Enhancement of rhizocompetence in pathogenic bacteria removal of a constructed wetland system

1 Enhancement of rhizocompetence in pathogenic bacteria removal of 1 constructed wetland system 2 Marwa BEN SAAD1, Myriam BEN SAID1, Isabel SANZ SÁEZ2, Olga SANCHEZ3, Jordi 3 MORATO4, Latifa BOUSSELMI1 and Ahmed GHRABI1 4 1Water Researches and Technologies Center, CERTE, BP 273 - 8020 Soliman Tunisia 5 2Institut de Ciències del Mar (ICM-CSIC), Pg. Marítim de la Barceloneta,08003 Barcelona, Spain 6 3Department of Genetics and Microbiology Faculty of Biosciences, UAB · 08193 Bellaterra Barcelona Spain 7 4UNESCO Chair in Sustainability, Polytechnic University of Catalunya, C1 Terrassa, 08222, Barcelona, Spain 8 *[email protected] 9 10 11 12 Abstract 13 The main goal of the present study was to enhance the rhizobacterium potential in horizontal 14 subsurface flow constructed wetland system planted by Phragmites australis through 15 environmentally friendly biological approaches. The bioinoculation of antagonist bacteria has 16 been used to promote higher rhizosphere competence in pathogenic bacteria removal. The 17 experience was performed with once and with sequential bio-inoculation. The results show 18 that the strain PFH1 played an active role in pathogenic bacteria removal. In fact, the 19 individual bioinoculated improves remarkably the inactivation kinetics of a pathogenic tested 20 bacteria; S. typhi in plant rizosphere by, 0.8 U-Log10 with once bio-injection and 21 approximately, 2.5 U-Log10 with sequential bio-injections.These results suggested that this 22 strain represents a promising candidate to improve the water purification by constructed 23 wetland. 24 Key words: Antagonism, Bioinoculation, Constructed Wetland, Rhizosphere. 25 Introduction 26 Constructed wetlands (CWs) have been used as a green technology to treat various 27 wastewaters for several decades. They offer a land-intensive, low-energy, and less 28 operational-requirements alternative to conventional treatment systems, especially for small 29 communities and remote locations(Ghrabi et al. 2011; Shen et al. 2015; Tee et al. 2016) 30 (Ghrabi et al. 2011; S. Wu et al. 2015) These engineered systems are designed to treat 31 contaminants in surface water, groundwater or waste streams by using natural functions of 32 2 wetland vegetation, soils and their microbial populations (Vymazal 2014). They have a great 1 potential for the treatment of wastewater of different origin (Wang et al. 2015; Zaytsev et al. 2 2011) such as domestic sewage, agricultural wastewater, industrial effluent, mine drainage, 3 landfill leachate, urban runoff, and polluted river water (Liu et al. 2015). CWs have been 4 successfully used to mitigate environmental pollution by removing of a wide variety of 5 pollutants from wastewater such as organic compounds, suspended solids, pathogens, metals, 6 and nutrients (Zhang et al. 2014). During wastewater treatment in CWs, pollutants are 7 removed through an integrated combination of biological, physical and chemical interactions 8 between the plants, the substrate and the inherent microbial community (Wang et al. 2015) . 9 CWs are typically classified into two types according to the wetland hydrology: free water 10 surface (FWS) CWs and subsurface flow (SSF) CWs. FWS systems are similar to natural 11 wetlands, with a shallow flow of wastewater over a saturated substrate. On SSF systems, 12 wastewater flows through the substrate which supports the growth of plants, and based on the 13 flow direction, SSF CWs could be further divided into vertical flow (VF) and horizontal flow 14 (HF) CWs. A hybrid CW, a combination of various wetland systems, was also introduced for 15 the treatment of wastewater (Wu et al. 2015). For the purpose of this paper, only a subsurface 16 flow constructed wetland and especially the horizontal subsurface flow constructed wetland 17 planted by Phragmites australis is considered. 18 Microorganisms play a vital role in degradation of multiple pollutants in CWs. It has been 19 recognized that the removal of most pollutants in CWs is due primarily to microbial activity 20 (Meng et al. 2014). Removal of a particular pollutant is typically associated with a specific 21 microbial functional group, therefore the employment of design and operational 22 methodologies that enhance the activity of that group will better optimize performance 23 (Faulwetter et al. 2009). 24 3 It has long been renowned that many naturally occurring rhizosphere bacteria and fungi may 1 offer a viable substitute for the use of chemicals and are antagonistic towards crop pathogens. 