Effect of pH, nitrogen sources and salts on the degradation of phenol by the bacterial consortium under saline conditions.
Hypersaline environments are important for both surface extension and ecological significance. As all other ecosystems, they are impacted by pollution. Marine and fresh water, soils and air have been impacted by the dispersion of contaminants. Contamination and biodegradation in extreme environments has received little attention although many contaminated ecosystems present high or low temperatures, extreme acidic or alkaline pH, high pressures or high salinity (1). Extremophilic microorganisms (extremophiles) are adapted to thrive in such hostile environments.
Halophiles are group of extremophiles that can not only grow in saline conditions but requires salinity for their growth. These halophiles grow optimally, in media containing between 3 % and 15 % NaCl (2), although the main characteristic of this group of organisms is their ability to grow in a very wide range of salt concentrations.
Saline and hyper-saline environments are frequently contaminated with organic compounds as a result of industrial activities (1, 3). Contamination of these habitats constitutes a serious environmental problem mainly due to the high toxicity exhibited by aromatic hydrocarbons. In most cases, biodegradation constitutes the primary mechanism for contaminant removal. However, biodegradation processes are difficult to perform under saline conditions (1, 3, 4). In addition, it is known that the traditional pollutant biodegradation is less efficient or does not function when salinity increases above that of the sea (3). Conventional microorganisms do not survive under these saline conditions. One remedy for the removal of these xenobiotic compounds in the saline environment is the use of halophiles which are adapted to live in such saline conditions. The degradation or transformation of organic pollutants by halophilic and halotolerant microorganisms has received little attention.
Phenols are major pollutants of industrial wastewaters since they are commonly used in many industries such as oil refining, coke conversion, pharmaceutical and resin manufacturing plants. Biodegradation of phenol under saline conditions have been reported by few authors (5, 6, 7, 8, 9, 10, 11). Most of these reports were mainly based on degradation of the compound by single strain. The saline wastewater containing phenolics when treated with pure species of bacterial cultures may suffer from contamination problems and thus will be impossible to maintain in the field condition.
Hence in the present study bacterial consortium capable of degrading phenolic compounds were isolated from saline environments and was employed in the degradation of phenol. It was suggested that halophiles have more demanding nutritional requirements at high salt concentrations, and hence, complex media containing growth promoting factors may help to stimulate growth of halophilic bacteria at high salt concentrations (3). Present study focuses on the effect of pH, low concentrations of nitrogen sources and effect of different salts on the degradation of phenol by the isolated bacterial consortium.
Materials and Methods
Bacterial consortium and Culture conditions
The bacterial consortium was isolated from soil samples from different habitats of Chennai, having proximity to saline environments. During the initial adaptation stage the consortium was enriched with 50 mg/L of phenol and the concentrations of phenol were increased up to 300 mg/L after acclimatization. The bacterial consortium was biochemically characterized, having 6 strains, of which 4 strains were gram positive and two strains are gram negative. Further molecular analyses by cloning the mixed DNA and 16S rRNA gene sequence analysis, it proved that the 6 bacterial isolates were Bacillus cereus, Arthrobacter sp., Bacillus licheniformis, Halomonas salina, Bacillus subtilis and Pseudomonas aeruginosa. The sequences of the isolates were deposited in Gen Bank and their accession numbers are EU780459, EU780460, EU780461, EU780462, EU780463, and EU780464.
During the enrichment period with phenol, the consortium was acclimatized with 50 mg/L of phenol as the sole carbon and energy source. The bacterial consortium was enriched in mineral salts medium (g/L) of NaCl, 10.0 to 150.0, K[H.sub.2]P[O.sub.4] 0.25, N[H.sub.4]Cl 1.0, [Na.sub.2]B[O.sub.7] 2.0, Fe[Cl.sub.3] 0.0125, Ca[Cl.sub.2] 0.06 and Mg[Cl.sub.2] 0.05. The medium was supplemented with a specified amount of added NaCl and 10 mg of yeast extract, adjusted to pH -7 (Alva and Peyton 2003). The medium was autoclaved, cooled to room temperature and was amended with phenol (100 mg/L) through a sterile filter (0.45 um) in 250 ml Erlenmeyer flasks. The chemicals and reagents used in the study were analytical grade.
