Environmental sanitation is a huge issue in whole world, day by days the problem of environmental pollution through industrial, agriculture, domestic contaminants is increasing. Phenol and its derivatives are toxic chemical contaminant in environment; it has negative impact on the biological environment. Because of the revolution of industrial field, many strange compounds have been introduced into the environment. Environment polluted due to the accumulation of these compounds and effects on ecosystems [1].

PHENOL is a toxic chemical compound. It comes into the environment through anthropogenic and natural resources. Phenol compounds contain –OH compounds to the benzene ring. It is in the form of white, pinkish crystalline solid or liquid. The crystals are wet and colorless, it smells like disinfectants, it should be handled carefully, and when it comes in contact with skin it causes irritation. High concentration of phenol can cause chronic exposure such as skin burns, liver damage and nervous disorder [2].Decomposition of organic matter and burning wood release phenol to the environmental[3]. Skin absorbs the vapor of phenol when Phenol is in solution form, also it can pass through the skin, and phenol metabolism happens in the liver, lung and kidney [4]. Phenol is one of the most important for many industries, a large amount of phenol are effused into streams and soil, and distributed as pollutants. It's also toxic to organisms and may cause cancer to humans [5]. Phenol is used in industries such as electrical, automotive and crude material, but it's considered as a toxic pollutant, constructions and the main organic constituents detect in petrochemical effluents, also it is used in plastic and varnish industries [6]. The World Health Organization (WHO) mentioned that phenol can enter drinking water in concentrations not more than 1 μg/L. [7]. Large amounts of wastewater generated by industries during the manufacturing and processing of major compounds. In these wastewaters phenol and ammonia are found, also it causes environmental pollution especially in water resources, it causes unfavorable changes in soil and water environment, presence of phenol inhibits the biodegradation of other contaminants. The instability of phenol and its affinity for liquid state makes phenol risky to humans when human drink water contains phenol [8]. Phenol may cause odor in drinking water and it's toxic to particular aquatic species [9]. Phenol is toxic to humans and foul odor in the water [10]. A number of microorganisms have been reported to degrade phenol at low concentration, including Alcaligenesentrophus, Bacillus stearothermophilus Pseudomonas sp., Rhodococcus sp., and Trichos-poron cutaneum. Degradation of phenolic compounds by P. putida CP1 [11]. And A (a) has been reported earlier [12]. Phenol biodegradation comes from bacteria more than the other microbes. The genus Pseudomonas is used for phenolic compounds degradation. The aims of the current study were to degrade microorganisms by phenol and use that microorganisms to reduce water pollution.

Sludge sample collected from municipal sewage treatment plant, placed in Al-Hamzah city, diwaniyah. Sludge is the residue obtained after treatment of sewage waste water, numbers of microorganisms are found in sewage sludge. This sludge sample was collected in polythene bag and was preserved at 4°C in fridge, then prepared one gram from the sample and put it in two conical flasks containing 100 ml of enrichment culture media, one flask contain sterile Nutrient Broth and the second flask contain Mineral Salt Broth , then kept on rotary shaker at 130 rpm at room temperature for 24-96h. Next step was isolated bacteria by using 0.1 ml of suspension from each flask, and then flasks kept in same condition as last time, and to isolate Microorganisms capable of utilizing phenol, used solution of Nutrient agar with 0.02gm Phenol used for isolation of phenol degrading microorganism, here one loopful of sample from enriched flask were streaked on nutrient agar plates which were containing phenol as a carbon source, plate kept 3 days in room temperature, the next step was Screening and selection of phenol tolerating microbial strains by using mineral salt broth. Mineral salt base agar with phenol media, then identification of microorganisms by Isolated colonies for Gram staining and identified on the basis of gram nature. Solutions required: 

• Hucker’s Crystal Violet

• Gram’s Iodine (Lugol’s Iodine)

• Ethyl Alcohol (95%)

