	4.17 ± 0.62*	1.15 ± 0.40	0.27 ± 0.12	36.39 ± 6.34*
NNP	3198.81 ± 144.87*	226.32 ± 25.63*	3.29 ± 0.68*	4.15 ± 0.71*	0.86 ± 0.25*	0.19 ± 0.08*	36.25 ± 6.20*
P	3010.65 ± 227.30	192.05 ± 17.88	2.71 ± 0.33*	3.95 ± 0.59*	1.24 ± 0.28	0.33 ± 0.10	28.26 ± 3.75*

Dissolved organic carbon (DOC), total dissolved nitrogen (TDN), total phosphorus (TP), total dissolved phosphorus (TDP) and soluble reactive phosphorus (SRP) concentrations in the pond's water and in each treatment after the nutrient enrichment. All values are in μM L−1 with the exception of N:P which represents the elemental stoichiometry of seston. Each value represents the average and one standard deviation. The “

” symbol denotes significant change from the control.

n class="Chemical">Nutrient concentrations.

Dissolved n class="Chemical">organic carbon (pan> class="Chemical">DOC), total dissolved nitrogen (TDN), total phosphorus (TP), total dissolved phosphorus (TDP) and soluble reactive phosphorus (SRP) concentrations in the pond's water and in each treatment after the nutrient enrichment. All values are in μM L−1 with the exception of N:P which represents the elemental stoichiometry of seston. Each value represents the average and one standard deviation. The “

Phylogenetic diversity and responses to treatments

The phylogenetic relationships of partial 16S rRn class="Chemical">NA genpan>e sequenpan>ces (700 bases) of the 152 strainpan>s were determinpan>ed by queries againpan>st the Ribosomal Database Project (Cole et al., 2009) usinpan>g SeqMatch anpan>d Classifier (Wanpan>g et al., 2007). The phylogenpan>etic results showed that the strainpan> collection was dominpan>ated by Proteobacteria (145 isolates/95.4%), with members from the gamma- (137/90.1%) anpan>d alpha- (8/5.3%) subdivisions (Table 1), consistenpan>t with the use of the pan> class="Chemical">PIA medium for isolation. We also found several Actinobacteria (7/4.6%). As described by King et al. (1954), this medium includes Irgasan®, a broad spectrum antimicrobial substance that is not active against Pseudomonas spp. The medium also enhances the formation of pigments by Pseudomonas by adding magnesium chloride and potassium sulfate. Pseudomonas species were found in all un-enriched water cultures as well as in almost all enriched samples, with the exception of the NP treatment. Pseudomonas was the most abundant genus for the water isolates and overall for all samples (35.5% of the isolates). Halomonas was the most abundant genus among the sediment strains, representing 30.9% of the isolates.

Using the aligned sequences, a maximum likelihood tree was constructed (Figure 1). For any given treatment, groups of closely related, almost identical strains dominated the samples, even though they were obtained from different replicate mesocosms. Previous to the experimental manipulations, only gamma-proteobacteria were retrieved from the n class="Chemical">water samples, which were dominpan>ated by pan> class="Species">Pseudomonas. Other genera were present, including Aeromonas, Shewanella, Citrobacter, Rheinheimera, and Halomonas. With the addition of phosphorus (P treatment), many closely related Pseudomonas were retrieved. In the NP treatment, all the obtained isolates belonged to the genus Aeromonas. For the high N:P treatment (NNP), a group of Pseudomonas became abundant and two Aeromonas isolates were obtained.

Figure 1

Phylogenetic relationships among 16S ribosomal RNA genes. Sequences from the 152 isolates (highlighted in colors) and 21 reference strains. The coded name of the strains corresponds to the treatments and the environment from which they were isolated. Water samples: #[A-E]#, mesocosm T0, XC#, control treatment, XP#, phosphorus enrichment, XNP#, N:P 16:1 treatment, XNNP#, and N:P 75:1 treatment. Sediment samples: JPXs_#, mesocosm T0, XCs_#, control treatment, XPs_#, phosphorus treatment, XNPs_#, N:P 16:1 treatment, XNNPs_#, and N:P 75:1 treatment, where # is a number and X a letter from A to E, each one represents a replicate.

