Hydrol. Earth Syst. Sci., 23, 139–154, 2019 https://doi.org/10.5194/hess-23-139-2019 © Author(s) 2019. This work is distributed under the Creative Commons Attribution 4.0 License. Microbial community changes induced by Managed Aquifer Recharge activities: linking hydrogeological and biological processes Carme Barba1,2 , Albert Folch1,2 , Núria Gaju3 , Xavier Sanchez-Vila1,2 , Marc Carrasquilla3 , Alba Grau-Martínez4 , and Maira Martínez-Alonso3 1 Department of Civil and Environmental Engineering, Universitat Politècnica de Catalunya (UPC), C/Jordi Girona 1–3, 08034 Barcelona, Spain 2 Associated unit: Hydrogeology Group (UPC-CSIC), Barcelona, Spain 3 Department of Genetics and Microbiology, Universitat Autònoma de Barcelona (UAB), 08193 Bellaterra, Spain 4 Grup de Mineralogia Aplicada i Geoquímica de Fluids, Departament de Mineralogia, Petrologia i Geologia Aplicada, Facultat de Ciències de la Terra, Universitat de Barcelona (UB), C/Martí i Franquès s/n, 08028 Barcelona, Spain Correspondence: Carme Barba (carme.barba@upc.edu) Received: 3 April 2018 – Discussion started: 17 April 2018 Revised: 13 December 2018 – Accepted: 14 December 2018 – Published: 11 January 2019 Abstract. Managed Aquifer Recharge (MAR) is a tech- a naturally based, passive and efficient technique with broad nique used worldwide to increase the availability of water implications for the biodegradation of pollutants dissolved in resources. We study how MAR modifies microbial ecosys- water. tems and its implications for enhancing biodegradation pro- cesses to eventually improve groundwater quality. We com- pare soil and groundwater samples taken from a MAR facil- 1 Introduction ity located in NE Spain during recharge (with the facility op- erating continuously for several months) and after 4 months As the Intergovernmental Panel on Climate Change has of no recharge. The study demonstrates a strong correlation stated for years, climate change is affecting and will con- between soil and water microbial prints with respect to sam- tinue to affect the availability and quality of freshwater re- pling location along the mapped infiltration path. In particu- sources, with severe consequences to humans and ecosys- lar, managed recharge practices disrupt groundwater ecosys- tems. In particular, the Mediterranean Basin is expected to tems by modifying diversity indices and the composition of become warmer and drier (Bates et al., 2008). Therefore, microbial communities, indicating that infiltration favors the among other actions, claiming a secure water supply should growth of certain populations. Analysis of the genetic pro- increase groundwater storage of quality water as a strategic files showed the presence of nine different bacterial phyla in management tool in times of scarcity. the facility, revealing high biological diversity at the high- Managed Aquifer Recharge (MAR) is a globally used, est taxonomic range. In fact, the microbial population pat- worldwide extended technology based on refilling aquifers terns under recharge conditions agree with the intermedi- with water from different sources (e.g., river, reclaimed or ate disturbance hypothesis (IDH). Moreover, DNA sequence opportunity water). MAR facilities are usually intended to re- analysis of excised denaturing gradient gel electrophoresis cover groundwater levels or to become water reservoirs, but (DGGE) band patterns revealed the existence of indicator other objectives can be targeted. It is quite common to take species linked to MAR, most notably Dehalogenimonas sp., advantage of the potential of soil as a biogeochemical reactor Nitrospira sp. and Vogesella sp.. Our real facility multidisci- to enhance the quality of water infiltrating the vadose zone, plinary study (hydrological, geochemical and microbial), in- especially in surface replenishment systems (Drewes et al., volving soil and groundwater samples, indicates that MAR is 2003; Nadav et al., 2012). Published by Copernicus Publications on behalf of the European Geosciences Union. 140 C. Barba et al.: Microbial communities in MAR The Llobregat River (Catalonia, NE Spain) is fed by about tion of laboratory experiments and a full-scale MAR facility. a hundred waste water treatment plants. While nitrogen, However, to our knowledge, there are no microbial finger- phosphorous and organic matter (chemical oxygen demand, printing studies of MAR surface infiltration basins that inte- COD) are eliminated below the legal limits before treated grate results from surface water, groundwater and soil sam- wastewater is discharged to the river, emerging organic con- ples and compare them in two different operational periods. taminants (EOCs) are not fully removed (Loos et al., 2013). The main goal of this study is to determine how MAR Consequently, significant concentrations of many EOCs have activities induce changes in the microbial communities in a been detected in the Llobregat River (López-Serna et al., real