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Biocidal Efficacy of Dissolved Ozone, Formaldehyde and Sodium Hypochlorite Against Total Planktonic Microorganisms in Produced Water



Y.T. Puyate and A. Rim-Rukeh
 
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ABSTRACT

The performance of three biocides (dissolved ozone, formaldehyde and sodium hypochlorite) in eliminating the bacteria and fungi in produced water is investigated experimentally. The analysis involves monitoring the microbial population in nine conical flasks each containing the same volume of a mixture of produced water, culture medium that sustains the growth of microorganisms and a known concentration of biocide. The concentrations of each biocide used in the study are 0.1, 0.2 and 0.5 ppm. It is shown that dissolved ozone exhibits the best biocidal characteristics and a concentration of 0.5 ppm eliminated all the microorganisms in the produced water after 150 min contact time.

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Y.T. Puyate and A. Rim-Rukeh, 2008. Biocidal Efficacy of Dissolved Ozone, Formaldehyde and Sodium Hypochlorite Against Total Planktonic Microorganisms in Produced Water. Journal of Applied Sciences, 8: 860-865.

DOI: 10.3923/jas.2008.860.865

URL: https://scialert.net/abstract/?doi=jas.2008.860.865

INTRODUCTION

Microorganisms such as sulphate reducing bacteria (Desulfovibrio species), sulphide oxidizing bacteria (Thiobacillus ferroxidans), iron bacteria (Gallionella species), nitrogen utilizing bacteria (Pseudomonas species) and filamentous fungi (Cladosporium resina), are detrimental and can be found in closed systems such as fuel storage tanks, heat exchangers, cooling towers and pipelines (Beech and Sunner, 2004). System failures resulting from corrosion influenced by these microorganisms have been widely reported in petrochemical and gas industries (Ibe, 1989; Abu, 1992), nuclear power stations (Videla, 1996), underground pipelines (Bowman, 2003; Rim-Rukeh and Awatefe, 2006), geothermal plants (Pryfogle, 2002), fire protection systems (Mittleman, 2003) and wastewater treatment plants (Iversen, 2001). It is estimated that about 20% of all corrosion damages are caused by microorganisms (Booth, 1971). Damages resulting from microbial corrosion in production, transport and oil storage facilities, amount to hundreds of million US dollars per year in United States of America (Costerton and Boivin, 1991).

Biofilms are slimy, mucous-like substances formed on the surfaces of materials in aquatic environment where nutrients are available for bacteria utilization. Biofilms consist of microbial cells immobilized on the surfaces of a material together with Extra-cellular Polymeric Substances (EPS) which bind the cells together (Characklis and Marshall, 1990; Costerton et al., 1995). Cases of contamination of surgical equipment by biofilms are reported in the literature (de Carvalho, 2007). It is estimated that about 750,000 surgical equipment are contaminated by biofilms annually in United States of America, resulting in more than 1.6 billion US dollars in hospital expenses (Gupta et al., 2002).

Microorganisms exist in the soil and are often found in oil pipelines (EIA, 2005). Strategies such as use of biocides (to counteract the actions of microorganisms), keeping the pipeline clean (pigging) and prevention of direct contact between the metal and its environment, have been employed to prevent biocorrosion of pipelines (Videla, 1996; Guiamet and de Saravia, 2005). Biocides are anti-microbial chemicals that have the potency to kill microorganisms or inhibit their growth and reproductive cycles by poisoning the microbial enzyme and causing protein denaturation, cell leakage and lysis (Gaylarde, 1995). Examples of biocides are chlorine, sodium azide, dissolved ozone, formaldehyde, sodium hypochlorite, chloramine, bromine, hydrogen peroxide, glutaraldehdye, peracetic acids, isothiazolones and quaternary ammonia compounds (Videla, 1996).

