Removal of Cadmium from Contaminated Soils by Multiple Washing with Iron (III) Chloride

In the present study, three concentrations of FeCl₃ were applied to the Cd-contaminated soils at two solid-to-solution ratios, and 1–3 washes were applied. The Cd removal percentages increased with the number of washes. An optimal treatment for removing Cd from the soil was a solid-to-solution ratio of 1/2 using 45 mM FeCl₃ and three washes. However, the application of FeCl₃ decreased the soil pH values, increased the soil electrical conductivity, and also changed the exchangeable concentrations of cations. These changes negatively influenced the germination percentage of pak choi (Brassica campestris L. ssp. chinensis) compared with control soils.     

Removal of Contaminating Metals from Soil by Sulfur-Based Bioleaching and Biogenic Sulfide-Based Precipitation

The potential of a hybrid process incorporating sulfur-based bioleaching and sulfide-based precipitation for treatment of metal-contaminated soil was examined in batch-type experiments. The sulfur-based soil bioleaching process with Acidithiobacillus sp. could be initiated at a wide range of initial pH from 4.0 to 6.3. After 15 days, 98% of Zn, 89% of Cu and 79% of Cd was bioleached. The gaseous sulfides recycling from Desulfovibrio sp.-mediated sulfate-reducing reactor via N₂ sparging efficiently treated metal-loaded soil leachate. With a sulfide/metal ratio of 3.0, 88% of Zn, 100% of Cu and 95% of Cd were precipitated, resulting in effluent metal concentrations of 3.5 mg Zn²⁺/L, 0.2 mg Cu²⁺/L and 0.03 mg Cd²⁺/L.

Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the supplemental file.     

Metal Leaching and Reductive Dissolution of Iron from Contaminated Soil and Sediment Samples by Indigenous Bacteria and Bacillus Isolates

The purpose of this study was to leach Cu, Zn, As, and Fe from contaminated soil and sediment samples with indigenous heterotrophic bacteria isolated from the study sites. The sediment contained Fe in the form of goethite and low concentrations of other metals. The soil contained hematite and high concentrations of other metals. The environmental conditions affected the bacterial activity in the metals dissolution. As and Fe were the major metals leached from the sediment sample while a minor fraction of Cu was solubilized. Cu and Zn were the major metals leached from the soil sample while only a minor fraction of Fe was dissolved. As a control, a disinfectant was used for partial inactivation of indigenous bacteria. This treatment had a negative effect on the leaching of Fe, Zn and As from soil and sediment samples, but it increased Cu dissolution from the sediment. Bacterial different dissolution of Fe during soil and sediment bioleaching was also investigated with ferrihydrite. The iron concentration was much higher during ferrihydrite dissolution when indigenous bacteria from sediment were used compared to indigenous bacteria isolated from soil. The indigenous bacterial inoculum provided more biological and metabolic diversity which may account for the difference in reductive iron reduction from ferrihydrite. The Bacillus cultures isolated from soil and sediment samples showed similar efficiencies in reductive dissolution of ferrihydrite. The synergetic bacterial inhibition effect created by the environmental conditions can influence bioremediation effect.     

Novel Hyper Antimony-Oxidizing Bacteria Isolated from Contaminated Mine Soils in China

Antimony (Sb)-oxidizing bacteria play an important role in environmental Sb bioremediation because of their ability to convert the more toxic Sb(III) to the less toxic Sb(V). So far, the information about the Sb(III)-oxidizing bacteria species is still limited. In this study, three highly Sb(III)-resistant bacterial strains were isolated from contaminated mine soils after aerobic enrichment culturing with Sb(III) (1 mM). The morphological, biochemical, and 16S rRNA gene sequencing analysis suggested that the three novel bacterial isolates fell within Cupriavidus, Moraxella, and Bacillus, respectively. Among the strains, Moraxella sp. S2 isolated from soils with the highest Sb content exhibited the highest minimum inhibitory concentration for Sb(III) but the lowest Sb(III) oxidation efficiency, which could not completely oxidize 50 μM Sb(III) in 15 days. Cupriavidus sp. S1 was able to oxidize 50 μM Sb(III) completely in 12 days, but could not oxidize 100 μM Sb(III) even with extended time of incubation, while Bacillus sp. S3 with the lowest resistance to Sb(III) could aerobically oxidize 100 µM Sb(III) within 2 days, showing high Sb(III) oxidation efficiency. Our research demonstrated that indigenous microorganisms associated with Sb mine soils were capable of Sb oxidation, and the novel bacteria isolated could represent good candidates for Sb remediation in heavily polluted sites.     