• Safranin

The Procedure was, Smear was prepared on a clean grease free glass slide and heat fixed. The smear was treated with solution

• A for 1 minute. Solution a was removed by solution 

• B and smear was allowed to react with solution b for some minutes, then washed with clean water, next smear treated with solution 

• C for several seconds, and smear washed with clean water, later smear treated with solution 

• D for half minutes, then washed with water again, finally dried and examined

10⁶ cells ml^-1 of each isolate were inoculated in flasks containing 100 ml sterile mineral salt media with 0.02gm of phenol, the glasses were kept on rotary shaker at 130 rpm for 1 day, after that effect of different conditions on microorganisms and phenol degradation efficiency were analyzed, then treated of these microorganisms was also given to sewage water and was studied for various physico-chemical analyses. Analysis of concentration of residual phenol by 4-amino antipyrine method [13]. Phenol concentration analysis by using 4-aminoantipyrine in sample is a photometric test method based on the reaction of stem distillate phenolic compounds which react with 4-aminoantipyrine at pH 10± 2 in the presence of potassium ferricyanide (K₃Fe(CN)₆) to form a coloured antipyrine dye, this antipyrine colour formed in solution is measured at 510 nm, and the concentration of phenolic compounds in the sample is expressed in terms of mg/L of phenol (C₆H₅OH). PH meter and EC-TDS meter used for measuring E.C , TDS and PH. 

Chemically TS estimated by using gravimetric method, the evaporating dish was dried at 104±1⁰C for 60 minutes and cooled in desiccators to take the weight of the dish, 25 ml of the sample was taken for the analysis in a pre-dried dish and was evaporated to dryness in an oven at 104 ±1⁰C , the dish was cooled in desiccators and final weight was taken for the analysis of TS content, and by using the equation ( TS-TDS ) got TSS (mg L^-1). Also COD determinate by added 10 ml of 0.25N potassium dichromate (K₂Cr₂O₇)and 30 ml concentration sulphuric acid - silver sulfate (H₂SO₄+Ag₂SO₄) reagent in 20 ml diluted sample, the mixture was refluxed for 2 h and was cooled to room temperature, then the solution diluted to 150 ml by using distilled water and excess K₂Cr₂O₇ remained was titrated with ferrous ammonium sulfate (FAS) using ferroin indicator, sharp color change from blue green to wine red indicated the end point of titration. And sulphate content was determined by taking 100 ml of sample, 20 ml of buffer solution was added and with continuous stirring spoonful of BaCl₂ (0.3g) crystals were added barium sulfate turbidity of the solution was measured on a Nephelometer and sulfate content was measured by comparing turbidity readings with calibration curve. In addition nitrogen also determined by taking 50 ml of sample in kjeldal flask initially sample was digested with digestion reagent for one hour, sample was cooled and diluted to 300ml with distilled water after that 50 ml of sodium hydroxide thiosulfate was added and is swirled, digestion is followed by distillation where 50ml of boric acid cum indicator is placed in 500ml flask and 200ml distillate were collected, This sample is titrated with 0.2n H2So4 solution. Same treatment is given to blank i.e. to distill water. Finally, heavy metals estimated by atomic absorption spectroscopy (AAS) instrument, 20ml of sample was boiled with concentrated Nitric acid till it become half of the volume, also High performance liquid chromatography technique (HPLC) was used to analyze phenol concentration after treatment of microorganism.

Effect of pH

PH is the very crucial factor for growth of bacterial cells, that is why efficiency and capability of isolates have been checked at different pH levels like pH-6 ,pH-7 and pH-8 i.e. at acidic, neutral and alkaline pH . From table no.1 phenol concentration obtain for Isolate Y are 5.5 mg/L, 6mg/L, 7mg/L at acidic, neutral and alkaline pH respectively out of initial concentration of 20mg/L, and for isolate C values observed are 4mg/L, 4mg/L and 2mg/L from initial concentration of 20mg/L at acidic, neutral and alkaline pH respectively.