Growth and degradation of phenol
Growth of the bacterial consortium on phenol was studied by determining the colony forming units (CFUs) per ml on nutrient agar medium. The mineral medium was substituted with different concentrations of NaCl concentrations from 10 g/L to 100 g/L were the optimum was 50 g/L of NaCl and at 100 mg/L of phenol maximum degradation was achieved. At 24 h time interval, the culture was centrifuged at 10,000 rpm for 15 minutes to remove the cells. The culture supernatant was collected and used for the estimation of residual phenol.
Total Protein analysis
For analysis of total cell protein, samples were centrifuged at 12000 rpm for 10 minutes and washed with fresh (substrate-free) mineral medium, then centrifuged and washed few times to remove the substrate. The pellet from each sample was then disrupted by sonication at 30 % amplitude for a total of 3 minutes (1.5 min x 2) in an ice-water bath. 0.5mL of sample was added to 0.5mL Coomassie Blue protein dye and the absorbance at 595nm was measured. Total protein concentration was determined by calibration with bovine serum albumin standards according to (12).
Biodegradation on phenol
For the degradation study, mineral salts medium containing phenol was inoculated with the bacterial consortium. Different conditions used for the degradation of Phenol were (i) medium + Phenol + bacterial consortium; (ii) medium + Phenol and (iii) medium + bacterial consortium, with (ii) and (iii) serving as controls. The bacterial consortium was added to the medium at concentrations of [10.sup.4] -[10.sup.5] cfu/mL. The culture, in duplicate, was incubated at 37 [degrees]C with shaking at 150 rpm. The cell suspensions were clarified by centrifugation at (10,000 rpm for 15 min, 6[degrees]C).
The culture supernatant was extracted with dichloromethane every 24 h interval for 5 days, was extracted twice with dichloromethane (v/v), after acidification to pH 2.5 with 1N HCl. The extracts were filtered through anhydrous sodium sulphate and condensed to 1 mL with a rota vapour (Buchi, Germany) for further gas chromatographic analysis. Gas chromatograph (Chemito GC Model No 1000) was equipped with FID detector and capillary column (Varian Chromopak capillary column CP SIL 8 CB, 30m X 0.32 mm. Nitrogen was used as a carrier gas, injector temperature was 220[degrees]C, detector temperature was 250[degrees]C and the oven temperature of the column was maintained at 150[degrees]C.
Effect of pH, salts and alternate nitrogen sources
The effect of different pH (5.5, 6, 6.5, 7, 7.5, 8 and 8.5) was examined with the bacterial consortium with 100 mg/L phenol at 5 % NaCl. The growth medium containing (0.01%) yeast extract was replaced with alternate nitrogen sources such as tryptone (0.01 %) and urea (0.01 %). Effects of alternate salts on the biodegradation of phenol were determined by addition of the 5 % individual salts such as KCl, [Na.sub.2]S[O.sub.4], [K.sub.2]S[O.sub.4] and NaN[O.sub.3] to the mineral salts medium.
Results and Discussion
In the degradation of phenol by the isolated bacterial consortium under saline conditions, six bacterial strains existed in the synergistic mixture. The molecular identification by cloning proved that the six strains were Bacillus cereus, Arthrobacter sp., Bacillus licheniformis, Halomonas salina, Bacillus subtilis and Pseudomonas aeruginosa. Experiments conducted with the individual bacterial strains and consortium proved that the bacterial consortium was most efficient in the degradation of phenol than the individual strains. Hence for further analyses to study the effect of pH, nitrogen sources and salts bacterial consortium was used as the inoculum.
