Environ. Sci. Technol. 2010, 44, 3123–3129
Combined Chemical-Biological Treatment for Prevention/ Rehabilitation of Clogged Wells by an Iron-Oxidizing Bacterium EFRAT GINO, JEANNA STAROSVETSKY, EYAL KURZBAUM, AND R O B E R T A R M O N * ,† Faculty of Civil & Environmental Engineering, Division of Environmental, Water and Agricultural Engineering, Technion, Haifa 32000, Israel
Received December 12, 2009. Revised manuscript received February 20, 2010. Accepted March 3, 2010.
Groundwater wells containing large concentrations of ferrous iron face serious clogging problems as a result of biotic iron oxidation. Following a short time after their start off, wells get clogged, and their production efficiency drop significantly up to a total obstruction, making cleanup and rehabilitation an economic burden. The present study was undertaken to test an experimental combined treatment (chemical and biological) for future prevention or rehabilitation of clogged wells. Sphaerotilus natans (an iron-oxidizing bacterium) freshly isolated from a deep well was grown to form biofilms on two systems: coupons and sand buried miniature wedge wire screen baskets. A combined chemical-biological treatment, applied at laboratory scale by use of glycolic acid (2%) and isolated bacteriophages against Sphaerotilus natans (SN1 and ER1-a newly isolated phage) at low multiplicity of infection (MOI), showed inhibition of biofilm formation and inactivation of the contaminant bacteria. In addition to complete inactivation of S. natans planktonic bacteria by the respective phages, earlier biofilm treatment with reduced glycolic acid concentration revealed efficient exopolysaccharide (EPS) digestion allowing phages to be increasingly efficient against biofilm matrix bacteria. Utilization of this combined treatment revealed clean surfaces of a model stainless steel wedge wire screen baskets (commonly used in wells) for up to 60 days.
Introduction Bacterial biofilms are a universal phenomena that affect almost all ecosystems (1). Development of biofilms on surfaces may cause a diversity of malfunctions in industrial equipment involved in water production such as the following: clogging of filtration media (2), corrosion of metal (3, 4) and biodegradation of rubber components (5), disinfection reduced performance (6, 7), energy losses (8), increased resistance to disinfectants through genetic exchange among biofilm species (9), production of secondary metabolites (10, 11), malodors and toxins (12, 13), and other health problems (14–18). Worldwide, groundwater is still the main supply source of drinking water to large populations in spite of intensive * Corresponding author phone: 972-4-8292377; fax: 972-4-8292377, 972-4-8293309; e-mail:
[email protected]. † Member of Grand Water Research Institute at Technion. 10.1021/es903703v
2010 American Chemical Society
Published on Web 03/18/2010
research and development in water reuse, desalination, and advanced treatment (19). Groundwater is exposed to a variety of contaminants originating from the surface and subsurface. Since groundwater contains a variety of minerals and in some cases organic pollutants (as nutrients), biofouling by autochthonous or contaminant bacteria will occur (20). The subsurface part of a typical well is made of a stainless steel (SS) screening device that prevents soil particles from being collected during pumping. The screening device has the greatest potential for fouling by inorganic particulate matter, oxidation of minerals (e.g., Fe2+ to Fe3+), and biomass production by free-living groundwater bacteria (21–23). Well clogging phenomena is not a new phenomenon and is mainly based on biofilm formation by iron bacteria (e.g. Thiobacillus, Gallionella, Leptothrix and Sphaerotilus sp.) in groundwater, due to oxygen shifts from anaerobic to aerobic through pumping activity. Clogged wells’ cleaning techniques, based on physicochemical principles, were tested on field studies such as the following: pressure acidization, heat application, shockwaves, fluid percussive methods, combination of jetted disinfectant, detergent and heat (32 to 100 °C, at 51 Atm), disinfectants (chlorine, hydrogen peroxide, brominated compounds, organic acids including chelators), carbon dioxide, phosphorus-based acids, polymers, and different blends of the above methods with variable results (24–27). Among these methods, combined biological-chemical treatment has never been applied in spite of its possible successful outcome since a major role in well clogging is attributed to bacterial activity. An overview of the recent scientific literature on “bacteriophages therapy” reveals several decades of “renaissance” in use of phages to combat different bacteria in many area of applications: slime and biofilm control, plant diseases, medicine, foodborne pathogen control, and detection (28–34). Since their discovery by D’Herrelle and Twort, bacteriophages are still a promising antibacterial form to combat infections due to their specificity, rapid multiplication properties, and their restriction to prokaryotic organisms (35, 36) Recently bacteriophages seem to have a large variety of uses in different areas: diabetic wound infections (foot ulcers), topical cleaning and disinfection (37), food industry (meat and poultry spraying against Salmonella sp.) (38), UF membrane clogging prevention (39), and many others (40). Historically, Doolittle et al. (41) were perhaps the first to show the potential use of T4 bacteriophage to act lytically on E. coli biofilms, and recently Lu and Collins (42) proved that biofilm EPS can be dispersed biocatalytically by enzymatic engineered bacteriophages. Goldman et al. (39), in a recent publication on effluents ultrafiltration, showed that simultaneous inoculation with specific lytic phages active against three contaminant bacteria can reduce biofilm formation on UF membrane and increase its flux by 30 to 60%. In comparison with effluent the diversity of well bacterial species is smaller (attributed to groundwater lack of organics, presence of metal ions such as Fe2+ and Mn2+ and variable oxic/anoxic conditions); therefore, only specific bacterial strains such as iron and manganese oxidizers/reducer and sulfate-reducing bacteria will be present, if external pollution is not present. Based on this concept, the present study focused on utilization of specific bacteriophages infecting iron-oxidizing bacterium Sphaerotilus natans sp., in order to prevent iron oxidation-precipitation and clogging of well’s screen. VOL. 44, NO. 8, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Physicochemical Parameters of Groundwater from Deep Well Idan 2a parameter
concentration
pH turbidity (NTU) temperature (°C) TDS (mg L-1) TCOD (mg L-1) TOC (mg L-1) TSS (mg L-1) VSS (mg L-1) MBAS (mg L-1) conductivity (µmho cm-1) hardness (mg L-1) CO2 (mg L-1) dissolved O2 (mg L-1) H2S (mg L-1) Cl- (mg L-1) NO2- (mg L-1) NO3- (mg L-1) PO4- (mg L-1) SO4- (mg L-1) Ca2+ (mg L-1) Mg2+ (mg L-1) Mn2+ (mg L-1) K (mg L-1) Na (mg L-1) Li (mg L-1) total iron (mg L-1) Fe2+ (mg L-1) Zn (mg L-1) B (mg L-1) Si as SiO2 (mg L-1) Ba (mg L-1)
6.87 ( 0.28 0.78 ( 0.13 34 ( 2 1584 ( 23 37.7 ( 0.7 1.3 ( 0.5 4.76 ( 0.67 2.36 ( 0.43 0.0148 ( 0.0024 2150 ( 720 842 ( 16 25 ( 3.2 5 ( 1.4 0.034 ( 0.008 561 ( 64 0.98 ( 0.03 18 ( 2.34 2.34 ( 0.33 289.6 ( 10.3 168.8 ( 5.5 101.5 ( 2.1 0.003 ( 0.001 10.08 ( 0.56 253 ( 10.4 0.096 ( 0.006 0.027 ( 0.005 0.008 ( 0.002 0.028 ( 0.004 0.230 ( 0.032 22 ( 6.7 0.033 ( 0.003
Materials and Methods Well Geophysical and Mechanical Parameters. Idan 2a well is located in the southern part of Israel, the central Arava Valley. The average yearly precipitation is 50 mm, and the potential evaporation exceeds 3000 mm. The average yearly temperature is 25 °C (43). Groundwater is pumped from the Graben fill aquifer with a chlorinity between 200-1800 mg L-1. Idan 2a well has a depth of 177 m, with a submerged pump located at 108 m and a Johnson type screen from 110.8 to 177 m (more details in Supporting Information, SI, Section 1). Well Water Physicochemical Parameters. The physicochemical parameters of Idan 2a’s water are presented in Table 1. All chemical tests were performed according to Standard Methods (44). Isolation and Enumeration of S. natans and Heterotrophic Bacteria from Well