Removal of Heavy Metals from Contaminated Soil and Sediments Using the Biosurfactant Surfactin

The feasibility of using a biodegradable surfactant, surfactin from Bacillus subtilis, for the removal of heavy metals from a contaminated soil (890?mg/kg zinc, 420?mg/kg copper, 12.6% oil and grease) and sediments (110?mg/kg copper, 3300?mg/kg zinc) was evaluated. Results showed that after one and five batch washings of the soil, 25 and 70% of the copper, 6 and 25% of the zinc, and 5 and 15% of the cadmium could be removed by 0.1% surfactin with 1% NaOH, respectively. From the sediment, 15% of the copper and 6% of the zinc could be removed after a single washing with 0.25% surfactin/1% NaOH. The geochemical speciation of the heavy metals among the exchangeable, oxide, carbonate, organic, and residual fractions was determined by selective sequential extraction procedure. For both matrices, the exchangeable fractions were minimal, while the carbonate and the oxide fractions accounted for over 90% of the zinc present and the organic fraction constituted over 70% of the copper. Results after washing indicated that surfactin with NaOH could remove copper from the organic fraction, zinc from the oxide, and cadmium from the carbonate fractions. The residual fraction remained untouched. These experiments indicate that the sequential extraction studies could be useful in designing soil-washing procedures.     

Evaluation of critical nutritional parameters and their significance in the production of rhamnolipid biosurfactants from Pseudomonas aeruginosa BS‐161R

Eleven biosurfactant producing bacteria were isolated from different petroleum‐contaminated soil and sludge samples. Among these 11 isolates, two were identified as promising, as they reduced the surface tension of culture medium to values below 27 mN m^?1. Besides biosurfactant production property, they exhibited good flocculating activity. Microbacterium sp. was identified as a new addition to the list of biosurfactant and bioflocculant‐producers. Optimization of various conditions for rhamnolipid production was carried out for one of the promising isolate, Pseudomonas aeruginosa BS‐161R. Bioglycerol (2.5%), as a cheap renewable carbon source, attained better rhamnolipid yield, while sodium nitrate appeared to be the preferable nitrogen source. The optimum carbon to nitrogen (C/N) and carbon to iron (C/Fe) ratios achieved were 15 and 28,350, respectively, which favored rhamnolipid production. Physical parameters like pH, temperature, and agitation speed also affected the production of rhamnolipids. Results from shake flask optimization indicated that the concentration of bioglycerol, sodium nitrate, and iron were the most significant factors affecting rhamnolipid production, which was supported by the results of central composite rotatable design. After optimization of the culture conditions, the production of rhamnolipids increased by ninefold from 0.369 to 3.312 g L^?1. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Removal of Nonvolatile Hydrophobic Compounds from Artificially and Naturally Contaminated Soils by Column Flotation

Removal of a nonvolatile paraffin oil from spiked soils using column flotation with countercurrent bubbles was explored at both ambient and elevated temperatures. Up to 80% of the contaminant was separated from the coarse fraction (250 to 800?µm) by flotation at 45°C using aqueous solutions of anionic and nonionic surfactants or alkali salt as collectors. With the 75 to 800?µm fraction, removal efficiencies of up to 65% was achieved. Sodium dodecyl-sulfate and Triton 100X at 50?ppm concentrations as well as sodium carbonate at pH 10 were found to yield similar removal efficiencies. Same surfactants were tested in soil washing experiments at similar and higher dosages. Removal efficiency by flotation was higher than those obtained by soil washing in all cases. In addition, as high surfactant dosage are not used in flotation, unlike in the case of soil washing, the problem of formation of stable emulsions was absent. Experiments with soil polluted by hydrocarbons from a contaminated site demonstrated the feasibility of the flotation process for decontamination of coarse (250 to 830?µm) fractions. A 70% reduction of petroleum hydrocarbon in soil was achieved as a result of flotation at 45°C using the above surfactants.     

