Carbon Dioxide as a Microbial Toxicity Enhancer of Some Antibacterial Agents: A New Potential Water Purification Tool

The aim of current paper was to investigate the possibility of increasing the toxicity of calcium hypochlorite (Ca(ClO)2) and hydrogen peroxide (H2O2) on Escherichia coli K-12 by preliminary enrichment of culture media by carbon dioxide (CO2). For this purpose, the microbes sensitivity to H2O2 or/and Ca(ClO)2 at normal and CO2-enriched medium was studied by spectrophotometric, radioisotopic, and electronmicroscopic methods. Ten-minute preincubation in CO2-enriched medium enhanced the toxic effect of both H2O2 or/and Ca(ClO)2 on bacteria as a result of induced growth inhibition, compared to noCO2 enriched group. Additionally, changes in cell morphology and proliferation were observed. It was demonstrated that the preliminary incubation of microbes in CO2-enriched culture media in nonsupercritical concentration elevate the toxic effect of H2O2 or/and Ca(ClO)2 on microbes. This can serve as a novel, effective, inexpensive, and environmentally friendly approach for water purification from bacteria, further improving the protection of the environment and human health.


Introduction
One of the global challenges of the 21st century is to find the best method for cleanup of aquatic ecosystems from microbes, which will be cheap, safe for human health, and environmentally friendly [1,2]. Chlorination is one of the most widely used processes for microbial control [3] in both drinking water and wastewater processing [4]. Chlorine is a powerful antimicrobial substance due to its potential oxidizing capacity [3]. In general, disinfectants like chlorine are used in very high concentrations for the purpose of attaining a rapid rate of killing. It is difficult for microorganisms to survive in such concentrations. However, the use of the chlorine increases the risk of the formation of potentially hazardous by-products or the production of foul tastes and odors, which are the main drawbacks of chlorination [5]. Conversely, at low chlorine levels, microorganisms that survive the treatment may be injured rather than inactivated [6]. Under suitable conditions, injured cells might repair cellular damage and recover [7]. An appreciation of the nature of sublethal injury and repair is, therefore, important in devising chlorination strategies and in developing combination treatments with synergistic actions against the target microorganisms. The mechanisms of action of chlorine on microorganisms have been widely investigated [8]. Nevertheless, the mechanism by which chlorine exerts its lethal effect has never been fully elucidated.
Currently, H 2 O 2 and supercritical carbon dioxide (SCCO 2 ) are being used as tools for water purification but separately. While the molecular mechanism of "killing" effect of H 2 O 2 on bacteria is well documented [9], the mechanism of "killing" effect of CO 2 on bacteria still remains discussable [10]. Although it is already documented that the combination of SCCO 2 and H 2 O 2 inactivates spores to high degree [11], the potential mechanism by which this inactivation takes place is still unknown. Moreover, this combination of SCCO 2 and H 2 O 2 has not yet been used as a water purification tool.
Our previous investigations showed 2 functionally different states of membrane proteins, active and inactive (reserve) 2 ISRN Biophysics in eukaryotic cells [12]. The ratio of these active and inactive molecules could be changed by the modulation of cell hydration [12][13][14]. It was also shown, that metabolic poisons cause cell hydration [15] resulting in the increase of the number of functionally active receptors in the membrane [12]. On the one hand, CO 2 , as a strong metabolic poison, that leads to the increase of cell hydration, could serve as a potential factor able to elevate membrane sensitivity to H 2 O 2 or/and Ca(ClO) 2 of microbes. Thus, it was suggested that CO 2 could elevate the toxic effect of H 2 O 2 or/and Ca(ClO) 2 on cell.
Therefore, the use of the activation of microbial metabolism induced by comparatively low concentrations of CO 2 to enhance the toxic effect of H 2 O 2 or/and Ca(ClO) 2 on microbes was checked. This fact and data obtained from our published patents [16,17] suggest that a certain combination of both of "killer" molecules may allow the development of a novel and safe method for water purification.