facility composed of a settling and infiltration pond ad- 2012) and its associated groundwater bodies (Jurado et al., jacent to the Llobregat River. We evaluate changes on diver- 2012). sity indices and we incorporate results of the DNA sequence Biodegradation of EOCs strongly depends on redox con- analysis of excised denaturing gradient gel electrophoresis ditions (Barbieri et al., 2011; Maeng et al., 2010). In this (DGGE) band patterns for samples taken from different en- regard, it has been shown that MAR is a feasible tech- vironments and locations within the site and under conditions nique capable of partially degrading some of these contami- of recharge and non-recharge. Additionally, we link our re- nants (Hellauer et al., 2017; Massmann et al., 2008), particu- sults with ecological principles and potential biogeochemi- larly when bioprocesses are enhanced (Grau-Martínez et al., cal processes (i.e., pollutants degradation) occurring due to 2018; Schaffer et al., 2015). Infiltration through the soil in- MAR activities. trinsically leads to two main consequences in groundwater recharge: 2 Material and methods 1. Development of different vertical and temporal redox zonations responding to organic matter availability as 2.1 The Llobregat MAR site electron acceptors are consumed (Greskowiak et al., 2006). The Llobregat MAR system is located 15 km inland from the Mediterranean Sea, close to Barcelona city (Fig. 1). The 2. Development of microbial communities according to aquifer thickness in the vicinity is 10–15 m, with alternating the flow paths. Fingering below the surface of the sands and gravels. Non-continuous fine-grained sediments recharge systems and preferential flow paths in the are widely present (Pedretti et al., 2012). The distance from saturated zone can create anaerobic microsites (e.g., the bottom of the pond to the water table oscillated from 9 m Bridgham et al., 2013) in which oxygen is consumed (July 2014) to 7 m (March 2015) in the period under study. faster than it can be diffused from oxic zones. Water entering is diverted from upstream of the river to a pre-sedimentation basin. After 2–4 days of residence time, Indeed, MAR implies groundwater quality modifications it is diverted to an infiltration basin of 6500 m2 . The infil- when compared to natural flow conditions. This includes sev- tration capacity has been estimated at 1 m3 m−2 d−1 on aver- eral parameters such as organic matter, dissolved oxygen age, and the local transmissivity of the aquifer is estimated content, temperature, pH, electrical conductivity and nutri- as 14 000 m2 d−1 (unpublished). ents (Rivett et al., 2008; Zhang et al., 2016). Such distur- In 2011, a reactive barrier was installed at the bottom of bances have ecological implications, as all these parameters the infiltration basin to increase the organic load of the in- affect the growth and activity of microorganisms and the cor- filtration water, thus promoting biological processes through responding degradation of emerging contaminants (Barbieri the soil and the vadose zone. The barrier was composed of or- et al., 2012; Regnery et al., 2017; Valhondo et al., 2018). ganic compost (50 % in volume) mixed with sand and gravel. Microbial studies linked to MAR practices involve mostly Small amounts of clay and iron oxides were added to fos- laboratory experiments (Alidina et al., 2014b; Freixa et al., ter adsorption and ion exchange. Previous studies demon- 2015; Li et al., 2013; Rubol et al., 2014). As for micro- strated that the reactive barrier enhanced the removal of some bial MAR field studies, most relevant research is limited to emergent contaminants, such as sulfamethoxazole or caffeine well-injection systems (Ginige et al., 2013; Reed et al., 2008; (Valhondo et al., 2014). More information about the site and Zhang et al., 2016) or riverbank filtration conditions (Huang the performance of the reactive barrier can be found in Val- et al., 2015). Onesios-Barry et al. (2014) compared results hondo et al. (2014). from a column experiment and soil samples in a MAR site There are six piezometers distributed in a 500 m transect in the US, focusing on the microbial populations linked to across the study area (Fig. 1). P1, P3, P2, P5 and P10 are fully pharmaceutical and personal care products removal, and con- screened. P8 is a multilevel piezometer drilled at three differ- cluded that microbial composition and structure of both sys- ent depths. Water from piezometer P1 represents background tems were comparable. Regnery et al. (2016) went one step conditions (not affected by recharge). further by relating the relative abundance of functional genes involved in xenobiotic pathways with attenuation of some trace organic chemicals and their byproducts in a combina- Hydrol. Earth Syst. Sci., 23, 139–154, 2019 www.hydrol-earth-syst-sci.net/23/139/2019/ C. Barba et al.: Microbial communities in MAR 141 Figure 1. Geographical location of