The PH concentration of the original sample was 6, which is slightly acidic. When this sample is treated with strains they show alkaline pH, results observed are 8.62, 7.18, 7.18 for strains Y,C and CY respectively Table 1-3.

At initial level the basic objective of the experiment was to isolate the phenol degrading microorganism from sewage sludge; from that sludge sample I got three different isolates after Gram method were named as C, Y and W_. These selected micro-organisms were examined for their ability to reduce the Phenol concentration of 20mg/L., it was observed that among these three isolates C and Y found to be more effective after the interval of 48 hours. Isolate W was not suited for the degradation phenol as shown in Figure 1.

Optimization of the process parameters

Phenol degradation was carried out with selected strains of C and Y. Effect of optimized conditions was also estimated on strains. Parameter studied under this are:

Effect of pH, Effect of temperature, Effect of Vitamins, Effect of sugars. As shown in above Figure 2. Isolate C has degraded around 90% of phenol at alkaline

Table 1: Efficiency of Phenol degradation of Isolates Y, C and W 

Table 2: Effect of Optimized Conditions on Phenol Degradation by Microorganisms

All values in mg/L, Initial phenol concentration was 20 mg/L

Table 3: Reduction efficiency of isolate C and Y and CY

Figure 1: Phenol reducing efficiency

Figure 2: Effect of pH

Figure 3: Effect of Temperature

Figure 4: Effect of Vitamins

pH; hence, it is observed that these isolates work efficiently at neutral pH, and slightly alkaline pH.

Effect of Temperature

Microorganisms are very sensitive to temperature. Here we observed the phenol degradation efficiency of microorganisms at temperature 30°C, 40°C and at 50°C.As shown in table no.2 activity of microorganism is highest at temperature range of 30°C to 40°C.They show lower activity at range 50°C.Phenol concentration after 48 hrs to these temperature range is 0.5mg/L, 0.5mg/L, 6mg/L for isolate Y out of 20mg/L of initial concentration. Isolate C shows 8mg/L, 8mg/L and 5.5 mg/L concentration out of 20mg/L initial concentration at temperature of 30°C, 40°C and at 50°C respectively.

As seen in the graph isolate Y shows high degradation at temperature 30°C and 40°C, which is 2.5% remaining phenol concentration, and for isolate C maximum degradation observed at temperature 50°C shows, it indicates that optimized temperature range for isolate Y is 30°C and 40°C and isolate C is very efficient at temperature 50°C shows 72.5% degradation within 48 hrs Figure 3.

Effect of Vitamins

Vitamins were used as supplementary nutrients for the enhanced phenol degradation through isolate C and Y. Biotin in concentration of 100ppm, 200ppm, 300ppm and 400ppm were fed to microorganism, it was shown 5mg/L, 5 mg/L ,4 mg/L, 4 mg/L phenol concentration respectively out of initial concentration of 20mg/L for isolate Y, for isolate C results obtained were 4mg/L , 3mg/L, 3mg/L,4mg/L concentration of 100ppm, 200ppm, 

Figure 5: Effect of Sugar

300ppm and 400ppm respectively out of initial concentration of 20mg/L as shown in Table no.2.

As seen in Figure 4 isolates C shows maximum degradation at vitamin dose of 200ppm and 300ppm while isolate Y shows maximum efficiency of phenol degradation at 300 ppm and 400ppm dose.

Effect of Sugars

Glucose and Lactose sugar were used as additional nutrient source to speed up the process of phenol degradation. Glucose is readily available and is a cheap source of sugar while Lactose is also the present in some amount in the sewage effluent. From Table 2 it seems that in presence of glucose isolate Y degrade 10mg/L of phenol concentration while isolate C degrade 4mg/L of phenol concentration. Lactose sugar help to degrade 4mg/L of phenol concentration.