Experimental Setup and Apparatus.
The processing apparatus used in this work contained 50 liter CO 2 vacuum tank, the airline tubing with regulator valve and the CellStar CO 2 incubator (USA, Model: SWJ500TV BA). The test tubes containing 100 mL bacterial culture were placed in the CellStar incubator at 20 • C. Carbon dioxide (air liquid, 99.7% purity) was injected into the individual test tubes in the incubator at the selected pressure (atmospheric pressure) for 10 min. The corresponding non-CO 2 controls were made the same way, except that the test tubes were not flushed by carbon dioxide. After 10 min of incubation, the H 2 O 2 and/or Ca(ClO) 2 in corresponding concentrations was added to the tubes, and then samples were closed and removed to thermostat at 37 • C for 18 h for the future processing.

Bacterial
Strain, Culture Media, Chemicals, and Estimation of the Bacterial Growth. Wild-type Escherichia coli K-12 (obtained from the Armenia Collection of Microorganisms (Institute of Microbiology of the Center of Microbiology and Microbial Depository of Armenian NAS, Yerevan)) was used for experiments. Prior to each test, the bacterial cultures (previously stored at 4 • C in nutrient-enriched agar slants (NEA, containing: nutrient enrich broth (NEB) + 1.5% agar (Difco), final pH 7.1 ± 0.2 at 37 • C) were grown in nutrientenriched broth (NEB, containing: peptone 15 g/L, sodium chloride 6.0 g/L, yeast extracts 3.0 g/L, final pH 7.5 ± 0.2 at 37 • C), at 37 • C in thermostat overnight (18 h) and used for the treatments.
For H 2 O 2 and Ca(ClO) 2 treatments, fresh hydrogen peroxide and calcium hypochlorite solutions were prepared immediately before the experiments and added to the bacterial cultures in the corresponding concentrations of the substances in aqua media. Microbial growth was evaluated spectrophotometrically at a wavelength of 600 nm using a CΦ-46 LOMO spectrophotometer (Russian Federation) for 18 h.

Electronmicroscopy
Assay. The damage of microbial cells was visualized by electron-microscopy. Negative contrasting was performed using 2% phosphoric-tungsten acid solution at pH 6.8-7.0. Microscopic preparations of bacterial suspensions were made after the centrifugation of bacterial suspension at 3000 rpm (CLR-1) for 10 min, then bacterial sediment was fixed by 2.5% solution of glutaraldehyde in 0.1 M cacodylate buffer under pH 7.4 during 2 h under room temperature. After three-phase washing in 0.1 M cacodylate buffer, the cells were fixed under room temperature by 1% of solution tetroxid osmium in 0.1 M cacodylate buffer under pH 7.4 during not less than 1 h. After washing in the same buffer, dehydration of sample was conducted in the ethanol increasing concentrations for 30, 50, 70, 96 and 100% or acetone increasing concentrations and potting by mixture of Araldite on Lufft. Then the bacterial sediment was polymerized in thermostat at 37 • C and 59 • C in Araldite preparations. Ultrathin sections were prepared using the ultra cut "Reichert" "Ultracut-Young" and stained in the uranyl acetat and lead citrate. The sections were checked by electronic microscope Tesla BS-500 (Czechoslovakia). The photomicrographies were scanned in permit of 900 pixels for inch and processed on computer programme Corel Draw versions 11 and Photoshop versions 8. Computer morphometric and stereometric analysis of electron microscopic imaging was performed according to the program "Video Test Structure-5, nanotechnology."

Radioactive Labeled [ 3 H]-Thymidine-Based Cell Proliferation Assay.
For the measurement of the cell proliferation, the bacteria cultures were grown in 100 mL of NEB at 37 • C overnight. One mL of overnight culture (10 10 cells H]-thymidine uptake was stopped by precipitation of each portion with ice-cold 10% trichloroacetic acid (TCA). The cells were harvested by centrifugation for 10 min at 5000 g and washed 2 times by TCA and then again harvested by centrifugation. Then 3 mL of Bray's scintillation cocktail was added to each sample, and the radioactivity was measured by using a Wallac 1450 liquid scintillation counter.

Statistical
Analysis. Statistical analyses were conducted by SPSS 17 software. The differences among the means of treatment were tested by using the paired sample t-test. All experiments were repeated at least three times. Values are shown as means ± std. error.  checked the potential of elevation of the toxic effect of comparatively low doses of H 2 O 2 on microbes by preliminary increase of the metabolic activity of microbes using preincubation of the latter in the aqua medium enriched with CO 2 (10 min). Since CO 2 dissolves in aqueous solutions, forming an acid and lower the pH, to avoid the additional effects of these, the pH was adjusted to the constant 6.0, supporting the maximal growth of E. coli [20] by addition of NaOH (0.1 mol/L) during CO 2 treatment.