Phylogenetic relationships among 16S ribosomal Rn class="Chemical">NA genpan>es. Sequenpan>ces from the 152 isolates (highlighted inpan> colors) anpan>d 21 referenpan>ce strainpan>s. The coded name of the strainpan>s corresponds to the treatmenpan>ts anpan>d the enpan>vironmenpan>t from which they were isolated. pan> class="Chemical">Water samples: #[A-E]#, mesocosm T0, XC#, control treatment, XP#, phosphorus enrichment, XNP#, N:P 16:1 treatment, XNNP#, and N:P 75:1 treatment. Sediment samples: JPXs_#, mesocosm T0, XCs_#, control treatment, XPs_#, phosphorus treatment, XNPs_#, N:P 16:1 treatment, XNNPs_#, and N:P 75:1 treatment, where # is a number and X a letter from A to E, each one represents a replicate.

Phylogenetic differences were observed between the abundant strains obtained from the different habitats (n class="Chemical">water vs. sedimenpan>t) but also betweenpan> the strainpan>s correspondinpan>g to the differenpan>t treatmenpan>ts. At the beginpan>ninpan>g of the experimenpan>t, the sedimenpan>t samples were dominpan>ated by anpan>other pan> class="Species">gamma-proteobacterium, Stenotrophomonas, as well as an alpha-proteobacterium, Rhizobium. Some Actinobacteria were also isolated from these initial samples, belonging to the genus Brachybacterium. With P enrichment, Rhizobium was also found, as well as an Actinobacterium, from the genus, Kocuria. However, a larger change in the sampled community was found in all treatments including nitrogen along with P (NP and NNP), where Halomonas (gamma-proteobacteria) was largely dominant.

Antibiotic resistance

We analyzed the prevalence of antibiotic resistances among the 960 isolates. The antibiotics analyzed were n class="Chemical">Carbenicillin, pan> class="Chemical">Kanamycin, Tetracycline, Streptomycin and Gentamicin (Supplementary Table 1; Supplementary Figures 1, 2). Carbenicillin was the most common form of resistance among the isolates. The Carbenicillin resistance was found in 55.2% of the water isolates at time zero and in 68.3% of the total water isolates after the experiment, and in 28.6 and 90.5% of the total sediment isolates, at time zero and after the experiment, respectively. Tetracycline was the least common form of resistance, being represented in 10.4 and 0.8% of the water isolates and 7.1 and 1.0% of the sediment isolates before and after the experiment, respectively. Kanamycin resistance was not observed in sediment samples after the experiment. Overall, there was a statistically significant decrease in the number of resistant strains after the experiment (Bernard's exact test; p < 0.05; Figure 2; Table 3), in all three fertilization treatments and in the control, with the marked exception of Carbenicillin, for which resistance increased significantly in NNP and P treatments in water and in all treatments in sediment.

Figure 2

Prevalence of antibiotic resistance among 960 isolates. The isolates analyzed for each treatment were as follows. T0—163, Control—87, NNP—186, NP—176, P—201, T0S—42, CS—11, NNPS—38, NPS—31, and PS—25.

Table 3

Antibiotic resistance.