the Llobregat MAR system and location of the established transect. (a) Transect section with piezometers (P1 and P2 are projected) and displaying sampling depths (red diamonds). (b) Blue line shows groundwater level in July 2014 (wet scenario – recharge) and March 2015 (dry scenario – non-recharge). www.hydrol-earth-syst-sci.net/23/139/2019/ Hydrol. Earth Syst. Sci., 23, 139–154, 2019 142 C. Barba et al.: Microbial communities in MAR 2.2 Hydrochemistry sampling surveys age of the two replicas. The evenness index is proportional to the equitable distribution of bands in the gel. Shannon is Two recharge situations were compared to evaluate the ef- an entropy index, reaching maximum (Hmax ) for the most fect of MAR on groundwater chemical signature. After equally distributed band patterns and with the higher value 6 months of continuous recharge operation, a sampling cam- of richness. paign took place in July 2014 (wet campaign). Samples were collected from surface water in both basins and in the exist- 2.4 Soil characterization ing piezometers at different depths (from −5 to 3 m a.s.l.; see diamonds in Fig. 1). The second sampling campaign was per- To complement the soil microbial community’s characteriza- formed in March 2015 after recharge had been discontinued tion, particle size measurements of soil samples were taken for 4 months. In this case, groundwater was also sampled. according to the ASTM guidelines. The soil was sampled in Water was analyzed for cations, anions (Cl− , NO− −2 the pre-sedimentation basin and at the entrance, middle and 3 , SO4 , − HCO3 ), dissolved organic carbon (DOC) and total organic end of the infiltration basin. Soil sampling was performed carbon (TOC). Analytical procedures are widely described close to the location where samples were taken for microbial in the Supplement. analyses. In both campaigns, temperature and electrical- conductivity vertical profiles were mapped along the 3 Results transect from data obtained at 50 cm intervals (MPS-D8, SEBA Hydrometrie). 3.1 Microbial differences in groundwater linked to recharge conditions 2.3 Microbial community characterization 3.1.1 Closing the conceptual flow model Water samples were extracted from the pre-sedimentation and infiltration basins at three locations (entrance, middle Understanding the flow pattern in MAR basins is essential and end) during recharge conditions (from now on, wet sce- to explain microbial community dynamics. In this regard, 2- nario). On the contrary, three soil samples were extracted D transects of temperature and conductivity alterations ob- at the same locations in the infiltration basin under non- tained at the time of the sampling campaigns (Figs. 2a and recharge conditions (termed dry scenario). Soil samples were 3a) based on vertical profiles indicate that (1) the vertical obtained from around 10 to 50 cm in depth. The sampling flow gradient pushes the existing groundwater downwards procedure for soil was done taking into account Lombard and forms a shallow front that travels approximately 120 m et al. (2011) recommendations, especially regarding the vari- downstream, eventually mixing with the background water; ability of microbial communities along a field transect. Soil (2) the background water is mostly found near the recharge samples were taken by means of cores, individually disas- pond and at the deepest sampling points below the pond. sembled and kept in a sterile bag. Groundwater samples were From this conceptual model, four main groups of ground- taken from −5 to 3 m a.s.l. depending on the piezometers (10 water can be defined under recharge conditions: samples for wet scenario and 7 for dry), at the same loca- tion as samples for the hydrochemical characterization. All – Type I water represents the background environment of soil and groundwater samples were taken in duplicate, kept the aquifer, unaffected by MAR activities. Water sam- in sterile conditions and preserved in the dark at −4 ◦ C until pled in P1 is an example of this type. being taken to the laboratory for molecular analyses. – Type II water is the infiltrating one (best observed in Protocols for molecular analyses of liquid and soil samples P8 at both sampling depths). It flows vertically through are thoroughly described in the Supplement. the vadose zone to the aquifer, creating a small water Once the main microbial communities were characterized, mound that pushes down the Type I water. three diversity indices were calculated. The first one is rich- ness (S), defined as the proportional number of microbial – Type III water, characteristic of points P2(3), P5(2.3) species present in a sample, i.e., equal to the total number of and P5(−2.2), is a mixture between types I and II wa- bands; the other two, Shannon (H ) and evenness (E), were ters, with a high proportion of the latter. calculated for each sample as follows: – Type IV water is again a mixture, but with a lower pro- S X portion of