Effect of the Antibacterial Properties of CO
The toxic effect of low concentrations of H 2 O 2 on E. coli K-12 was increased in cell growth medium enriched with CO 2 for 10 min (maximum) (Figure 1, (•)), compared to non-CO 2 enriched medium (Figure 1, ( )).

Effect of the Antibacterial Properties of CO 2 /Ca(ClO) 2
Combination on the Growth of E. coli K-12. The response of E. coli to chlorine highly depends on its concentration [21,22]. As the aim of present work is to find out the possibility of the modulation of the effect of CO 2 on the toxic effect of relatively low doses of chlorine on microbes, the dose-dependent effect of Ca(ClO) 2 (mg/L) on the growth of E. coli K-12 in normal and after 10 min incubation microbes in CO 2 -enriched culture medium was studied (Figure 2).
Preliminary treatment by CO 2 enhanced the "killing effect of Ca(ClO) 2 on the bacteria observed in all checked Ca(ClO) 2 concentrations. However, this effect was more pronounced at low concentration of Ca(ClO) 2 (>0.1 mg/L) and decreased parallel to the concentration increase up to constant level, about 15%. These data suggests the existence of the minimum two pathways through which the CO 2 could potentiate the toxic effect of chlorine. The observed CO 2induced enhancement of Ca(ClO) 2 toxicity at low doses can be explained by CO 2 -induced stimulation of oxidative metabolisms, which is a target for poisoning effect of Ca(ClO) 2 . Probably the CO 2 -induced poisoning effect of Ca(ClO) 2 is a reason for the increase of the number of cellular targets (receptors) for Ca(ClO) 2 attack. Our previous work showed that cell swelling, probably the result of cell metabolism poisoning, could serve as one of such pathways [13]. Therefore, according to the fact that CO 2 is metabolic poison, there is theoretical possibility that CO 2 -induced cell swelling could serve as a mechanism responsible for the increase of microbes' sensitivity to Ca(ClO) 2 .

Electronmicroscopic Study of CO 2 , H 2 O 2 , and Ca(ClO) 2
Effects on E. coli K-12. Electron-microscopic study of the untreated E. coli K-12 culture showed that cells have an ultrastructure, typical for gram-negative bacteria (Figures  3(a) and 3(b)). Ultrastructural analysis of E. coli K-12 has showed that effect of different biocides, such as CO 2 , H 2 O 2 , or/and Ca(ClO) 2 , leads to the ultrastructural changes in membranes, cytoplasm, and nucleotide. Treatment by CO 2 leads to the destruction of cellular wall and the removal of external membrane of wall of bacteria (Figures 4(a), 4(b), 5(a), 5(b), and 5(c)). An addition to the similar effect of H 2 O 2 and its derivatives, for example, removal of external membrane of wall, increase of the transparency of citoplasmatic membrane was also demonstrated ( Figures  6(a) and 6(b)). The treatment of E. coli K-12 by Ca(ClO) 2 destructs the cellular wall, which leaves from citoplasmatic membrane. Moreover, the cytoplasm had small granular structures (Figures 7(a) and 7(b)). control group. H 2 O 2 alone inhibited the proliferation by 27% and its combination with CO 2 (CO 2 /H 2 O 2 ) showed the proliferation decrease by 68%, Ca(ClO) 2 inhibited the proliferation by 19%, but the combined effect of CO 2 /Ca(ClO) 2 couple showed higher inhibition of 30% inhibition effect ( Figure 8).