Produced water (or formation water) is one that accompanies crude oil and gas from a producing well. It is an integral component of hydrocarbon recovery process and is usually produced during drilling and production phases of a well. Naturally, the crude oil and produced water contain various microorganisms which must be removed to avoid microbial corrosion of internal surfaces of pipes in which the crude oil is transported after separating the oil from the produced water. The produced water obtained from the separation process is discharged into the receiving water body after treatment, which necessitates elimination of the microorganisms in the produced water. Thus, the presence of microorganisms in both the crude oil and produced water is detrimental to the operations of oil companies and the ecosystem. In this paper, we compare the biocidal efficacy of sodium hypochlorite, formaldehyde and dissolved ozone, against total planktonic microorganisms in produced water; the results which may be applied in biocorrosion prevention and control programmes (Total planktonic microorganisms is the total number of free swimming microorganisms in water environment). The choice of these biocides is based on their reported environmental friendliness and stability over a wide range of pH (Videla, 1996).

MATERIALS AND METHODS

Characteristics of biocides used in the study: Sodium hypochlorite (NaOCI) is a pale yellow liquid. In its pure form, sodium hypochlorite is unstable and reacts with water to form hypochlorous acid (HOCI) and sodium hydroxide (NaOH). At a pH value ranging from 7 to 9, hypochlorous acid can be converted to hydrogen ion (H+) which is not an effective biocide and hypochlorite anion (OCl). At a pH of 7.5, about equal amount of hydrogen ion and hypochlorite anion are present. At a pH of 8.5, the split is about 10% hydrogen ion and 90% hypochlorite anion (Aieta et al., 1980).

Ozone is a bluish gas with a characteristic pungent odour. It is an oxidizing biocide that is partially soluble in water and highly unstable as it readily reverts to oxygen. The solubility of ozone in water is related to the amount of ozone in the carrier gas stream. Thus, it is important to produce a gas stream containing a relatively high amount of ozone. For example, the maximum solubilities at 25°C for gas stream containing 1 and 3% ozone are 2.7 and 8.1 ppm, respectively (Rice and Wilkes, 1991). These maximum levels are not obtained in practice because of out-gassing of the carrier gas which removes some of the dissolved ozone. Solubility of ozone also decreases with increasing temperature. Ozone degrades with pH, being fairly stable under certain conditions at pH of 6 and stable at pH of 10 (Rice and Wilkes, 1991).

Formaldehyde (HCHO) is a non-oxidizing biocide. It is a liquid that is readily soluble in water because of the presence of polar carbonyl group. It is an effective biocide that acts both on proteins and lipopolysaccharides of the bacterial cell envelope. It is known to be effective even at low concentration of 0.1 ppm (Longley et al., 1980).

Samples preparation
Culture medium:
The culture medium (petroleum agar) used in the experiments contains 0.5 g of ammonium chloride (NH4CI), 2.5 g of hydrated sodium hydrophosphate (NaHPO4•12H2O), 5 g of potassium hydrophosphate (K2HPO4,) and 15 g of Diefo agar powder, per litre of engine oil. The pH of the culture medium was adjusted to a range from 7.0 to 7.2 for stability of the medium by addition of 50 mL of 1 M NaOH solution. The culture medium was autoclaved at a temperature of 121°C and pressure of 1.2x104 Pa. After sterilization, the medium was allowed to cool to room temperature of the laboratory (about 28°C). This method of preparation of the culture medium is reported in the literature (Steelhammer et al., 1995) and the choice of petroleum agar is due to its suitability as substrate for a broad spectrum of microorganisms (Steelhammer et al., 1995).

Biocide concentrations: The concentrations of each biocide used in the study are 0.1, 0.2 and 0.5 ppm. In a typical experimental design, the concentration of biocide should increase or decrease gradually, perhaps with equal intervals. We note that 0.2-0.4 ppm give very close results, hence the choice of the three concentrations (0.1, 0.2 and 0.5 ppm) used in the present analysis. Higher dosage of biocide (i.e., concentration greater than 0.5 ppm) has a lot of environmental consequences including greenhouse effects (Meitz, 1991). Effective concentration of biocide for control of microbial growth in a system ranges from 0.1 to 0.5 ppm. (Guiamet and de Saravia, 2005; Rice and Wilkes, 1991; Videla et al., 1995). The various concentrations of dissolved ozone used in the study were prepared by NEK Technical, Port Harcourt, Nigeria; 0.1 ppm of sodium hypochlorite and 0.1 ppm formaldehyde were prepared by mixing 0. 1 mL of the pure liquid with 1 L of water; 0.2 ppm was prepared by mixing 0.2 mL of the pure liquid with 1 L of water and 0.5 ppm was prepared by mixing 0.5 mL of the pure liquid with 1 L of water. The pH of the prepared sodium hypochlorite solutions were adjusted to 8.5 (as indicated in the preceding section) by the addition of 50 mL of 1 M NaOH solution in order to obtain high concentration of hypochlorite anion in the medium (Aieta et al., 1980) and consequently high concentration of sodium hypochlorite.