Treatment	n	Carbenicillin		Kanamycin		Tetracycline		Streptomycin		Gentamicin
		Resistant (%)	p	Resistant (%)	P	Resistant (%)	p	Resistant (%)	p	Resistant (%)	p
Water T0	163	55.21		22.09		10.43		20.25		12.88
Water Control	87	70.11	0.09	3.45	4.60E-4	0.00	7.23E-3	13.79	0.93	1.15	7.23E-3
Water NNP	186	75.27	3.30E-4	1.61	4.30E-9	2.15	4.94E-3	3.23	1.81E-6	1.08	3.00E-5
Water NP	176	58.52	1.00	0.57	6.04E-10	0.00	4.00E-5	2.84	1.40E-6	0.57	2.00E-5
Water P	201	69.62	0.02	1.49	7.91E-10	0.50	5.00E-5	9.95	0.02	0.50	2.97E-6
Sediment T0	42	28.57		26.19		7.14		33.33		21.43
Sediment Control	11	100	3.00E-4	0.00	0.38	0.00	1.00	0.00	0.10	0.00	0.38
Sediment NNP	38	100	1.53E-11	0.00	2.54E-3	0.00	0.41	5.26	6.99E-3	0.00	0.01
Sediment NP	31	93.55	4.38E-8	0.00	8.15E-3	0.00	0.63	0.00	1.31E-3	0.00	0.02
Sediment P	25	68.00	6.64E-3	0.00	0.02	4.00	1.00	28.00	1.00	4.00	0.27

Percentages of strain resistant for different antibiotics, and their significance with respect to the initial condition (T0) in both water and sediment, according to Barnard's exact tests, with Bonferroni correction for multiple tests. Percentages with p-values less than 0.05 are considered significantly different than the corresponding to T0 values.

Prevalence of antibiotic resistance among 960 isolates. The isolates analyzed for each treatment were as follows. T0—163, Control—87, n class="Chemical">Npan> class="Chemical">NP—186, NP—176, P—201, T0S—42, CS—11, NNPS—38, NPS—31, and PS—25.

Percentages of strain resistant for different antibiotics, and their significance with respect to the initial condition (T0) in both n class="Chemical">water anpan>d sedimenpan>t, according to Barnpan>ard's exact tests, with Bonferroni correction for multiple tests. Percenpan>tages with p-values less thanpan> 0.05 are considered signpan>ificanpan>tly differenpan>t thanpan> the corresponding to T0 values.

Biofilm formation

To further characterize the ecological traits of the studied strains, biofilm formation was analyzed for 923 isolates. In the n class="Chemical">water samples, the enpan>riched isolates exhibited anpan> inpan>crease inpan> the proportion of the ability to form biofilm (Figure 3; Supplemenpan>tary Figure 1). Inpan> a structured enpan>vironmenpan>t, inpan>teractions canpan> be enpan>hanpan>ced by biofilm formation. As expected, sedimenpan>t strainpan>s genpan>erally had a greater tenpan>denpan>cy to produce biofilm that those isolated from pan> class="Chemical">water. However, in sediment, differences between before and after the treatments were not significant (Barnard's exact test; p > 0.05; Table 4), except for the NNP treatment, for which no biofilm-producing strains were detected (Figure 3; Supplementary Figure 2). In the case of water, a significant increase in the percentage of biofilm-producing strains was observed for all treatments involving nutrient additions, namely P, NP, and NNP (Barnard's exact test; p < 0.05; Table 4).

Figure 3

Relative frequency of biofilm forming strains among 923 isolates. The isolates analyzed for each treatment were as follows. T0—157, Control—84, NNP—174, NP—167, P—196, T0S—42, CS—11, NNPS—38, NPS—29, and PS—25.

Table 4

Biofilm formation.

Treatment	n	Biofilm forming (%)	P
Water T0	155	27.74
Water Control	82	41.46	0.13
Water NNP	174	54.60	3.07E-6
Water NP	167	54.49	4.37E-6
Water P	196	55.61	5.78E-7
Sediment T0	43	57.14
Sediment Control	11	90.91	0.16
Sediment NNP	38	0.00	5.82E-8
Sediment NP	31	75.86	0.50
Sediment P	26	53.85	1.00

Percentages of strain forming biofilm, and their significance with respect to the initial condition (T0) in both water and sediment, according to Barnard's exact tests, with Bonferroni correction for multiple tests. Percentages with p-values less than 0.05 are considered significantly different than the corresponding to T0 values.