Discussion
The search for novel alternatives of water disinfection is of immense interest, and much effort is concentrated to address this issue. The current paper suggests a novel tool for enhancement of the efficiency of the two most common disinfecting agents, such as H 2 O 2 and Ca(ClO) 2 by combing them with CO 2 .
Our data clearly showed that CO 2 increased the toxic effect of H 2 O 2 and/or Ca(ClO) 2 on microbes, and it could be used as a promising tool for water purification.
As the toxic effect of H 2 O 2 [19] and Ca(ClO) 2 [23] is realized by its oxidative properties, consequently, their inhibition effects depend on the initial metabolic activity of microbes. It is suggested that the factor having stimulatory effect on cell metabolism could enhance the toxic effect of H 2 O 2 and/or Ca(ClO) 2 on microbes.
On the basis of the fact that CO 2 at comparatively low concentrations [10] has strong stimulatory effect on cell metabolism, it could be suggested that cells pretreated by CO 2 could have high sensitivity to toxic effect of H 2 O 2 ( Figure 1) and Ca(ClO) 2 ( Figure 2). It should be mentioned that stimulation of growth occurs because some anabolic reactions involve CO 2 fixation, and in the absence of an external source of the gas, CO 2 concentration in cell can be rate limiting for these reactions, with resultant decreased growth rates [10]. The basis of CO 2 inhibition has not been clearly established. Carbon dioxide has a dual physiological role in microorganisms since it can both stimulate and inhibit cell development [24]. Various hypotheses have been proposed to explain the microbicidal activity of carbon dioxide. CO 2 dissolves in water to form carbonic acid. Thus, dissolved CO 2 acts by lowering the pH of the medium, and the resulting acidity leads to a disturbance of some biological systems within cells. It was, therefore, suggested that microbial inhibition was due to an alteration in the properties of cell (membrane, cytoplasm, enzymes, etc.) [25]. However, the reduction in the pH of the medium is not sufficient to account for the antimicrobial action of CO 2 , since it shows a specific inhibitory effect, which is greater than that of the other acids used to lower medium acidity (hydrochloric acid, phosphoric acid, etc.). These acids do not penetrate the microbial cells as easily as carbon dioxide [26]. Therefore, it was suggested that the comparatively low concentration of CO 2 -induced activation of the metabolism of microbes could serve as a convenient method for the increase of the sensitivity of microbes to the toxic effect of low concentration of H 2 O 2 and/or Ca(ClO) 2 .
The data obtained in the current study demonstrated that the toxic effect of H 2 O 2 and/or Ca(ClO) 2 on E. coli K-12 increased significantly when cell bathing medium was enriched with CO 2 10 min beforehand. The combination effect of CO 2 /H 2 O 2 and CO 2 /Ca(ClO) 2 was higher than in case of CO 2 , H 2 O 2 , and Ca(ClO) 2 treatments separately. It is also important to mention that combined inhabitation effect of CO 2 /H 2 O 2 (68%) is more higher than combined inhibition effect of CO 2 /Ca(ClO) 2 (30%).
Although our data did not allow us to make final conclusion on the nature of the mechanism through which the CO 2 poisoning metabolism could increase cell membrane sensitivity to H 2 O 2 and/or Ca(ClO) 2 , it was comparable to the reported CO 2 -induced swelling of microbes [27]. This suggests that the CO 2 -induced cell hydration could serve as one of fundamental mechanisms responsible for CO 2 -induced enhancement of the toxic effect of these substances , which can be realized in two ways: increasing the number of membrane chemoreceptors [12] and activating intracellular macromolecules (folding) activity [14].
Additionally, the electronmicroscopic measurement of cell volume in the above-mentioned conditions demonstrated that the cell swelling is not the only mechanism through which the cell inhibition is realized by CO 2 /H 2 O 2 and CO 2 /Ca/ClO) 2 . It is suggested that the ultrastructural visualizations could provide valuable data for correction of 6 ISRN Biophysics cytopathologic characteristics of mechanism of actions of CO 2 , H 2 O 2 , and Ca(ClO) 2 biocides on the bacterial culture. The main conclusion that is drawn from the conducted studies so far is that the noncritical concentration of CO 2 elevate the toxic effect of H 2 O 2 and/or Ca(ClO) 2 on E. coli K-12.
The current paper proves grounds for a new approach for decreasing microbial pollution in water, as well as for better understanding of the fundamental mechanisms controlling the microbial growth inhibition depending on environmental factors. We believe that this work will represent the first systematic investigation of the effect of CO 2 /H 2 O 2 and CO 2 /Ca(ClO) 2 combination at comparatively low concentrations on the reduction of microbial pollution of water. The suggested method will be free from the most negative properties of water purification methods, which are currently in use.