Simultaneous and Repetitious Removal of 2,4-Dichlorophenol and Copper from Soils Using an Aqueous Solution of Ethyl-Lactate-Amended EDDS

Heavy metals and the transformation products of herbicides, such as 2,4-dichlorophenol (2,4-DCP), are toxic soil pollutants. We assessed the ability of an aqueous solution of the “green solvent” ethyl lactate alone and combined with [S,S]-ethylenediaminedisuccinic acid (EDDS) to remove 2,4-DCP and copper simultaneously from soils. Ethyl lactate extracted 2,4-DCP from contaminated soil comparable to Triton X-100. Ethyl lactate/EDDS extracted more 2,4-DCP and Cu from contaminated soils than ethyl lactate alone. The enhanced extraction of Cu increased slightly with an increase in the EDDS/Cu molar ratio; the maximum Cu extraction efficiency was about 32.3% at an EDDS/Cu ratio of 5. An increase in the ionic strength (NaCl) of the ethyl lactate/EDDS solution decreased the amount of 2,4-DCP extracted by maximally 12% but increased the amount of Cu extracted by >500%. We tested the recycling of the ethyl lactate/EDDS solution with the cation-exchange resin 001×7 and the hyper-cross-linked polymer resin NDA-150. Fresh ethyl lactate/EDDS solution and two sequentially recycled solutions removed 31.4, 28.3, and 26.7% of the Cu in Cu-contaminated soil and 77.7, 62.9, and 56.8% of the 2,4-DCP in 2,4-DCP-contaminated soils, respectively. The ethyl lactate/EDDS solution removed 31.8% of the Cu and 73.0% of the 2,4-DCP in Cu- and 2,4-DCP-contaminated soils, and the solution remained effective even after two recyclings. The aqueous solution of ethyl lactate/EDDS can be used to effectively remove Cu and 2,4-DCP from complex contaminated soils and can be reactivated.     

Isolation and Characterization of Biosurfactant-Producing Bacteria from Oil-Contaminated Soil

Two biosurfactant-producing Pseudomonas aeruginosa strains (KISR C1 and KISR B1) were isolated from Kuwaiti oil-contaminated soil, which resulted from the Gulf War. The optimum environmental conditions that supported the growth and surfactant production of both isolates were examined. The two isolates differed in their biosurfactant-stimu-lating carbon source, nitrogen concentration, and the pH of the medium. C-1 isolate produced two types of rhamnolipids with a final concentration of 98.4?g/l after spiking the nitrogen-limited medium with 10?mg/ml olive oil. The other isolate (B-1) produced only one type of rhamnolipid (5.9?g/l) after spiking the medium with crude oil. The biosurfactant produced by this strain was found to be very effective in the emulsifica-tion of crude oil. The result suggests that this isolate can potentially be used to enhance bioremediation of oil-contamination and enhanced oil recovery.     

Comparison of Extractants for Removing Heavy Metals from Contaminated Clayey Soils

This article describes the removal of heavy metals from contaminated clayey soils by soil washing using various extractants. Two clayey soils, kaolin, a low buffering soil with pH of 5, and glacial till, a high buffering soil with pH of 8, were used to represent various soil conditions. These soils were spiked with chromium (Cr), nickel (Ni), and cadmium (Cd) to simulate improper disposal of typical electroplating waste constituents. The following extracting solutions were investigated for the removal of heavy metals from the soils: deionized water, distilled water, and tap water; acetic acid and phosphoric acid; chelating agents ethylenediaminetetraacetic acid (EDTA) and citric acid; and the oxidizing agents potassium permanganate and hydrogen peroxide. The effect of extractant concentration on removal of heavy metals was also investigated. Complete removal of Cr was achieved using 0.1?M potassium permanganate for kaolin, while a maximum of 54% was removed from glacial till. A maximum Ni removal of 80% was achieved using tapwater for kaolin, while a maximum removal of 48 to 52% was achieved using either 1?M acetic acid or 0.1?M citric acid for glacial till. A maximum Cd removal of 50% was achieved using any of the extractants for kaolin, while a maximum removal of 45 to 48% was obtained using either acids or chelating agents for glacial till. Overall, this study showed that complete removal of Cr, Ni, and Cd from clayey soils is difficult to achieve using the soil-washing process, and also the use of one extractant may not be effective in removing all metals. A sequential extraction using different extractants may be needed for the removal of multiple metal contaminants from clayey soils.     