As shown in Figure 5, it is clearly observed that in presence of glucose isolate Y Shows 50% reduction in phenol concentration and isolate C shows 20% reduction, with the help of lactose sugar isolate Y shows 20% reduction while isolate C shows 25% reduction. This implies that isolate Y act more efficiently in presence of glucose [14]. While isolated C has good efficiency with lactose. As seen in the table, used microorganisms shows good results for the reduction in parameters such as Electrical conductivity, Nitrogen, COD, Sulfate, TSS, TDS, TS and Phenol, Heavy metals .It indicates that along with the degradation of phenol these microorganism also helps in reduction of concentration of pollutants.

Electrical Conductivity

As shown in the table it can be seen that electrical conductivity of the original sample was 370.,which was reduced to 306 micro semen by all the strains. It shows that all the three microorganisms show similar efficiency for lowering the electrical conductivity. Conductivity is the ability of a substance to conduct electricity, the conductivity of water is a more-or-less linear function of the concentration of dissolved ions, conductivity is a numerical expression of the ability of water to carry an electrical current and varies with the number and types of ions, the solution contains, most dissolved inorganic substances in water are in the ionized form and hence contribute to the conductance.

Nitrogen

As seen in the table , the original concentration of total nitrogen in the sample was 71.2 mg/L. This is reduced to 6.72 by microbial strain Y, 8.96 by C and 7.16 by strain CY. Other than carbon source microorganisms need nitrogen source, available nitrogen in wastewater has been used by microorganisms for their growth. Strain Y required a high concentration of Nitrogen for its growth and metabolism.

COD

The reduction in COD content of the sewage waste water sample is recorded in table no. and depicted in following figure from this it is clearly seen that original COD content of the sample was 429mg/L it was reduced to 100.1 mg/L, 131.1mg/L, 136.1mg/L by strains Y,C and CY respectively. It indicates that strain Y shows good ability for COD removal. Chemical oxygen demand is a measure of the capacity of water to consume oxygen during the decomposition of organic matter and the oxidation of inorganic chemicals such as ammonia and nitrite. Higher the COD, higher the amount of pollution in the test sample. This test is commonly used to indirectly measure the amount of organic compounds present in water. 

Sulphate

The initial sample concentration was 9.65 which was reduced to below detectable level i.e. to 0mg/L by strain C. It indicates that strain C shows higher efficiency than strain Y and C which has reduced sulfate to 0.09 mg/L and 0.04 mg/L respectively.Sulphate is second to bicarbonate as the major anion in hard water reservoirs, sulfates (SO₄^2-) can be naturally occurring or the result of municipal or industrial discharges, hen naturally occurring, they are often the result of the breakdown of leaves that fall into a stream, of water passing through rock or soil containing gypsum and other common minerals, or of atmospheric deposition, point sources include sewage treatment plants and industrial discharges such as tanneries, pulp mills and textile mills. Runoff from fertilized agricultural lands also contributes sulfates to water bodies; sulfate is one of the major dissolved components of rain. 

TSS 

As seen in the table after treatment with microbial strains initial concentration of total suspended solids which was 182mg/L has reduced to 50mg/L,190mg/L and 50mg/L by strain Y,C and CY respectively it clearly shows that strain Y and CY shows same effect which is very significant as compared to strain C. , total suspended solids are those that can be retained on a water filter and are capable of settling out of the water column onto the stream bottom when stream velocities are low. They include silt, clay, plankton, organic wastes, and inorganic precipitates. Total suspended solids are solids in water that can be trapped by a filter. High concentrations of suspended solids can cause many problems for stream health and aquatic life. TSS cuts down light transmission through water and so lowers the rate of photosynthesis in aquatic flora. It is a major water parameter used to evaluate the strength of domestic wastewater and to determine the efficiency of the treatment unit. The reduction in TSS might be due to the use of organic and inorganic ions by the micro-organisms for their growth and development.