Relative frequency of biofilm forming strains among 923 isolates. The isolates analyzed for each treatment were as follows. T0—157, Control—84, n class="Chemical">Npan> class="Chemical">NP—174, NP—167, P—196, T0S—42, CS—11, NNPS—38, NPS—29, and PS—25.

Percentages of strain forming biofilm, and their significance with respect to the initial condition (T0) in both n class="Chemical">water anpan>d sedimenpan>t, according to Barnpan>ard's exact tests, with Bonferroni correction for multiple tests. Percenpan>tages with p-values less thanpan> 0.05 are considered signpan>ificanpan>tly differenpan>t thanpan> the corresponding to T0 values.

Discussion

In this study, the effect of experimental nutrient enrichment in a shallow, nutrient-deficient pond was analyzed to assess changes in the composition of the cultivable microbial community as a function of the n class="Chemical">N:P ratio of enpan>richmenpan>t, as well as the modifications inpan> the inpan>teraction network among cultivable isolates. The selective medium pan> class="Chemical">PIA was found to be highly selective to gamma-proteobacteria, a class previously reported as abundant at Churince as well as other sites at CCB (Souza et al., 2006; Escalante et al., 2009; Bonilla-Rosso et al., 2012) and it has not been analyzed in detail. Unfortunately, a great amount of the isolates could not be identified, as it seems that Cuatro Ciénegas bacteria produce compounds that inhibit the PCR reaction, compounds that remain despite the use of several DNA extraction methods. To analyze the community response to the nutrient enrichment without the cultivation bias, 16S libraries from water and sediment samples, from May to June 2011, were also sequenced. The analysis of the community showed, in agreement with this study, a shift in the community composition with the disappearance of several bacterial genera after the increased nutrient availability (Elser et al., 2014, in preparation).

Our hypothesis that Lagunita is P-limited was supported, as it was found that all added P that remained in the n class="Chemical">water column was immobilized inpan>to seston (Table 2). This is reflected by the fact that TDP anpan>d pan> class="Chemical">SRP were not significantly different respect to the control, but TP and phosphorus in seston were. This difference was also observed in the N:P ratio. With respect to total dissolved nitrogen, it was found that both nitrogen-amended treatments were significantly different but the phosphorus-amended treatment was not. This supports that our observations were not due to an isolation bias.

Based on the 16S rRn class="Chemical">NA sequenpan>ces from the cultivated strainpan>s, we found that two differenpan>t groups of gamma-proteobacteria responded to inpan>creases of nutrienpan>t availability inpan> pan> class="Chemical">water and sediment environments, Pseudomonas and Halomonas, respectively. The strains characterized differed among the different treatments in the physiological characteristics analyzed, biofilm formation and antibiotic resistance. In contrast, genera such as Shewanella, Citrobacter, and Rheinheimera in water, as well as Stenotrophomonas and Brachybacterium in sediment were not isolated from the fertilized mesocosms, although they were present at time zero. Moreover, some rare genera, such as Kocuria, which was not documented at time zero, was found after the enrichment. However, we note that this rare genus of Actinobacteria has been obtained in past work at CCB (Cerritos et al., 2011), so it is not foreign to this environment. Several genera isolated in this study, had not been previously reported to grow in PIA medium (McCaig et al., 2001; Rajkowski and Rice, 2001; Falcone-Dias et al., 2012; Weiser et al., 2014).