Pulsed Electrokinetic Removal of Chromium,Mercury and Cadmium from Contaminated Mixed Clay Soils

Electrokinetic remediation (EKR) processes are energy intense systems as they are mainly run under continuous constant current supply mode. In this study, pulsed electrokinetic remediation (PEKR) technique was employed for the removal of Cd, Hg and Cr from mixed contaminated natural clay and bentonite soils. The effects of voltage gradient, pulse duty cycle and bentonite/clay ratio on the simultaneous removal efficiencies of the heavy metals and specific energy consumption were investigated. Fifteen (15) PEKR experiments were conducted according to Box–Behnken design (BBD) with each experiment allowed to continuously run for 21 days. Increase in the proportion of the bentonite significantly decreased the removal efficiency of the heavy metals while having insignificant effect on the energy consumption. Conversely, increase in both voltage gradient and pulse duty cycle increased the heavy metals removal efficiencies, though at the expense of increase in energy consumption due to combine effects of increase in the soil electrical conductivity, amount of current needed to sustain the applied voltage gradient as well as the raise in the soil pH. The maximum achievable removal efficiencies for Cd, Hg and Cr were 21.87, 78.06 and 89.64% respectively. The specific energy consumption significantly increased from the range of 91.67–154.17 kwh/m³ to 1700–2441.67 kwh/m³ as a result of combined effect of increasing voltage gradient and pulse duty cycle. This demonstrated that effective PEKR could be achieved with significance reduction in the energy consumption via appropriate selection of pulse duty and voltage gradient for clay soils of different proportion of montmorillonite.     

Continuous rhamnolipid production with integrated product removal by foam fractionation and magnetic separation of immobilized Pseudomonas aeruginosa

Increasing interest in biological surfactants has led to intensified research directed at more cost-efficient production of biosurfactants, relative to traditional surface-active components based on petrochemical feedstocks. This publication will focus on a new integrated process for continuous rhamnolipid (RL) production. RL was synthesized by Pseudomonas aeruginosa DSM 2874 and was continuously removed in situ by foam fractionation. To prevent loss of the biocatalyst through foaming, bacteria were entrapped in magnetic alginate beads. Immobilizates were retained from the foam by high-gradient magnetic separation and back-flushed in the bioreactor at constant intervals. It was demonstrated that continuous RL production in a 10-L bioreactor over several cycles with intermediate growth periods is feasible. Complete separation of RLs from the production medium with an average enrichment ratio of 15 in the collapsed foam was demonstrated, yielding a final RL amount of 70 g after four production cycles.     

Molecular Characterization of Endophytic Bacteria from Metal Hyperaccumulator Aquatic Plant (Eichhornia crassipes) and Its Role in Heavy Metal Removal

In this study, among a collection of heavy metals resistant endophytic bacterial strains isolated from aquatic hyperaccumulator plant (Eichhornia crassipes), one plant growth promoting endophytic bacteria (PGPE), SVUB4 was selected for its ability to utilize 1-aminocyclopropane-1-carboxylic acid (ACC) as the sole N source and accumulate different heavy metals. The SVUB4 strain was characterized as Enterobacter sp. on the basis of its 16S rDNA sequences. Assessment of the parameters of plant growth promotion revealed the intrinsic ability of the strain for the production of IAA, siderophore and solubilization of insoluble phosphate. Furthermore, plasmid DNA analysis of Enterobacter sp. strain SVUB4 indicated the presence of a single large plasmid element. The results of plasmid curing experiments demonstrated that the ability of this strain to grow in presence of Cd and Zn was encoded by the 98 kb plasmid, whereas the ability to grow in the presence of Pb appeared to be encoded by the chromosome. The Cd and Zn removal capacity of the respective metal sensitive strain (plasmidless) were about 36 and 45 μg/g-1 DW, respectively, while the removal capacity of the both metal by metal resistant strain (p SVUB4) showed a significantly higher Cd and Zn removal capacity of 153 and 228 μg/g^?1 DW, respectively. However, both strains exhibited a similar pattern of Pb accumulation. The present observation also showed that for wild-type strain SVUB4 (pSVUB4), the overall level of IAA production in the absence and in the presence of Cd²⁺ or Zn²⁺was approximately the same. Nevertheless, strain SVUB4M in this respect appeared to be more sensitive to heavy metals: a noticeable decrease in IAA production was observed under the effect of both metals, especially with Cd²⁺.     

Metal Distribution and Characterization of Cultivable Lead-Resistant Bacteria in Shooting Range Soils

Shooting ranges represent about 10% of polluted sites in Switzerland. This pollution, mainly consisting of lead (Pb) and antimony (Sb), can spread into the local groundwater, wildlife and plants. In this study, shooting range stop butt soils (elevated soil mount behind the target) of three sites (Versoix, Cartigny and Nyon, Western Switzerland) were investigated for metal contamination and culturable lead-resistant bacteria in contaminated soils. The inductively coupled plasma mass spectrometry analysis of surface stop butt soils (0–5 cm) indicated high metal concentrations especially for Pb and Sb, e.g., the Pb concentration ranging from 61,135 to 395,651, 23,821 to 201,268 and 120 to 27,517 mg kg^?1 in the soil from the sites of Versoix, Cartigny and Nyon, respectively. Molecular analysis of 16S rRNA gene demonstrates the presence of bacteria from diverse classes: Flavobacteriia, α-Proteobacteria, γ-Proteobacteria, Actinobacteria and Bacilli that exhibited high Pb minimum inhibitory concentration value from 1,036 to 2,694 mg kg^?1. The isolated strains could be the subject of further studies to evaluate their role in bioremediation of lead-contaminated soil.     