Experimental method: The produced water used in the study was obtained from a gas plant in Rivers State, Nigeria, in October 2006 and taken to Postgraduate Microbiology Laboratory, University of Port Harcourt, Nigeria, for analysis. Microbial population in the produced water sample contain four groups of planktonic microorganisms (EIA, 2005): (i) Hydrocarbon Utilizing Bacteria (HUB) such as Pseudomonas sp. and Bacillus sp., (ii) Heterotrophic Bacteria (HB) such as Pseudomonas sp., Bacillus sp. and Norcadia sp., (iii) Hydrocarbon Utilizing Fungi (HUF) such Saccharomyces sp., Penicillium sp. and Candida sp. and (iv) Heterotrophic Fungi (HF) such as Saccharomyces sp., Penicillium sp. and Candida species.

Two grams of petroleum agar were added to 900 mL of produced water to sustain the growth of microorganisms and the microbial population of the mixture was determined after 1 h using the rapid agar dipstick (time-kill) technique which is widely used in the literature (Bloomfield et al., 1998; Wang et al., 2006; Willinger et al., 2005; Nato et al., 2003; Olsen et al., 2004). The rapid agar dipstick method simply involves dipping the dipstick in the produced water such that appropriate volume of the liquid adheres to it. After resealing the wet stick in its container, it is incubated at a temperature of about 30°C for 24 h. The number of microorganisms in the produced water was determined by matching the microbial growth on the incubated agar dipstick against a calibration chart provided by the manufacturer (Boots Micro-Check Company, Nottingham, UK). The inoculated medium (that is, the mixture of petroleum agar, produced water and microorganisms) was then divided equally into nine (9) conical flasks labeled according to the different concentrations of each biocide (i.e., 0.1, 0.2 and 0.5 ppm of sodium hypochlorite; 0.1, 0.2 and 0.5 ppm of dissolved ozone and 0.1, 0.2 and 0.5 ppm of formaldehyde). The same volume of biocide was added to each flask as labeled and the microbial population in each flask was determined at intervals of 30 min using the rapid agar dipstick technique.

RESULTS AND DISCUSSION

Figure 1-3 show the effects of different concentrations of each biocide on total planktonic microorganisms in the produced water.

It may be seen from Fig. 1-3 that the total planktonic microorganisms (or microbial population) in the produced water is 109 cells mL-1 initially; that is, in the absence of biocide. On application of a biocide, the total planktonic microorganisms in the produced water decreases with time due to the effect of the biocide. The degree of reduction of total planktonic microorganisms increases as the concentration of each biocide increases and this effect is more pronounced with dissolved ozone, followed by sodium hypochlorite and then formaldehyde. For example, Fig. 1 indicates that after 120 min contact time, 0.1 ppm dissolved ozone reduces the total planktonic microorganisms in the produced water from 109 cells mL-1 to 103 cells mL-1 (i.e., 6 log reduction of total planktonic microorganisms); 0.2 ppm reduces the microorganisms from 109 to 101 cells mL-1 (8 log reduction) and 0.5 ppm reduces the microorganisms from 109 to about 100.5 cells mL-1 (8.5 log reduction). After 150 min contact time, 0.1 and 0.2 ppm dissolved ozone reduce the total planktonic microorganisms in the produced water to about 100.5 cells mL-1 while 0.5 ppm eliminated all the planktonic microorganisms in the produced water.