Type II water. It is present in piezometers H= pi ln(pi ), P3(0.8), P3(−4.2), P2(−5) and P10(−1). i=1 Apart from temperature and conductivity, major ion com- H position does not show any significant trend related with E= , (1) Hmax groundwater zonation below the pond (see Table S1 in the where Hmax equals lnS and pi is the relative intensity of each Supplement). The role of nitrate and DOC in microbial com- band of the sample. Values reported correspond to the aver- munity patterns is discussed further below. Hydrol. Earth Syst. Sci., 23, 139–154, 2019 www.hydrol-earth-syst-sci.net/23/139/2019/ C. Barba et al.: Microbial communities in MAR 143 Figure 2. Temperature distribution at the local scale in (a) July 2014 (wet) and (b) March 2015 (dry). Red diamonds indicate sampling points for microbial and water analysis in each campaign. www.hydrol-earth-syst-sci.net/23/139/2019/ Hydrol. Earth Syst. Sci., 23, 139–154, 2019 144 C. Barba et al.: Microbial communities in MAR Figure 3. Electrical-conductivity distribution at the local scale in (a) July 2014 (wet) and (b) March 2015 (dry). Hydrol. Earth Syst. Sci., 23, 139–154, 2019 www.hydrol-earth-syst-sci.net/23/139/2019/ C. Barba et al.: Microbial communities in MAR 145 Table 1. Summary of values of the Shannon, richness and evenness indices at the Llobregat MAR site in different scenarios. Sampling location Shannnon (SD)∗ Richness (SD)∗ E (SD)∗ Environment (depth, m a.s.l.) Wet Dry Wet Dry Wet Dry T.I P1(−0.6) 2.57 (0.04) 2.73 22 (4.1) 19 0.61 (0.01) 0.64 P8(1) 1.43 (0.05) 1.73 10 (2.0) 11 0.34 (0.01) 0.41 Groundwater T.II P8(−3) 1.69 (0.20) 2.03 (0.14) 11 10 (2.0) 0.40 (0.05) 0.48 (0.03) P2(3) 2.29 (0.13) 18.5 (7.2) 0.54 (0.03) P2(−2) 2.67 (0.13) 18 (2.0) 0.63 (0.03) T.III P5(2.3) 1.91 (0.01) 2.41 14.5 (1.0) 14 0.45 (1 × 10−3 ) 0.56 P5(−2.2) 2.22 (0.01) 12.5 (1.0) 0.52 (2 × 10−3 ) P3(0.8) 2.65 (0.09) 2.01 (0.28) 21 10.5 (3.07) 0.63 (0.02) 0.48 (0.07) P3(−4.2) 2.65 (0.13) 20 0.63 (0.03) T.IV P2(−5) 2.80 (0.12) 23 (4.1) 0.66 (0.12) P10(−1) 2.51 (0.20) 2.49 (0.09) 17 16 (4.1) 0.59 (0.01) 0.59 (0.02) Pre-sedimentation 2.93 (0.28) 29 (14.3) 0.69 (0.07) Infiltration entrance 2.78 (0.53) 28.5 (17.4) 0.66 (0.12) Water of basins Infiltration midfield 2.66 (0.14) 25.5 (3.1) 0.63 (0.03) Infiltration end 2.58 (0.08) 24 (2.0) 0.61 (0.02) Pre-sedimentation 2.89 (0.16) 25.5 (7.2) 0.68 (0.04) Infiltration entrance 3.22 (0.09) 2.93 (0.21) 35.5 (1.0) 25 (7.6) 0.76 (0.02) 0.69 (0.08) Soil of basins Infiltration midfield 3.36 (0.14) 2.90 (0.05) 30.5 (3.1) 21 0.79 (0.03) 0.68 (0.01) Infiltration end 3.29 (0.05) 2.40 (0.07) 34 (2.0) 16 0.78 (0.01) 0.57 (0.02) ∗ Numbers in parenthesis after each index value indicate standard deviation, not reported whenever one replica was damaged. 3.1.2 Clustering groundwater microbial communities Figure 5 shows DGGE profiles and UPGMA clustering according to presence and abundance analysis of groundwater samples. The genetic fingerprints revealed high dissimilarities in the bacterial assemblage of about 70 % and 80 % during the active recharge period and To characterize differences in microbial communities due to the dry campaign, respectively (Fig. 5). Moreover, most recharge, groundwater samples were subjected to molecu- replicas grouped together, indicating sampling quality. Under lar analysis. Post-processing of DGGE gels allowed for non- active recharge (wet) conditions (Fig. 5a), the dendrogram metric multidimensional scaling (NMDS), showing similar- reproduces the water types postulated by the conceptual flow ities among band patterns (Fig. 4) and strong clustering of model quite well: in the first group, we can include four out microbial communities. Samples from both scenarios were of the five samples that were strongly influenced by recharge completely separated; blue squares (dry) and triangles (wet) (P5(−2.2), P5(2.3), P8(1) and P8(−3)), while in the second represent groundwater samples, and are clearly clustered in group, P2(3), P3(−4.2) and P3(0.8) clustered together with top and bottom halves of the plot, respectively. Moreover, P1 (not affected by recharge). In the dry campaign (Fig. 5b), samples from the wet scenario grouped according to water although no infiltration occurred, P8 appears separated from types. Types I and II are displayed on opposite sides; types the other piezometers, indicating the still marked influence III and IV (mixed) are displayed in between. The two green of the water infiltrated during the wet period, which occurred triangles in the center of the plot correspond to groundwater over 4 months earlier. samples from P10, which are samples that are slightly af- fected by recharge. These Type IV samples remain among 3.1.3 Variations in microbial diversity indices in different sets of groundwater samples from the wet and dry groundwater scenario. It seems that the theory of P10 as a location with a high proportion of mixing is also valid for the microbial The structure and processes