Effect of pH on the biodegradation of phenol
For optimum microbial activity in the environment, the preferred range of pH is between pH 6 to 8 (13). Therefore, it is not surprising to find that most microorganisms have evolved with pH tolerances within this range (14). Most heterotrophic bacteria favour a pH near neutrality (15). Nevertheless there are bacterial strains which can thrive outside this limit which belongs to the group of acidophiles (grows in lower pH) or alkaliphiles (grows in higher pH). The following experiment was performed to study the effect of pH range (5.5, 6, 6.5, 7, 7.5, 8 and 8.5) on phenol (100 mg/L) degradation by the bacterial consortium with the optimum salinity of 50 g/L.
Figure 1 shows the effect of pH on the growth and degradation of phenol (50 mg/L) at 5 % NaCl. The consortium was able to grow on a wide range of pH from 5.5 to 8.5 achieving the phenol degradation efficiency in a range of 5% to 99 %.
The maximum degradation efficiency of 99 % was achieved at 7. The consortium was able to grow on phenol with high degradation efficiency from pH 7.0-8.0. This pH range serves to be the optimum for proper growth of microorganisms (16, 17). However, when the pH values were lower than 7 and higher than 8, biodegradation efficiency was affected significantly.
During the range of optimum growth at pH 7, 7.5 and 8, the removal efficiencies were 99 %, 95 % and 74 % respectively. Above the optimum range at pH 8.5 the removal efficiency of phenol reduced to 42 %. In the lower range of pH 6.5 and 6 the efficiency lowered to 24 % and 9 %. The efficiency was least at pH 5.5 of about 5 %.
[FIGURE 1 OMITTED]
The optimum pH conditions for the growth of the consortium was comparable to phenol-degrading microorganisms of bacterial origin such as Pseudomonas sp., Arthrobacter sp. Bacillus cereus, Citrobacter freundii, Micrococcus agilis and Pseudomonas putida biovar B, Nocardiodes sp. and Alcaligenes faecalis performed at pH range of 7.0 to 10 (7, 18,19, 20). This shows that bacterial consortium usually prefers neutral pH as compared to other bacterial strains in phenol degradation. This was in accord with the results of Woolard and Irvine 1995, which showed the mixed culture degraded phenol (with 99 % phenol removal efficiency) in 140 g/L NaCl at neutral pH. An unidentified Halomonas sp. was able to completely degrade phenol at pH 7 in a bubble reactor where the concentration of phenol was 100 mg/L at 140 g/L of NaCl. Alva et al (2003) reported the degradation of phenol by a haloalkaliphilic bacteria Halomonas campisalis, which was able to degrade the substrate at pH 8- 11, where the maximum removal efficiency maximum at pH 9.5.
Effect of alternate nitrogen sources on the degradation of phenol (100 mg/L)
It is well-known that nutrients, particularly nitrogen and phosphorus, are required for to improve the aromatic compounds biodegradation. Yeast extract was used as one of the component in the mineral salts medium, to check alternative nitrogen sources tryptone and urea was used in the medium. Tryptone is the assortment of peptides formed by digestion of casein used as source of aminoacids by microorganisms. Urea is a compound of two amine groups, which acts as a good source of nitrogen.
The effect of alternate nitrogen sources on the degradation of optimum concentration of 100 mg/L phenol at 50 g/L of NaCl was determined by replacing 0.01 % of yeast extract with 0.01 % tryptone or 0.01 % urea. The removal efficiency of phenol (100 mg/L) by the addition of alternate nitrogen source is given in the Figure 2. The results showed that addition of tryptone in the place of yeast extract gave only 87 % removal in 4 days, with the maximum protein content of 39.3 mg/L. When urea was substituted for yeast extract the efficiency still reduced to 75 % with the maximum protein yield of 32 mg/L. The experiment results from the addition of yeast extract in the medium showed highest phenol removal efficiency of 99 % and it also enhanced the growth of the consortium which was shown from the previous experiment. Altering the nitrogen source with tryptone or urea didn't play an important role in improving the removal efficiency of the substrate; this might be due to the structure of yeast extract as it is readily available as aminoacids in the mineral salts medium. Hence it proved that using yeast extract as nitrogen source proved to be more efficient in degrading phenol.