Water. S. natans was isolated on solid Winogradsky medium containing (g L-1): 0.5 MgSO4 · 7H2O, 0.5 K2HPO4, 0.5 NaNO3, 0.5 NH4NO3, 0.5 ammonium ferric citrate, 0.2 CaCl2 · 6H2O, and 1.8% agar (pH4.8 ( 0.1) (45, 46). Average counts were 8.3 ( 1.6 × 105 CFU mL-1. Total heterotrophic bacterial count was performed by direct plating of well samples (0.1 mL/plate) on solid LB medium (1.5% agar) and incubated at 20 and 36 °C for 48 h (44). Average counts were 5 ( 0.6 × 107 CFU mL-1. Ferrous Iron Oxidation. Ferrous iron was measured by two methods: ferrozine and 1,10-phenanthroline (47–49). Briefly, the presence of Fe2+ was detected by magenta color development through a ferrous iron-ferrozine complex measured spectroscopically at 562 nm or orange color development through a ferrous iron-1,10-phenantroline complex measured spectroscopically at 515 nm against controls. Both methods were sufficiently accurate at the iron concentrations used in the present study. 3124
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Sphaerotilus natans Identification. Briefly, fluorescence in situ hybridization (FISH) was performed by use of FISH oligonucleotide probe (16S rRNA) for the target organism (Sphaerotilus natans) labeled with tetramethyl rhodamine isothyocynate (TRITC) as previously described (50, 51). Sphaerotilus natans DSM 565 (from our collection) was used as positive control for comparison in the FISH application (SI, Section 1). Sphaerotilus natans Growth and Enumeration. S. natans sp. isolated from well Idan 2a was grown and enumerated in Winogradsky liquid and solid (1.8% agar) medium, respectively, for 7-10 days at 30 °C (46). All counts were performed in triplicates. S. natans Bacteriophages Isolation and Enumeration. Bacteriophages were isolated from Haifa STP (sewage treatment plant) effluents by a second-step concentration method and double layer agar method (SI, Section 2, Figure S3) (52). Two major bacteriophage types were isolated (both containing DNA): one morphologically very similar to SN1 already described by Winston and Thompson (53) as belonging to the siphoviridae group (54) and a second (temporary named ER1) belonging to the myoviridae Figure 2 (SI Section 2, Figure S4). Enumeration of Biofilm Bacteria. Biofilm bacteria were enumerated according to a previously described method (7). Briefly, coupons from system 2 were transferred to a 50 mL sterile test tube containing phosphate buffer solution (0.1 mM, pH 7.0) and gently rinsed, to wash planktonic bacteria. Thereafter coupons were transferred to 50 mL sterile plastic tube containing phosphate buffer saline (0.1 mM, pH 7.5) and 0.1% Tween 80 (v/v) (Rohm & Haas, USA). The coupons were scrapped with a sterile rubber stick (Miniplast, USA) and vigorously vortexed, to remove attached bacteria. Final suspension dilutions were plated on Winogradsky solid medium and incubated at 30 °C for 5 to 7 days. Experimental miniature wedge wire screen baskets (system 1) were subjected to similar treatment. Experimental Setup Systems. System 1: comprised a miniature stainless steel (SS) screen covered with medium size sand (∼0.4 mm) up to a volume of 200 mL. The container was filled with 900 mL of original well water (from Idan 2a site) and the peristaltic pump turned on at different flow velocities as required. Each experiment was carried out for 60 days at room temperature (24 ( 2 °C) and in order to compensate for water evaporation, every 2-3 days groundwater from the same batch was added to keep the water level even (SI, Section 1, Figure S1). System 2: A Pyrex glass hose (Ø of 10 cm) was built with top 12 screw type openings and autoclavable screw-caps and two main outlets was the main biofilm reactor (SI, Section 1, Figure S2). The hose was connected on both sides with a Tygon flexible tube (autoclavable). Well water flow was regulated by a peristaltic pump. SS coupons (the size of microscope slides) were suspended into the hose by a plastic wire and left for biofilm formation as a function of time. At certain time intervals, coupons were taken out and biofilm bacteria enumerated in parallel with volume planktonic bacteria as mentioned below.