of ecosystems change when community composition behavior. a disturbance occurs (Grimm, 1994). Such changes in mi- Discrete bands are also portrayed (circles), allowing link- crobial communities have been quantified and described by age of the bands’ contribution to sample assemblages. Filled means of diversity indices (Table 1). Such indices, grouped circles report the class and genus of the sequenced bands, according to water types during wet conditions, were ordered whereas empty circles symbolize non-sequenced bands. along an imaginary line from low to highly perturbed as a www.hydrol-earth-syst-sci.net/23/139/2019/ Hydrol. Earth Syst. Sci., 23, 139–154, 2019 146 C. Barba et al.: Microbial communities in MAR Figure 4. Non-metric multidimensional scaling clustering for all groundwater samples. Blue squares and triangles represent samples in dry and wet scenarios, respectively. Colors in triangles represent water types. Black circles correspond to band migration numbers in DGGE gels that were sequenced (phylogenetic affiliation corresponding to each black circle). Non-sequenced bands are also portrayed (empty circles). Hydrol. Earth Syst. Sci., 23, 139–154, 2019 www.hydrol-earth-syst-sci.net/23/139/2019/ C. Barba et al.: Microbial communities in MAR 147 Figure 5. DGGE band patterns of bacterial 16S rRNA gene fragments and UPGMA cluster analysis for fingerprints obtained from wet (July 2014) (a) and dry (March 2015) (b) periods. The bar indicates 9 % divergence. Each sample is defined by a code indicating piezometer number and sampling depth (see Table 1). Black triangles indicate the position of bands recovered and sequenced, and the numbers correspond to their phylogenetic affiliation (see Table S2). consequence of water infiltration (Fig. 6). The lowest diver- Overall, sequences fell into nine different bacterial phyla sity indices were obtained for the recharging water (Type II), and eleven classes: Proteobacteria (Alphaproteobacteria, Be- indicating low species richness and a highly dissimilar pro- taproteobacteria and Gammaproteobacteria), Cyanobacteria, portion. In contrast, Type IV water, only slightly affected by Chloroflexi (Dehalococcoidia), Chlorobi (Chlorobia), Ni- water infiltration, displayed higher Shannon and evenness in- trospirae (Nitrospira), Acidobacteria, Actinobacteria, Firmi- dices, similar to Type I (unaffected by recharge). cutes (Bacilli) and Bacteroidetes (Cytophagia) (Fig. 7). The group designated as “others” includes unclassified and non- 3.1.4 Role of MAR activities for the microbial sequenced fine bands. community structure The two main classes displaying the largest differences be- tween the two scenarios are Betaproteobacteria and Dehalo- Prominent bands were recovered from the DGGE gels coccoidia, which were favored under recharge conditions. (Fig. 5) and sequenced. Table S2 shows the sequenced In particular, Dehalococcoidia is present in moderately and bands, their similarity values compared to the closest re- weakly influenced waters, and it is absent from the highly lated GenBank sequences and their phylogenetic affiliations. www.hydrol-earth-syst-sci.net/23/139/2019/ Hydrol. Earth Syst. Sci., 23, 139–154, 2019 148 C. Barba et al.: Microbial communities in MAR 3.2 Microbial community indicators of MAR in soil and surface water To study the impact of MAR on microbial community struc- ture, recharge water from pre-sedimentation and infiltration basins, as well as soils, were analyzed. Figures 8 and 9 show the relative abundance of bacterial phylotypes at the taxo- nomical level of classes for surface water and soil samples. The results are displayed according to the distance to the recharge basin inlet. Microbial richness in soil samples was controlled by wa- ter content. Non-recharge conditions had a primarily negative effect on the populations of Dehalococcoidia, Acidobacteria and Chlorobia, but favored the presence of Nitrospira, Cy- tophagia and Actinobacteria (Fig. 8). Shannon and evenness indices demonstrated that soils were more diverse under wet conditions than under dry ones (Table 1). For surface water samples (Fig. 9), there was a decreas- ing gradient in community complexity along the ponds. Aci- dobacteria, Betaproteobacteria and Cyanobacteria were the Figure 6. Average of Shannon indices in piezometer samples under main phylotypes present. wet conditions. Standard deviation is shown in error bars. Horizon- tal axis reflects the degree of perturbation of original groundwater due to recharge. 