TDS

The present results from Table and Fig 3.10. shown below, indicate that initial TDS concentration was 198 has been reduced to 150mg/L, 110mg/L, 160mg/L by strain Y, C and CY respectively. Strain Y has proved efficient for TDS decrease in sewage waste water. Strain CY shows very low efficiency for lowering TDS concentration. Total dissolved solids is an expression for the combined content of all inorganic and organic substances contained in a liquid which are present in a molecular, ionized or micro-granular suspended form. A large number of solids are found dissolved in natural waters, the common ones are carbonate, bicarbonates, chlorides, sulphates, phosphates and nitrates of calcium, magnesium, sodium, potassium, iron, magnesium etc. In other words, TDS is simply the sum of the cations and anions expressed in mgL^-1, a high content of TDS elevates the density of water, influences osmoregulation of freshwater organisms, reduces solubility of gases and in turn reduces the utility of water for drinking, irrigation and industrial purposes.

TS

The total solids content of the treated sewage water with micro-organisms was found to be 200mg/L,300mg/L and 210mg/L by isolating Y,C and CY respectively. Y bought highest reduction in total solids. C shows least reduction in lowering the concentration as compare to other isolates which is 300mg/L. Total solids (total residue) is related to turbidity, except that it includes not just suspended solids, but also dissolved solids such as the mineral ions calcium, phosphorus, iron, a certain level of these ions is essential for life, also cells depends on the density of total solids to determine the amount of water that flows in and out of the cell. Organic and inorganic present in the effluent might have been used by the micro-organisms for their survival and might have proved to be of better nutrient value for them.

Phenol

AS shown in table and depicted in the Figure 4 initial phenol concentration in sewage sample was 7mg/L. This is reduced to 3.5mg/L, 4mg/L and 5mg/L. by the isolate Y, C and CY respectively. Isolate Y found to be highly efficient for degradation of phenol. It shows 50% reduction, isolate C as compared with Y shows low efficiency for lowering the phenol concentration, and CY has efficiency near about 71% which is very less as compared to other two isolates. Phenol is rich in carbon content [15]. High phenol concentration is lethal to life, so it is called a xenobiotics substance . Microorganisms may have utilized phenol as a carbon source for their growth and metabolism.

Heavy Metals

For heavy metals estimation Cr, Cd, Zn, Fe, Cu, Mn, Co, Pb, Ni was analyzed initially. Then treatment of microorganisms given to samples and their effect for lowering the concentration of Cr, Zn and Fe were estimated. It has shown from the result that present isolates are capable of lowering the concentration of heavy metals. For chromium isolate C and Y shows good reduction efficiency, Initial concentration of Cr was 0.829 ppm was reduced to below detectable level it shows that these organisms with the help of other microorganisms of sewage water reduces chromium. This results obtained show that microorganisms utilized these elements and might have stored in microbial biomass, some of its quantity for enzymatic reactions during metabolism Figure 6.

One of the most affected pollutants in the environment is phenol, which enters the environment due to, petroleum refineries, industrial, natural activities, pesticide industries, and paint industries contribute to phenolic liquid waste, phenol presents in water imparts carbolic odor to receiving water bodies and may cause toxic effects on human, animals and plants [16]. So, it is necessary to remove phenol from industrial release into the environment, biological methods for the removal of phenol are possible because some microorganisms have the ability to degrade phenol. Researchers isolated microorganisms from nature and found good degradation yields [17]. Data are also available on the possibility of use of microorganisms for treatment of phenol bearing industrial wastewater [18]. So, in this research paper, attempts were made to isolate phenol degrading microorganisms after enrichment of soils collected from different locations and to select the most efficient strain. The process parameters for efficient biodegradation were also optimized.