[FIGURE 2 OMITTED]
Increase in salinity and depletion of nutrients inhibit the growth of the bacterial cells. To support the growth of bacterial cells, nutrients such as glucose, yeast extract and acetate were added as additional carbon source (20). Carla et al (21) reported the degradation of BTEX compounds by a halophilic and halotolerant microcosms, where they studied the rate of degradation of the benzene with addition of yeast extract, vitamins or trace elements. Benzene was completely degraded in 8 days after the additions of the nutritional factors, in the absence of the stimulants it took 15 days. An archaea (strain EH4) isolated from a salt marsh was able to degrade a higher percentage of eicosane ([C.sub.20][H.sub.42]) in the presence of yeast extract, peptone, and casaminoacids (22). Woolard and Irvine 1995 also reported that addition of yeast extract in the medium enhanced the removal efficiency of phenol.
Effect of complex salts on the degradation of phenol (100 mg/L) by the consortium at 50 g/L NaCl
Removal of phenolic compounds by microorganisms is also affected by some external factors such as salt ions, pH and temperature of the wastewater. Especially, high salt concentration is the most important factor which reduces microbial activity. Biodegradation of phenol in the presence of high salt concentration, particularly sodium chloride, has been reported. Kargi and Uygur 1996 (23) reported lower rate of phenol degradation at 3 % NaCl. Saline wastewater contains phenolic compounds, along with different salts other than NaCl. To study the effect of different salts along with NaCl; experiments were conducted with different salts [K.sub.2]S[O.sub.4], KCl,[Na.sub.2]S[O.sub.4], and NaN[O.sub.3] which is presented in the Figure 4. From the results it was observed that the bacterial consortium was able to grow on phenol in the presence of different salts at 50 g/L of NaCl. The removal efficiency of phenol (100 mg/L) was 92 % with [Na.sub.2]S[O.sub.4]. The presence of these salts influenced the degradation of phenol, where it reduced to 78 % and 72 % with KCl and [K.sub.2]S[O.sub.4]. The least removal efficiency was obtained with NaNO3 of 67 %. The protein concentration of the bacterial consortium during the degradation of different salts is given in the Figure 4. In the log- phase the protein concentration was in the range of 46.4 to 36.5 at the end of 2 days for [Na.sub.2]S[O.sub.4] and NaN[O.sub.3] respectively. In the case potassium salts, the proteins levels were 38.2 and 32.5 for KCl and [K.sub.2]S[O.sub.4] at the end of 2 days. This was in accordance with the results of Wang et-al., (2009) where they studied the effects of salts on the degradation of phenol-cresol mixtures. [Na.sub.2]S[O.sub.4] gave relatively higher removal efficiency of 90% than the other salts such as KCl and [K.sub.2]S[O.sub.4].
[FIGURE 3 OMITTED]
The present study on the degradation of phenol has showed that the bacterial consortium was able to utilize phenol (100 mg/L) at optimum pH of 7 with 99 % removal efficiency, and when yeast extract was replaced with tryptone and urea the degradation efficiency reduced markedly. This showed that presence of yeast extract proved to be a better nitrogen source for the degradation of phenol. In the addition of complex salts to the mineral medium the removal efficiency of phenol (100 mg/L) was not much affected in the presence of [Na.sub.2]S[O.sub.4]. The presence of complex salts did not have any effect on the microbial growth and biodegradation. The results suggested that the bacterial consortium can be used in the treatment of phenol containing saline wastewater in the presence of complex salts.
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Krishnaswamy Veenagayathri * and Namasivayam Vasudevan
Research Scholar and Professor Centre for Environmental Studies, Anna University, Chennai, India * Corresponding Author: E-mail: firstname.lastname@example.org