4 Discussion We contend that interdisciplinary analysis of geochemical characterization, recharge evaluation and microbial finger- recharge-influenced groundwater (P8(1) and P8(−3)). This printing, can provide relevant information about the fate of phylotype was identified at the genus level as Dehalogeni- microbial ecosystems in soil and groundwater. monas sp. (Table S2). Similar behavior was found in the Ni- trospira class, appearing in weakly influenced groundwater 4.1 Matching groundwater model, ecological in the wet scenario. disturbance principle and microbial communities Patterns in the structure of microbial populations corre- lated with water types. For Type I, differences in the bacte- Groundwater is a quite stable aquatic environment (Griebler rial assemblage between both campaigns were attributed to and Lueders, 2009) as it is shown by the low variability in seasonal changes (Table S1). Dehalococcoidia and Chloro- the hydrochemical data (Table S1). One could expect that bia were only detected in the wet scenario, while Cytophagia microbial communities in groundwater should also display and Nitrospira could only be detected under dry conditions. low variability that could be reflected in the diversity indices. During the active recharge period and for Type IV water, In this way, piezometer P1, which is unaffected by recharge, Dehalococcoidia was found in three out of four sampling showed stable diversity indices regardless of the sampling points and was the most abundant phylotype. For Type III campaign. However, disturbances produced by recharge, ev- water, significant differences were observed among in the idenced by temperature and conductivity gradients below the samples analyzed. Populations with the highest relative abun- pond (Figs. 2 and 3), favor the highest Shannon values for dance in P5(2.3) were Betaproteobacteria and Bacilli. The the moderately disturbed samples. As a result, the average former was also prominent in P5(−2.2), together with Cy- of diversity indices remains constant between both scenarios tophagia, while Dehalococcoidia were dominant in P2(3). but with a higher standard deviation during the wet scenario. Finally, in the case of groundwater Type II (recharge wa- These two facts combined suggest that perturbations caused ter), the bacterial assemblage was dominated by members by recharge influence much more the composition of micro- of the Betaproteobacteria class. During the dry period, no bial communities in groundwater than the natural variability clear distribution patterns in the bacterial relative abundances of the background aquifer water between scenarios. at the phylum and class level were observed, in part due to MAR is a passive treatment technique that can provide the DGGE profiles, mainly composed by fine bands (Fig. 5); simultaneously oxic and anoxic conditions (Maeng et al., these were difficult to recover and purify and thus could 2011). This has wide implications for the potential biolog- not be characterized. However, it should be mentioned that ical removal of selected emerging contaminants, as each mi- Betaproteobacteria were dominant in both P8 samples, con- cropollutant is most efficiently removed under specific re- tributing more than 50 % to the relative abundance. dox conditions (Schmidt et al., 2007). Some can even be de- Hydrol. Earth Syst. Sci., 23, 139–154, 2019 www.hydrol-earth-syst-sci.net/23/139/2019/ C. Barba et al.: Microbial communities in MAR 149 Figure 7. Bacterial community structure of groundwater samples. Class relative abundances calculated for wet (a) and dry (b) scenarios. Figure 8. Bacterial community structure of soil samples from pre-sedimentation and infiltration basins. Class relative abundances calculated for wet (a) and dry (b) scenarios. www.hydrol-earth-syst-sci.net/23/139/2019/ Hydrol. Earth Syst. Sci., 23, 139–154, 2019 150 C. Barba et al.: Microbial communities in MAR (Type II). In ecological terms, Type III and Type IV waters represent different proportions of perturbation. In the most altered groundwater zone (represented by P8 samples), Betaproteobacteria grew above 50 % of the rela- tive abundance (Fig. 7). The main phylogenetic affiliation of this phylotype at the genus level is Vogesella. Strains of this genus are able to catabolize monosaccharides under aer- obic conditions, but not under low-oxygen conditions. Fur- thermore, all Vogesella strains are denitrifiers (Grimes et al., 1997). Indeed, P8, located below the pond, receives oxygen- rich water during the recharge process, driven by fingering in the vadose zone. Although dissolved oxygen was not mea- sured in the present study, data from other campaigns con- firm this behavior for oxygen in P8 samples (data not shown). Moreover, nitrate concentration in the surface water was low (Table S1), and thus most denitrification is expected to occur under the pond. Grau-Martínez et al. (2018) recently con- firmed that nitrate was consumed via the denitrification path- Figure 9. Bacterial community structure from water samples of pre- way under the infiltration pond in the Llobregat MAR sys- sedimentation and infiltration basins. Class relative abundance cal- tem, supporting the idea that Vogesella sp. could be one of the culated for wet scenario. genera involved in nitrate consumption. Likely, depending on the oxygen content, Vogesella sp. will adapt its metabolic function