All these changes in bacterial groups, which were isolated during different times in the experiment, suggest that an important community change at the bacterial level took place after the enrichment, however it should be taken carefully given the sampling method. It can be speculated that the original community in Lagunita was dominated by K-strategists, which rely on long-term survival on limited resources (n class="Chemical">Pianka, 1970; Fuchs et al., 2000; Sinpan>ger et al., 2011). After the enpan>richmenpan>t, the microbial community was dominpan>ated by faster-growinpan>g gamma-proteobacteria, which canpan> be considered r-strategists, rapidly exploipan> class="Chemical">ting nutrient patches and then dying or becoming dormant after substrate exhaustion. Indeed, Pseudomonas has been previously characterized in general as a r-strategist, as pseudomonads rapidly colonize and grow on nutrient-rich environments (Juteau et al., 1999; Margesin et al., 2003), due to its metabolic versatility (Clarke, 1982; Hallsworth et al., 2003; Domínguez-Cuevas et al., 2006). This shift is important as it has been reported that K-strategists are expected to allocate more energy and interact in a broader way with their environment, for example developing strategies to cope with their environment, than to grow (Fontaine et al., 2003).

Although the experiment showed a reduction in diversity of the isolated strains and interactions as a whole, details of these responses of strains to the individual treatments were largely idiosyncratic and seemed independent of their phylum. For example, the group that includes strain AP29, affiliated with n class="Species">Pseudomonas, responded to the P treatmenpan>t, while anpan>other group of pan> class="Species">Pseudomonas, which includes strain ENNP10, responded to the NNP treatment. However, another Pseudomonas group was abundant at the beginning of the experiment and remained abundant in the control, as well as in the P treatment (group including strains AP9, AC10, and 1C11) (Figure 1). The sudden availability of P in P-deficient environment could favor the dominance of the Pseudomonas genus, which has been reported to solubilize phosphate (Park et al., 2009; Parani and Saha, 2012). In sediment samples it was observed in both N+P treatments that Halomonas exhibited the greatest response. This observation is consistent with the fact that the Halomonas genus has been identified as capable of denitrification and may have taken advantage of the added NO3 in the NP and NNP treatments (Mormille et al., 1999; Guo et al., 2013).

The production of chemical compounds as bacteriocins and/or antibiotics is a common mechanism of antagonism among microorganisms (Riley and Gordon, 1999; Lenski and Riley, 2002; Riley and Wertz, 2002; Kirkup and Riley, 2004; Hibbing et al., 2010; Kohanski et al., 2010; Majeed et al., 2011, 2013; Pérez-Gutiérrez et al., 2013; Aguirre-von-Wobeser et al., 2014). In this study, we observed a general decrease in the antibiotic resistance in both the n class="Chemical">water anpan>d sedimenpan>t enpan>vironmenpan>t, for all anpan>tibiotics except pan> class="Chemical">Carbenicillin, after the experiment (Table 3). This decrease likely reflects modifications in microbial survival strategies under different conditions, including the control were wind movement of both water and sediment, was restricted by the mesocosm tubes. Given the antagonistic network previously documented among Pseudomonas bacterial isolates from CCB (Aguirre-von-Wobeser et al., 2014), this antagonism could be due to the competition for resources. Following “microbial market logic” (Werner et al., 2014), without the acute nutrient limitation, the cost of producing antibiotics to repel competitors for a scarce resource is no longer beneficial in the case of increased P and the ideal N:P ratio, while in the NNP treatment, the community is so perturbed by the further limitation of P in relation to N that antagonism or cooperation through biofilm formation is no longer an economic option. On the other hand, in a rock—paper—scissors (RPS) model behavior scenario, that can be applied only for structured environment (Kirkup and Riley, 2004; Nahum et al., 2011) such as sediment or biofilm, the strains that produce toxins (C) kill sensitive strains (S), which outcompete resistant strains (R), which in turn outcompete C (Czarán et al., 2002; Kirkup and Riley, 2004). In this RPS game, the resistant and producer strains spend resources to keep the resistance, which in an enriched environment may be no longer needed, thus those strains are outcompeted by the sensitive strains (Kerr et al., 2002). Interestingly, this shift from a collection of isolates with a high prevalence of resistance to several antibiotic tested, to a collection dominated by sensitive strains was observed in both habitats: water and sediment (Figure 2), suggesting that the fact of predicting the neighborhood, as required for the rock-paper-scissor model, is not a requisite for antagonism, at least in the analyzed system.