2 Thus, Plant growth promoting rhizobacteria (PGPR) has been shown beneficial to plant 3 growth and health by emancipating their activity on nitrogen fixation, the production of 4 phytohormones and antifungal compounds, and induced systemic resistance (Sindhu, 5 Rakshiya, and Sahu 2009). 6 Based on the importance of rhizosphere competence or root colonization in beneficial plant– 7 microbe interactions (Ben Saad et al. 2016), the main goal of the present study was to enhance 8 the inactivation of pathogenic bacteria rates in horizontal subsurface flow constructed wetland 9 system planted by Phragmites australis using antagonistic bacteria. This work aimed to 10 demonstrate the beneficial application of biotechnology to confer higher rhizosphere 11 competence in the removal of pathogenic bacteria; S. typhi ATCC 560 by environment 12 friendly biological approaches. 13 Methods 14 1. Sampling and isolation of bacterial strains from different environments 15 Bacterial strains were isolated from different ecological niches: wastewater, soil, Phragmites 16 australis roots and sheets from the Technical Demonstration Center (TDC) that treats sewage 17 from the university home located at the Agronomic Institute of Tunis (INAT). Rhizosphere 18 samples were collected from each wetland at the entrance, middle, and exit at a depth of 19 approximately 30 cm under the gravel surface. All samples were processed in the laboratory. 20 To isolate bacteria from the rhizosphere, the roots were initially separated from the rhizomes, 21 and then small pieces of roots were immersed in sterile saline solution (0.85 g/L NaCl) and 22 vortexes 15min in order to release the bacteria attached to roots into the solution. 23 4 The same protocol was followed to isolate bacteria from the sets of reeds. Concerning the 1 wastewater samples, these samples underwent decimal dilutions in sterile saline solution and 2 spread out over selective medium. 3 2. Identification of the strain and detection of siderophores production 4 The identification of selected bacteria was based on the phenotypical aspect of colonies, the 5 microscopic examination standard microbiological and biochemical tests. Siderophore was 6 detected by the method of Jalal and Vander Helm (1990) using a spectrophotometric assay 7 where a peak at 495 nm on the addition of 2% aqueous solution of FeCl3 to 1 mL of 8 supernatant indicated the presence of siderophores. 9 3. Antagonism test between isolated bacterial strains and against pathogenic bacteria 10 S. typhi ATCC 560 11 Antagonism test had been performed between isolated bacterial to avoid negative interaction 12 between them after their bioinoculation. The Petri dish surface was seeded by an indicative 13 strain and then the blank discs deposited on the culture medium had been drenched with 50µL 14 of filtered supernatant of a putative antagonist strain, collected after centrifugation at 4000 15 r.p.m for 15 min. The diffusion of the antimicrobial agents was enhanced by incubation at 16 37°C for 24hr. Antagonist activity was revealed by the appearance of inhibition zone around 17 the discs. 18 4. Study of motility of isolated bacteria and biofilm production. 19 The different types of mobility (swimming, swarming and twitching) were determined by the 20 method of Reimmann et al. (2002). The biofilm production of bacterial isolates was detected 21 by two methods: the first described by Freeman and al. (1989), consisted of plating the test 22 strains on solid medium contained brain heart infusion broth 37 g/L, sucrose 50 g/L, agar 10 23 g/L and Congo Red indicator 8 g/L (Sujatha N. 2013). After incubation at 30°C for 24hr, 24 5 black colonies indicate biofilm production. After this qualitative study, we proceeded in a 1 quantitative study describes by O’Toole (1998). This method uses the dye crystal violet (CV). 2 biofilm production was estimated by spectrophotometric measurement of the OD 600nm. 3 5. Molecular identification of the selected strain 4 Bacterial DNA was extracted and purified using the v-DNA reagent (GenIUL) according to 5 the manufacturer’s instructions. The concentration of the extracted DNA was measured using 6 a spectrophotometer at 260nm. DNA purity was estimated from the A260/A280 ratio. The 7 complete 16S rRNA gene was amplified using bacterial primers 27F (5’-TAC GGY TAC 8 CTT GTT AYG ACT T-3’) and 1492Rmod (5’-AGR GTT TGA TCM TGG CTC AG-3’). 