At initial level the basic objective of the experiment was to isolate the phenol degrading microorganism from sewage sludge; from that sludge sample I got three different isolates after, Gram method and microscopic observation characteristic specification of the Sludge Isolated was carried out, the three isolates samples were named as C, Y and W_. These selected micro-organisms were studied their ability to reduce the Phenol concentration of 20mg/L during initial phase. It was observed that among these three isolates C and Y found to be more effective after the interval of 48 hours. Isolate W was not suited for the degradation phenol. After adaptation of microorganism to 200ppm phenol concentration these isolated dosed for 10,000ppm of phenol concentration analysis was carried on HPLC, it is observed that Isolate Y has reduced 8190.2 ppm of phenol whereas Isolate C reduced 8256.6ppm of phenol. It indicates that both microorganisms are capable of high phenol degradation.

As isolate Y and C found to be more effective attention was given towards them. Isolate Y and C are more efficient than the isolate W. So isolate W has not been used for further determination. Further concentration is given on the effect of optimized conditions on microorganisms for efficient phenol degradation, effect of pH, temperature, and vitamin dose and sugar concentration, and have been observed for phenol degradation.

Figure 6: Heavy Metals

PH Optima

The effect of pH of the medium on phenol degradation by C and Y was assessed. The data revealed that the highest degradation obtained 14.5mg/L by isolate Y was highest as compared with isolate C when pH was adjusted to 7.0, indicating pH preference of the bacterium, also observed that phenol degradation by Candida tropicalis was at its peak at pH 7.0.

Vitamins are required for synthesis of an array of enzymes and coenzymes involved in metabolic activities. Hence, the effect of biotin on the degradation of phenol was studied. It was observed that biotin stimulated growth of Isolate C and Y resulting in statistically significant increase in phenol degradation. At concentrations of 300 and 400ppm isolate Y shows highest degradation that is about 60% reduction. Isolate C showed good efficiency at 200 and 300 ppm and reduced about 70% of phenol. Kafkewitz et al. observed that the availability of biotin in the culture medium would increase the aerobic degradation and dechlorination of 2-chlorophenol and 4-chlorophenol by [11-16 ] per cent.

Temperature plays an important role in growth of microorganisms in present investigation. Isolate Y shows noticeable phenol temperature in the range of 30°C to 40°C and isolate C was effective in the temperature range of 50°C.In about 80% of reduction is seen at high temperature.

In the present investigation, the effect of sugars like Glucose and Lactose have also been analyzed. In case of isolate Y glucose addition shows good efficiency for phenol degradation it is observed that 50% of phenol is degraded in presence of glucose sugar. Isolate C reduced 75% of phenol in the presence of Lactose sugar. It indicates that in presence of Glucose Isolate Y acts more efficiently. and isolate C shows efficient activity in presence of Lactose. 

Phenol degrading microorganisms were also used for treatment of Sewage waste water. Effect of these microorganisms on lowering the pollution because of the addition of impurities and physico chemical factors estimated. In sewage wastewater reduction of Sulphates, Chemical oxygen demand, Total solids, Total suspended solids, Total dissolved solids, Phenol, pH, Electrical conductivity and heavy metals was observed. Accept pH and Electrical conductivity all factors are considered as nutrients within the permissible limits. In excess they are responsible for the water pollution.Treatment of microorganisms found to be helpful in reducing the level of water pollution by removing excess amount of Sulphates, Chemical oxygen demand, Total solids, Total suspended solids, Total dissolved solids, Phenol.

In the present study, an attempt was made to isolate, screen and select the most efficient microorganisms capable of degrading phenol. It has been seen that microorganisms isolated from sewage sludge are found to be excellent for phenol degradation. They show good adaptability in phenol concentration. 

Under the optimized conditions they act effectively for phenol degradation

Optimized conditions are

• pH-Neutral
• Temperature range-30°C to 40°C
• Vitamin- concentration of Biotin in range of 200 to 300ppm
• Sugar-Glucose for isolate Y and Lactose for isolate C

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