Market logic suggests that local environments determine trade connections (Werner et al., 2014), and biofilm formation is a way to ensure a local environment both in n class="Chemical">water anpan>d sedimenpan>t. The mechanpan>isms of bacterial biofilm formation are processes by which sinpan>gle cells coordinpan>ate anpan>d implemenpan>t the formation of complex surface-attached communities (Davey anpan>d O'Toole, 2000). Bacteria that reside withinpan> the biofilm are to some extenpan>t isolated from enpan>vironmenpan>tal stresses, such as desiccation or nutrienpan>t limitation (Danpan>hornpan> et al., 2004). Complex inpan>teractions, inpan> particular mutualistic behaviors, are expected inpan> a biofilm, sinpan>ce the secretion of the matrix that forms the biofilm is a form of public good that will inpan>crease the survival of the coexispan> class="Chemical">ting partners and will avoid the presence of cheaters (Werner et al., 2014).

Biofilm-forming strains were found in our experiment in both habitats at time zero and in most of the enrichment treatments, suggesn class="Chemical">ting that this cooperation strategy is more inpan>grainpan>ed inpan> the whole pan> class="Chemical">CCB microbial community than antagonistic interactions promoted by antibiotics, where rare species cannot afford to pay its costs. Biofilm formation was not present only in the sediments of the NNP treatment where P was further limited in relation to N. This treatment was dominated by strain of Halomonas. The NP treatment was also dominated by closely related strains of Halomonas that were biofilm-forming (Supplementary Figure 2). This differential response suggests that a wide range of strategies to cope with environmental limitations is present in CCB microbes, even within a single genus.

As the Black Queen Hypothesis (BQH) suggests, certain biological functions are not only expensive, but are also broadly distributed in the community, since they are public goods. Hence, the majority of the community can afford to lose those functions if they are at least a proportion of helpers that produce such public good (Morris et al., 2012). As a consequence, for a microbial market, we also need “pn class="Species">rice” differenpan>ces anpan>d supply, as well as “demanpan>d” variation. Inpan> this study, we observed a shift inpan> the composition of the bacterial community, from a diverse community prior the enpan>richmenpan>t to a community dominpan>ated by few genpan>era such as pan> class="Species">Pseudomonas in water or Halomonas in sediment after the addition of N+P. As previously stated, the change was not only related to the community composition, but also to its physiological characteristics as antibiotic resistances and the ability of biofilm formation. These changes could be explained with this microbial market theory as the supplies changed with the experiment the balance between cost and benefit.

According to the market theory, the benefit of trade depends not only on the interacn class="Chemical">ting partnpan>ers but also on the available supply of commodities from other sources anpan>d it is expected that biotic or abiotic conditions inpan>fluenpan>ce the demanpan>d for a particular service (Wernpan>er et al., 2014). Based on anpan> anpan>alysis of Illuminpan>a tags of 16S rRpan> class="Chemical">NA (Elser et al., 2014, in preparation), our study site exhibits a large bacterial diversity dominated by alpha-proteobacteria and bacteroidetes. In the original community, we expected to find a strong competition among the members of this community. Under the original condition of extreme low P and N availabilities, it is predicted that specialization will be favored. All this considering, it is not surprising that when this particularly diverse and fragile network of interactions was perturbed by the mesocosm conditions and the nutrient input, the community structure and its biological market equilibrium changed, reducing its overall diversity, not only in the few cultured genera that we could follow, but in the overall community (Elser et al., 2014), suggesting that the resilience of this extremely oligotrophic oasis depends precisely on the permanence of such unbalanced stoichiometry.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.