The Removal of Pb and Cd from Heavily Contaminated Soil in Kayseri,Turkey by a Combined Process of Soil Washing and Electrodeposition

In this study, a combined system of soil washing and electrodeposition was designed to remove Pb (16381±643 mg/kg) and Cd (34347±1310 mg/kg) from contaminated soil. 0.05 M Na₂EDTA was used as a chelating agent for the remediation of soil, taken from the nearby city Kayseri, Turkey. As a result of the batch extraction tests, maximum removals were determined as; at the 20:1 liquid: soil ratio for Pb is 60.7%, for Cd at the 30:1 liquid: soil ratio is 67.4%. An electrochemical treatment was applied to the waste washing solution which appeared to be the second pollutant after the Na₂EDTA extraction from the soil. With extraction tests of Pb and Cd, being transformed from the solid phase to the liquid phase. The electrochemical treatment (electrodeposition), performed in three different potential (6 V, 8 V and 10 V) and maximum removal efficiencies, were found 99.7% and 80.3% at 10 V for Pb and Cd, respectively.

Speciation tests (BCR) were carried out, both before and after the soil washing process, to evaluate the redistribution of metal fraction in the soil. The fraction, associated with the organic substance, was found as 10.67% for Pb and 1.81% for Cd. The metal bioavailability factor increased after soil washing, which indicates that EDTA could enhance the mobility of Pb and Cd.     

Remediation of Copper from Copper Mine Wastes and Contaminated Soils Using (S,S)-Ethylenediaminedisuccinic Acid and Acidophilic Bacteria

The biodegradable chelating agent (S,S)-Ehylenediaminedisuccinic acid (EDDS), autochthonous acidophilic bacteria, and a combination of the two means were investigated for the removal of pseudo-total and ethylenediaminetetraacetic acid (EDTA)-available content of Cu from surface layers of three soil categories in the Bor copper mining area. Their efficiencies were compared at mine overburden, flotation tailings, and agricultural land sites in order to determine the potential role of these approaches in the soil remediation process. The most effective removal of Cu was achieved on flotation tailings, where combined treatment showed significant reduction of pseudo-total and EDTA-available concentrations of Cu (40.5?±?27.3% and 99.6?±?0.2%, respectively). Acidophilic bacteria treatment showed high efficiency on flotation tailings, removing 94.1?±?1.2% of EDTA-available Cu. EDDS treatment showed discernible results in the removal of EDTA-available Cu from agricultural land soil (44.4?±?13.9%). In the case of overburden soil material, selected agents did not have statistically significant results in the removal of pseudo-total or EDTA-available fraction of Cu. Chosen remediation approaches showed diverse efficiency for soil categories on investigated sites. Combined approach showed synergistic results in the case of EDTA-available Cu removal from flotation tailings soils, suggesting that this combination deserves further attention as a potentially promising environmentally friendly remediation option.     

Application of Box-Behnken Design to Hybrid Electrokinetic-Adsorption Removal of Mercury from Contaminated Saline-Sodic Clay Soil

A hybrid electrokinetic-adsorption (HEKA) technique using uniform electric field and granular activated carbon (GAC) produced from date palm pits was investigated for the removal of mercury from natural saline-sodic clay heavily contaminated with heavy metals, phenol, and kerosene. Response surface methodology (RSM) was employed to model, optimize, and interpret the results obtained with the aid of Design Expert software. According to the Box-Behnken experimental design, 15 experiments were conducted each with residence time of three weeks. The effects of voltage gradient (0.2–1 V/cm), initial Hg concentration (mg/Kg), and polarity reversal interval (0-48 hours) on Hg removal efficiency and energy consumed for Hg removal were investigated. Respectively, the responses fitted reduced cubic (R² = 99.3%) and quadratic models (R² = 92.3%) with the overall relative contributions of the investigated parameters on the responses following the order: voltage gradient > initial Hg concentration > polarity reversal interval based on analysis of variance (ANOVA). The optimal conditions obtained with desirability of 90% aimed at maximizing Hg removal were 24 hours polarity reversal interval, 0.2 V/cm voltage gradient, and 100 mg/kg initial Hg concentration. This optimum operating condition yielded good removal of Hg (99.5%) at reduced energy consumption of 50.1kWh.m^?3mg^?1. Experimental validation of the models showed good prediction of Hg removal efficiency (0.0368% prediction error). The results presented herein suggest that HEKA technology could be utilized effectively for the removal of Hg from contaminated, low permeable soils under extreme soil and contamination conditions.     