Fig. 1: Effects of different concentrations of dissolved ozone on total planktonic microorganisms in produced water

Fig. 2: Effects of different concentrations of sodium hypochlorite on total planktonic microorganisms in produced water

In the case of sodium hypochlorite as shown in Fig. 2, 0.1 and 0.2 ppm reduce the total planktonic microorganisms in the produced water to 103 cells mL-1 after 120 min contact time, while 0.5 ppm reduces the total planktonic microorganisms to 102 cells mL-1 for the same contact time. After 150 min contact time, 0.1 and 0.2 ppm sodium hypochlorite reduce the total planktonic microorganisms in the produced water to 102 cells mL-1 while 0.5 ppm reduces the total planktonic microorganisms to 101 cells mL-1. Figure 3 shows that after 120 min contact time, 0.1 ppm formaldehyde reduces the total planktonic microorganisms in the produced water to 104 cells mL-1; 0.2 ppm reduces the total planktonic microorganisms to 103 cells mL-1 and 0.5 ppm reduces the microorganisms to 102 cells mL-1. After 150 min contact time, 0.1 ppm formaldehyde reduces the total planktonic microorganisms to 103 cells mL-1, 0.2 ppm reduces the microorganisms to 102 cells mL-1 and 0.5 ppm reduces the microorganisms to 101 cells mL-1.

Fig. 3:
Effects of different concentrations of formaldehyde on total planktonic microorganisms in produced water

Fig. 4: Effects of 0.1 ppm of dissolved ozone, sodium hypochlorite and formaldehyde, on total planktonic microorganisms in produced water

Figure 4-6 show the performance of the same concentration of the three biocides on the total planktonic microorganisms in the produced water. Thus, at a given concentration of the biocides, dissolved ozone exhibits the best biocidal efficacy against the total planktonic microorganisms in the produced water, followed by sodium hypochlorite, with formaldehyde as the least effective of the three biocides, which is consistent with Fig. 1-3. As the concentration of a biocide increases, its performance also increases while the contact time decreases and conversely.

Fig. 5:
Effects of 0.2 ppm of dissolved ozone, sodium hypochlorite and formaldehyde, on total planktonic microorganisms in produced water

Fig. 6: Effects of 0.5 ppm of dissolved ozone, sodium hypochlorite and formaldehyde, on total planktonic microorganisms in produced water

Although 0.5 ppm dissolved ozone eliminated all the planktonic microorganisms in the produced water after 150 min contact time, 0.1 and 0.2 ppm dissolved ozone would also eliminate the microorganisms in the produced water but would require longer contact time depending on the concentration used. Since a biocide is said to be effective if it can achieve at least 4 log reduction of total planktonic microorganisms (Costerton and Lashen, 1984; Gaylarde and Videla, 1992), 0.1, 0.2 and 0.5 ppm dissolved ozone became effective as biocide after about 60, 50 and 40 min contact time respectively as may be read from Fig. 4-6. Dissolved ozone as low as 0.01 ppm can result to between 2 and 4 log reduction of E. coli bacteria (Brooke and Puckorius, 1991; Videla, 1996). It is also indicated that a constant concentration of ozone within a range from 0.02 to 0.05 ppm in cooling water systems will avoid formation of biofilms (Brooke and Puckorius, 1991). For biofouled metal surfaces, the presence of 0.2 to 1.0 ppm dissolved ozone results in rapid separation of sessile bacteria. Ozone is also said to be environmentally friendly because of its rapid decomposition to oxygen (Meitz, 1991).

CONCLUSION

Laboratory investigations of the effects of different concentrations (0.1, 0.2 and 0.5 ppm) of each of three biocides (dissolved ozone, sodium hypochlorite and formaldehyde) on the total planktonic microorganisms (bacteria and fungi) in produced water have been presented. It is shown that the performance of each biocide increases as its concentration increases resulting in a shorter contact time of the biocide. Dissolved ozone exhibits the best biocidal efficacy against total planktonic microorganisms in the produced water, followed by sodium hypochlorite, with formaldehyde as the least effective of the three biocides.

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