in favor of aerobic oxidation of organic matter or by means of denitrification, thus becoming a good indicator of graded by co-metabolism, involving different redox states in highly disturbed MAR environments. the process (Rodriguez-Escales and Sanchez-Vila, 2016). In For Type III and Type IV waters, Dehalogenimonas this sense, MAR is an efficient remediation system. In ad- sp., within the Dehalococcoidia class, is characteristic of dition, many sequenced phylotypes, such as Nitrospira sp., medium-disturbance groundwater (Figs. 4 and 7). Dehalo- Stenotrophomonas sp. and Methylobacterium sp., have been genimonas sp. has been studied in recent years because some associated with degradation capabilities (Cycoń et al., 2017; strains are associated with dechlorination in contaminated Daims et al., 2001; Wang et al., 2015). In short, the MAR mi- sites. This genus is strictly anaerobic and mesophilic, and crobial ecosystem studied in this work presents many more some species can reductively dehalogenate polychlorinated phylotypes than previous studies reported in groundwater aliphatic alkanes (Martín-González et al., 2015; Moe et al., systems (Logue et al., 2015), and thus MAR can be consid- 2009). As a result, recharge creates reducing conditions, ered an efficient remediation system. likely indicating the existence of microzones or microsites We further tested the intermediate disturbance hypothesis (Bridgham et al., 2013; Hamersley and Howes, 2002), de- (IDH) for microbial communities in groundwater (Fig. 6) re- fined as local anoxic areas that coexist with fast-traveling lated to MAR activities. IDH was originally proposed for oxygen-rich paths. Thus, microbial analysis can be used tropical rain forests and coral reefs (Connell, 1978) and to unmask the apparent mishap of water samples that are supports the idea that small perturbations create new ac- oxic and display some typical anaerobic species. Moreover, cess to resources for species which have overlapping niches, some species of Dehalogenimonas can dechlorinate some allowing their coexistence. This mechanism, known as a trichloroethane isomers (Dillehay et al., 2014), a pollutant competition–colonization trade-off, can explain IDH in local reported in the Llobregat lower valley at levels as high as communities, leading to an increase in diversity. However, 300 µg L−1 (Valhondo et al., 2014), thus opening the door when the degree of disturbance rises, only eurytolerant popu- for the development of enhanced remediation activities. lations can survive and grow. Thus, an inverse correlation be- The evenness index is an indicator of the equity of a com- tween diversity and the degree of disturbance (reflected in the munity and can be quite informative for observing pertur- temperature and conductivity profiles) was expected (see Ta- bations to microbial communities. In the wet scenario, the ble 1). Such correlations have also been reported in recharge lowest values of E were obtained for the samples most af- wells and snowmelt-influenced aquifers (Ginige et al., 2013; fected by recharge (Table 1), indicating that some species de- Zhou et al., 2012). veloped into predominant members of the microbial assem- In the Llobregat MAR system, the initial diversity in the blage. Groundwater samples displayed the highest evenness microbial community increased with perturbation caused by values in the area less affected by recharge and in the dry sce- recharge (Fig. 6), with maximum diversity associated with nario. In the latter case, values indicate the recovery of micro- Type IV water, and was lowest for the most disturbed water bial communities from the disruption caused by recharge. In Hydrol. Earth Syst. Sci., 23, 139–154, 2019 www.hydrol-earth-syst-sci.net/23/139/2019/ C. Barba et al.: Microbial communities in MAR 151 fact, P8(1) samples in the dry scenario were not fully consis- bution changes among scenarios. Whereas Dehalococcoidia tent with this conceptual model, with a low evenness index and Chlorobia classes appear in wet soils, Nitrospira, Cy- and very low nitrate concentration. Furthermore, the pres- tophagia and Actinobacteria are favored under dry condi- ence of Methylotenera mobilis (Betaproteobacteria class) in tions. both P8 sampling points was more than 40 %, on average, The role of the reactive layer at the infiltration pond could of the relative abundance. Methylotenera mobilis is a methy- be extrapolated as a system fed with a considerable organic lotroph species with denitrification abilities (Chistoserdova, carbon load. Laboratory experiments and constructed wet- 2011). These results suggest that P8 denitrification processes lands demonstrate that concentration of microbial activity occur below the basin even when it is empty, indicating that and TOC degradation is concentrated in the first centimeters 4 months is not enough time to revert back to natural con- of the filter material (Ragusa et al., 2004; Sleytr et