蛋壳内膜分解菌所产蛋白酶的纯化及其N-末端氨基酸序列测定

从土壤中分离得到的1株产蛋壳内膜分解酶(ESM protease)的铜绿假单胞菌(Pseudomonasaeruginosa).通过对其发酵液进行饱和硫酸铵盐析,二次离子交换层析得到蛋壳内膜分解活性达到304.5 U/mg的目标蛋白,SDS-PAGE电泳显示该酶分子量约为32 kD,通过测定其N-末端15个氨基酸残基为:Ala、Glu、Ala、Gly、Gly、Val、Ala、Gly、Lys、Glu、Asp、Ala、Ala、Glu和Leu.     

The Co-application of Willow and Earthworms/Horseradish for Removal of Pentachlorophenol from Contaminated Soils

Pentachlorophenol (PCP) was once commonly used as a pesticide worldwide, and is now a toxic and recalcitrant environmental pollutant. To explore a practical approach in the remediation of PCP-contaminated soils in China, we evaluated the efficacy of a local willow (Salix × aureo-pendula CL “J1011”) on removing PCP and assessed the potential of a native earthworm (Metaphire guillelmi) and horseradish (Armoracia rusticana Gaerth), alone and in combination with willow, to enhance the efficiency of removing PCP from spiked field soils. Willow, horseradish, and earthworms alone significantly increased PCP removal from soil. After 45 days, only 47.2% of the PCP remained in the presence of willow alone; 68.4, 51.4, and 46.3% of the PCP remained with 50, 100, and 200 g horseradish m^?2, respectively; and 41.1% of the PCP remained in the presence of earthworms. The removal of PCP from soils significantly increased with co-application of willow and horseradish (23.1% remaining) and especially with the co-application of willow and earthworms (2.2% remaining). Our results indicate that in terms of remediation efficiency, economic value, ecological benefits, and ease of use, a system consisting of this autochthonous willow and indigenous earthworms has great potential for the remediation of PCP-polluted soil in China.     

Combined treatment of Pseudomonas aeruginosa biofilms with bacteriophages and chlorine

Bacterial biofilms are a growing concern in a broad range of areas. In this study, a mixture of RNA bacteriophages isolated from municipal wastewater was used to control and remove biofilms. At the concentrations of 400 and 4 × 10⁷ PFU/mL, the phages inhibited Pseudomonas aeruginosa biofilm formation by 45 ± 15% and 73 ± 8%, respectively. At the concentrations of 6,000 and 6 × 10⁷ PFU/mL, the phages removed 45 ± 9% and 75 ± 5% of pre‐existing P. aeruginosa biofilms, respectively. Chlorine reduced biofilm growth by 86 ± 3% at the concentration of 210 mg/L, but it did not remove pre‐existing biofilms. However, a combination of phages (3 × 10⁷ PFU/mL) and chlorine at this concentration reduced biofilm growth by 94 ± 2% and removed 88 ± 6% of existing biofilms. In a continuous flow system with continued biofilm growth, a combination of phages (a one‐time treatment at the concentration of 1.9 × 10⁸ PFU/mL for 1 h first) with chlorine removed 97 ± 1% of biofilms after Day 5 while phage and chlorine treatment alone removed 89 ± 1% and 40 ± 5%, respectively. For existing biofilms, a combined use of a lower phage concentration (3.8 × 10⁵ PFU/mL) and chlorination with a shorter time duration (12 h) followed by continuous water flushing removed 96 ± 1% of biofilms in less than 2 days. Laser scanning confocal microscopy supplemented with electron microscopy indicated that the combination treatment resulted in biofilms with lowest cell density and viability. These results suggest that the combination treatment of phages and chlorine is a promising method to control and remove bacterial biofilms from various surfaces. Biotechnol. Bioeng. 2013; 110: 286–295. © 2012 Wiley Periodicals, Inc.