9 Each PCR reaction with a final volume of 25 µl contained: 2µl of template DNA, 0.5 µl of 10 each deoxynucleoside triphosphate at a concentration of 10 µM, 0.75 µl of MgCl2 1.5 mM, 11 0.5 µl of each primer at a concentration of 10 µM, 0.125 µl of Taq DNA polymerase 12 (Invitrogen), 2.5 µl of PCR buffer supplied by the manufacturer (Invitrogen, Paisley, UK) and 13 Milli-Q water up to the final volume. Reactions were carried out in a Biorad thermocycler 14 using the following program: initial denaturation at 94ºC for 5 min, followed by 30 cycles of 15 1 min at 94ºC, 1 min at 55 ºC and 2 min at 72 ºC, and a final extension step of 10 min at 72ºC. 16 PCR products were verified and quantified by agarose gel electrophoresis with standard low 17 DNA mass ladder (Invitrogen). Purification and One Shot Sanger sequencing of 16S rRNA 18 gene products was performed by Genoscreen (Lille, France) with primers 27F and 19 1492Rmod. 20 6. Conception and construction of the pilot-scale systems 21 The experimental system designed for the bio-inoculation, included two small parallel 22 identical horizontal subsurface flow constructed wetlands. Both basins were filled with gravel 23 and planted with reed. The first one served as a control and the second served for the different 24 6 bio-assays. The size of each constructed wetland bed was 0.3 x 0.44 x 0.28. The treatment 1 area was packed with 8-12mm diameter pea gravel while bigger and larger gravel of 302 80mm diameter was used at the inlet and outlet areas in order to prevent clogging of the filter 3 media. The relative porosity has been calculated in 0.26 (n = Vv/Vt where Vv is the void 4 volume and Vt is the total volume (Meng et al. 2014)). The pilote constructed wetlands had 5 bottom slope of 1% to facilate the flow of water by gravity. The plants were allowed to grow 6 and multiply over three months. There was a peridioc application of wastewater to serve as a 7 source of nutrients for the plants. The main characteristics of the experimental system are as 8 follows: Surface area: 13.2dm2, Hydraulic Residence Time HRT (theoretical): 0.385, Gravel 9 depth: 2dm, Average starting reed heights: 57cm.The figures 1 and 2 show the conception of 10 horizontal subsurface flow constructed wetland systems adopted in this study. 11 12 Figure1. The representative figure of the pilot-scale of subsurface flow constructed wetland 13 7. Monitoring of bacteria removal 14 To optimize the experience, the kinetic growth of the selected strain was determined using a 15 spectrophotometer to estimate absorbance of cell suspensions (DO600). Series tests were 16 conducted to determine the lag time that made the selected bacteria to adapt to new 17 conditions. 18 The experience in situ was done as follows: Phase I start from the sowing of interest bacteria 19 into the rizosphere environment. The main events are activation of the antagonist inoculum 20 7 and establishment of an antagonist population in the plant rizosphere. Phase II is the process 1 of the introduced antagonist and native root-associated microbes to establish a population 2 density and persist in the rhizoplane, rhizosphere or inside the root. 3 Therefore, the antagonist bacterium was inoculated into the rhizosphere of the pilot-scale (F). 4 After an adaptation period (depends on growth kinetic parameters), a contaminated effluent 5 by 104 UFC/ml of an indicator bacteria; S. typhi ATCC 560 was added for both pilotscales (F 6 and T). 7 Based on growth kinetic parameters of interest bacteria (PFH1), sequential bio-injections were 8 performed in pilot scale (F) to test of the spatio-temporal dynamics and microbial ecological 9 processes of root colonization by an antagonist and to explore the impact of accumulation 10 effect of sequential bioinoculation of antagonist bacteria to promote inhibition of pathogenic 11 bacteria. 12 The monitoring of pathogenic bacteria removal after bioinoculation was determined by 13 culture on selective medium (SS: Selmonella Shigella agar). 14 15 Results and discussion 16 1. Isolation and Screening of bacterial strains 17 After sampling, isolation and purification stages, 19 bacterial strains were isolated from 18 different ecological niches from the Technical Demonstration Center (TDC) that treats 19 sewage from university home located at the Agronomic Institute of Tunis (INAT). The isolats 20 strains were selected and screened for general functional properties of plant promoting 21 rhizobacteria; namely, siderophore production and antagonist activity against pathogenic 22 bacteria in addition of the bacterial motility (swimming, swarming and twitching motilities) 23 and biofilm production. 24 8 Bacterial biofilm formation is important for root colonization. Indeed, root-associated bacteria 1 have been studied extensively, and many of these promote the growth of host plants or are 2 used as biocontrol agents (Dekkers et al. 1998). The plant-growth-promoting bacteria have 3 been reported to discontinuously colonize the root surface, developing as small biofilms along 4 epidermal fissures. 