al., 2007; ditions at this sampling point. This assumption is consistent Tietz et al., 2007) in response to oxygen concentration verti- with nitrate isotopic data presented in Grau-Martínez et al. cal distribution. Although rapid oxygen depletion and conse- (2018) and is also in agreement with the study of Rodriguez- quent denitrification conditions have been evidenced in lab- Escales et al. (2016) in which biomass decay acted as an en- scale MAR experiments (Alidina et al., 2014a; Dutta et al., dogenous carbon source for respiration once the input carbon 2015), this effect may not happen rapidly under real infiltra- was reduced, maintaining denitrification rates. tion conditions, where entrapped gas (Heilweil et al., 2004) or fingering processes (Kung, 1990) may provide higher oxy- 4.2 Microbial community structure in soils and surface gen concentrations than in lower-dimension systems (e.g., waters columns). Lab experiments are doubtlessly useful to eluci- date the behavior of microbial communities under controlled We analyzed the heterogeneity of the microbial community conditions. However, sometimes it may be difficult to trans- structure in soil and surface water in terms of the distance fer conclusions obtained from lab samples to real sites. to the infiltration basin entry point (Fig. 9 and Table 1). Pat- terns in surface water microbial composition are linked to 4.3 Transferability of results and future work sequential sedimentation processes as revealed by granulo- metric analyses of soil samples (Table S3). The result was Field studies are indeed realistic, but their transferability to that surface water became poorer in terms of the presence of other areas becomes challenging. Following an ecological microbial communities between the pre-sedimentation basin argument, this study evidences that the intermediate distur- and the end of the infiltration basin. The main reason could bance hypothesis has been accomplished in the Llobregat be the decrease in solids suspended throughout the system MAR site. Therefore, we could expect the same behavior in due to the sequential decantation of particles and their at- other impacted areas under similar recharge conditions. De- tached biomass. Furthermore, surface water displays rel- spite the novelty and transferability of this study being quite atively higher values of Cyanobacteria and Acidobacteria clear, future work is needed to keep evaluating the relation- classes compared to groundwater. Cyanobacteria constitute ship between different ecological, microbial, hydrochemical the largest, most diverse, and most widely distributed group and physical variables in different sites worldwide. An im- of photosynthetic prokaryotes, which are capable of conduct- provement of this multidisciplinary understanding of pro- ing nitrogen fixation (Stanier and Bazine, 1977). However, cesses could be achieved by combining statistical techniques members of the phylum Acidobacteria are physiologically with process-based models. This would allow the direct ex- diverse and ubiquitous in soils, degrade a wide range of car- tension of the results from one experiment to other sites. bon sources (from substances with a wide range of complex- ity), and are capable of reducing nitrates and nitrites (Kielak et al., 2016). This heterogeneous effect with distance to the 5 Conclusions entry point of the basin is also observed in the diversity in- dices, which lose diversity with distance and are inversely This study aims at integrating different fields such as hy- correlated to the proportion of fine particles. Similar behavior drogeology, ecology and microbiology applied to a real for richness correlated to soil texture was reported elsewhere MAR facility, relating flow (infiltration) conditions, physic- (Chau et al., 2011). ochemical water parameters and microbial changes induced Differences in the microbial communities in soils between by managed recharge along vertical transect. We observed the two basins were concentrated in the large organic matter that infiltration ponds modify the hydrochemistry and ecol- content provided by the reactive barrier present in the lat- ogy of the groundwater environment, especially in terms ter, being a source for the growth of bacterial communities of microbial communities. Comparing recharge and non- and enhanced diversity under recharge conditions. The role recharge scenarios, we found that microbial diversity indices of the humidity on microbial diversity is also significant, as (Shannon) correlate inversely with the degree of perturba- was previously reported in horizontal subsurface constructed tion caused by the induced recharge, substantiating an inter- wetlands (Nurk et al., 2005). Furthermore, phylotype distri- mediate disturbance hypothesis distribution. In fact, MAR www.hydrol-earth-syst-sci.net/23/139/2019/ Hydrol. Earth Syst. Sci., 23, 139–154, 2019 152 C. 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