Results and Discussion In order to examine the new approach of combined treatment (chemical-biological) on removal of iron oxidizing bacteria, a S. natans bacterium was isolated from deep water well and subjected to a series of experiments. Ferrous iron oxidation by S. natans in the absence and presence of its specific bacteriophages was tested in the original well water supplemented with ∼0.7 mg L-1 ferrous iron (FeSO4 0.7H2O) due to the initial low iron concentration (0.008 ( 0.002 mg L-1, Table 1). Figure 1A,B represents 4 days batch culture oxidation of ferrous iron by S. natans without and with bacteriophages
FIGURE 1. Batch ferrous iron oxidation by S. natans without (A) and with (B) addition of bacteriophages.
FIGURE 2. Fe2+ oxidation at high concentrations (47 mg/L initial concentration) by abiotic and biotic (S. natans alone, S. natans and bacteriophages SN1 and ER1) processes as a function of time. Note: (black line- abiotic oxidation); (green line- S. natans + SN1 phage); (red green line- S. natans + ER1 phage); (light blue line- S. natans DSM 565); (dark blue line- S. natans this study isolate). present. S. natans culture without bacteriophages revealed an increased of 4 logs with a constant ferrous iron reduction of 85.7% (Figure 1A). With the addition of specific bacteriophages (ER1 and SN1, at 105 PFU mL-1) on the second day, S. natans growth ceased and a reduction of ∼5 logs was observed on viable cells, while ferrous iron remained almost unchanged (range 0.5 to 0.48 mg L-1) for the remaining time interval (Figure 1B). The oxidation of higher ferrous iron concentration was also tested for 10 days under the same experimental conditions (Figure 2). Abiotic ferrous iron oxidation was 8.5% (4 ( 0.5 mg L-1), biotic oxidation by S. natans was 99.57% (from 47 to 0.2 mg L-1), while introduction of the bacteriophage (SN1 or ER1 at 103 PFU mL-1) on the second day halted ferrous iron oxidation. Glycolic acid addition to clogged wells at concentrations >3% (v/v) is a common practice in Israel. Consequently, S. natans isolate was exposed to various glycolic acid concentrations (0.5 to 7%, v/v) for up to 72 h in well water (SI, Section 2 Figure S5). S. natans bacterium exposed to glycolate concentrations of 0.5 to 1.5% for 24 h revealed a 3 to 4 logs reduction; however, after 72 h a regrowth of ∼2 logs was observed, while at >3% glycolate no regrowth was observed. At 5 and 7% glycolate concentrations complete inactivation was observed after 72 h. These results are important in view of the following effects: enhanced corrosion at high acid concentrations, maintenance treatment cost, and possible regrowth due to assimilable organic carbon (AOC) utilization (20).
FIGURE 3. Inactivation of S. natans by ER1 bacteriophages at different MOI values. S. natans bacteriophages were also tested for viability following exposure to various glycolate concentrations. First, the newly isolated bacteriophage ER1 was exposed to different concentrations of glycolic acid, pH range from 3 to 7 (SI Table S1). Except 0.5% glycolic acid (that reduced phage titer by one log) all other acid concentrations increased bacteriophage count by 0.5 to 1.5 log after 24 h of exposure. The increase can be related to glycolic acid ability to open bacteriophage clamps as experimental stocks were generated directly from infected and lysed bacterial cells, therefore containing bacterial debris. Similar results were obtained with phage SN1 (data not shown). ER1 phages showed temperature resistance too (SI, Section 2 Figure S6). Between 20 to 60 °C there was no significant reduction in phage counts (p e 0.05). Only at 70 °C a 3 logs reduction was observed. As well water in the subtropic dry climate can reach elevated temperatures (>30 °C) this resistance is an advantageous. ER1 bacteriophage was also tested for its ability to inactivate S. natans at different multiplicity of infection (MOI) (Figure 3). S. natans cells at an initial concentration of 106 CFU mL-1 were reduced by 2.5 logs in 5 days by 10 PFU (MOI ) 1 × 10-5). At MOI-s of 1 × 10-3 and 1 a complete reduction in bacterial cells was observed after 3 days. Regardless of faster inactivation by MOI value of 1, a MOI of 1 × 10-3 was used in all other experiments with excellent results. In real life, iron oxidizing bacteria numbers can differ according to environmental conditions (mainly Fe2+ concentration). Consequently, different S. natans concentrations