5 Among the isolates, we were screened PFH1 strain to apply as an interest to be inoculated into 6 the rizospheric zone to enhance the reduction of pathogenic bacteria. Indeed, the antagonism 7 test had revealed the strain PFH1 as the most antagonist bacteria against S. typhi. Indeed, The 8 ability of microbes to produce a wide range of antimicrobial compounds, including lytic 9 agents, antibiotics, bacteriocins, protein exotoxins and other secondary metabolites, is critical 10 to their success in antagonistic activities (W.-Y. Liu et al. 2013). 11 12 2. Molecular identification of the selected strain 13 The 16S rRNA gene sequences of the selected bacteria were determined. The sequence 14 analysis revealed that this bacteria has 99% of similarity with Enterobacter cloaceae. 15 Enterobacter cloacae are a gram-negative Proteobacterium belonging to the 16 Enterobacteriaceae family. Within this family, Enterobacter is most closely related to, and is 17 grouped in a sub-clade with, Klebsiella. The E. cloacae species comprises an extremely 18 diverse group of bacteria that has been found in diverse environments, ranging from plants to 19 soil to humans (Liu et al., 2013). 20 Enterobacter species have been reported as both plant pathogens and human opportunistic 21 pathogens (Nishijima et al., 2007), and also as important engineering and plant growth22 promoting bacteria (Nie et al., 2002). Some Enterobacter strains may play important roles in 23 plant–microbe interactions and hence in biocontrol mechanisms. In this sense, we are used the 24 9 selected bacteria to control the pathogenic density and its application to improve the water 1 treatment by constructed wetland. 2 3. Application bioinoculation of selected bacteria. 3 To optimize the experience and before inoculation into the rhizosphere, the kinetic growth of 4 PFH1 strain was investigated in saline water and autoclaved wastewater at room temperature 5 in order to determine the specific growth characteristics of PFH1 namely the lag time, (ʎt) the 6 maximum specific growth rate (µmax), the bacterium generation time (tG) (Figure 2). 7 8 9 Figure 2. Kinetics growth of PFH1 in different growth temperature conditions. 10 Data are averages of three experiments 11 12 3.1. Removal of pathogenic bacteria with single bioinoculation 13 The figure 3 shows the kinetic of bacteria removal with an initial concentration of indictor 14 bacteria (S. typhi) equal to 106 UFC/ml in presence of PFH1. 15 0 0,2 0,4 0,6 0,8 1 1,2 0 15 30 45 60 75 90 105 120 135 OD600 Time (min) Saline Water Autoclaved wastewater Saline Water µmax = 0. 25 min-1 tG= 2.77 min ʎ = 60 min Autoclaved WW µmax = 0.31 min-1 tG= 2.23 min ʎ =75 min 16 developing a commercial bioinoculant to be applied in biological water treatment process to 1 improve the treated water quality. 2 3 CONCLUSION 4 From the present research, we can conclude that application of bioinoculation has a potential 5 to enhance the pathogenic bacteria removal process. Indeed, the preliminary results show the 6 beneficial effect of the bioinoculated strain (PFH1) in the rhizosphere to increase remarkably 7 the efficiency of the water treatment system for the reduction of pathogenic bacteria with a 8 reduction in contact time. 9 This study has contributed with an eco-friendly strategy to improve water treatment process 10 by constructed wetland, and highlighted the fact that better yields can be obtained thought 11 bio-inoculation. 12 As a perspective of this study, the application of this strategy in field conditions with multi13 inoculation of antagonists substances protected by natural polymers to inactivate pathogenic 14 bacteria in treated water without chemical addition, extension in the retention time or addition 15 of a complement water treatment stages. 16 Acknowledgment 17 This work is supported by CERTE contract programs funded by the Ministry of Higher 18 Education and Scientific Research of Tunisia. 19 20 References 21 Ben Saad M, Ben Said M, Bousselmi L, and Ghrabi A (2016) Application of Bioinoculation to 22 Enhance Rhizocompetence of Horizontal Subsurface Flow Constructed Wetland System, Desal 23 and Wat Treat 57 (46): 22133–39. doi:10.1080/19443994.2016.1141713. 24 Dekkers, L C, C C Phoelich, L van der Fits, and B J Lugtenberg. 1998. “A Site-Specific Recombinase 25 Is Required for Competitive Root Colonization by Pseudomonas Fluorescens WCS365.” 26 17 Proceedings of the National Academy of Sciences of the United States of America 95 (12): 7051– 1 56. doi:10.1073/pnas.95.12.7051. 2 Faulwetter, Jennifer L., Vincent Gagnon, Carina Sundberg, Florent Chazarenc, Mark D. 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