were tested in batch well water samples for inactivation by ER1 bacteriophage at a constant concentration of 103 PFU mL-1 (Figure 4). S. natans at initial concentration of 103 CFU mL-1 and phage ER1 at MOI)1 was completely inactivated after 3 days. S. natans initial larger numbers (105 to 109 CFU mL-1 and MOI values from 0.01 to 1 × 10-6, respectively) VOL. 44, NO. 8, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 4. S. natans inactivation by phage ER1 (103 PFU mL-1) at different initial bacterial concentrations. showed similar inactivation rates after 4 days of incubation. Interestingly, at initial concentrations of 105 and 107 CFU mL-1 a growth of 1.5 logs and 1 log correspondingly were observed after 3 days from bacteriophage addition. At 109 CFU mL-1, which seems to be the maximal growth potential of S. natans under the present experimental circumstances, no further growth was observed, and inactivation started at day one. Control, without bacteriophage, remained essentially constant throughout the experiment. The proposed treatment is intended to be applied in soilgroundwater environment; therefore, possible phages adsorption to clay particles may impact their transport and infectivity in soils (55–57). Three soil types containing various clay content (from low to high): loam, loess, and grumosol were tested for phages adsorption at different pH-s (SI, Section 2, Table S2). After 24 h, loam soil (10% clay) revealed the highest adsorption of 45-90.5%, followed by loess (29.4% clay) 0-66.1%, and grumosol (61.5% clay) 0-25%. Gerba and Goyal (55) already suggested that the prediction potential of animal viruses as well bacteriophages to adsorb to soil is too difficult to model and should be calculated merely experimentally. Armon and Cabelli (56) testing f2 phage-clay (kaolinite and bentonite) interaction found an adsorption of 93 to 99.7%, while Blanc and Nasser (57) reported on reduced adsorption of bacteriophages (15%) in 12% clay content soil compared to 30-99% in clayless soil. In this study experimental phages ER1 and SN1 followed the pattern described by Blanc and Nasser (57). Therefore increased clay content does not seem to be a diffusion barrier of these experimental phages. Based on these preliminary results, it was suggested to use glycolic acid against biofilm EPS to be followed by specific phages to inactivate released planktonic bacteria.
From previous experiments with SS metal coupons, S. natans cells and FeSO4 0.7H2O (0.7 mg L-1) it was found that two days is the required time interval to form a stable biofilm (data not shown). Using system 1, S. natans planktonic cells were enumerated following various treatments (SI, Section 2, Figure S7). After two days of continuous well water recirculation, glycolic acid (2%, v/v) was added and recirculated for one hour, then ER1 bacteriophages (103 PFU mL-1) were added, and the system recirculated for an additional 60 days. S. natans solely (control) showed a slight increase (approximately 0.5 log) due to biofilm detachment during the 60 days of the experiment. When phages ER1 (103 PFU mL-1) were introduced on the second day, S. natans count was reduced by 3 logs after 5 days and remained stable (700-800 CFU mL-1) for the rest of the time. Sole addition of glycolic acid (2%) revealed a similar reduction in bacterial numbers (3 logs); however, starting on the seventh day a regrowth of 2 logs was recorded (103 to 105 CFU mL-1) without any further changes. As previously shown, S. natans could be inactivated by >3% glycolate, but at lower concentrations it was able to recover and even regrowth (SI, Section 2, Figure S5). Beside glycolate itself, regrowth is probably also due to partially acid decomposed EPS. After 60 days of recirculation the S. natans count dropped to only a few bacterial cells without practical regrowth. Using system set 2, it was possible to enumerate individually planktonic and biofilm S. natans cells exposed to combined treatment (Figure 5A,B). Free cells counts (Figure 5A) revealed very similar inactivation patterns already observed with system 1 (SI, Section 2, Figure S5). Glycolate alone reduced planktonic cells count by 5 logs in 7 days, but later an enhanced regrowth of ∼4 orders of magnitudes occurred. ER1 phages solely (MOI of 0.0002) reduced planktonic cells by ∼6 logs during 30 days, without any additional reduction. Glycolate and ER1 phages combination reduced free cells count to
