Microbial Degradation of Reactive Red 195 by Three Bacterial Isolates in Anaerobic-Aerobic Bioprocess

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2.2. Composition of Synthetic Wastewater

The basic composition of a synthetic dye wastewater medium was (g L^-1): Na₂HPO₄(3.6), KH₂PO₄(1.0), (NH₄)₂SO₄(1.0), MgSO₄(1.0), CaCl₂(0.1), FeC₆H₅O₇(0.01), and 10 ml of trace element solution per liter was used for all the studies. The trace element solution used was of the following composition (mg L^-1): ZnSO₄.7H₂O (10.0), MnCl₂.4H₂O (3.0), CoCl₂.6H₂O (1.0), NiCl₂.6H₂O (2.0), Na₂MoO₄.2H₂O (3.0), H₃BO₃(30.0) and CuCl₂.2H₂O (1.0). The medium was supplemented with 10 mg/L. Soluble starch and yeast extract were added to the synthetic dye media to a final concentration of 0.5% (w/v) and 0.01% (w/v) respectively and sterilized along together with the media. Sodium carbonate, N₂CO₃, (25% w/v) was separately sterilized and added to the media in order to maintain an alkaline pH.

2.3. Configuration of the Continuous Reactor System

The basic configuration of the continuous reactor system consists of anaerobic and aerobic reactors respectively. In the continuous reactor system, the synthetic wastewater containing a reactive Red azo dye (0.01 g/L) were continuously pumped with a flow rate of 1 ml/min into an anaerobic reactor consisting of anaerobic consortia of alkalophilic mud samples (10% w/v) which are collected from common effluent treatment plant of Ankleshwar, Gujarat, India. The retention time of reactive Red azo dye in the anaerobic reactor was 2 h. The treated dye along together with some consortia of alkalophilic mud samples were then pumped into the aerobic reactor with a flow rate of 1 ml/min. The aerobic reactor was continuously supplied with air by using a pump. The continuous anaerobic/aerobic reactor also consists of three sampling ports (before the anaerobic reactor, after the anaerobic reactor and after the aerobic reactor) for the High performance Liquid Chromatography (HPLC), Thin Layer Chromatography (TLC) and Uv-Visible Spectrophotometry analysis.

2.4. Decolorization and Growth Measurement

In order to measure decolorization, sampling was done for every 24 h for 5 days from the three sampling sites of the continuous reactor (before the anaerobic reactor, after anaerobic treatment and after aerobic treatment). Then the samples were centrifuged at 8000 rpm for 10 min and were analyzed by UV-Vis spectrophtometry. Similarly, sampling was also done for every 24 h from the anaerobic batch decolorization test by isolates (E1, E2 and E3) from the continuous reactor, clarified and analyzed by UV-Vis spectrophotometry. For both tests, an uninoculated culture media with and without added dyes were used as a control and blank respectively. Decolorization efficiency of the sediment consortia in the anaerobic/aerobic reactor and the different isolates (E1, E2 and E3) in an anaerobic batch decolorization was expressed as

Decolorization (%) = (A₀ - A)/A₀ x 100,

Where, A₀ = the initial absorbance and

A = the absorbance after decolorization at the λ_max(nm) of the dye under study.

The average decolorization rate (μgh^-1) was calculated as

C x % D x 1000/(100 x t)

Where C is the initial dye concentration and %D is the dye decolorization (%) after time t (h).

The bacterial growth or turbidity after decolorization of Reactive Red under batch test by three isolates isolated from the continuous reactor was calculated by determining the difference between the absorbance of the culture samples before and after centrifugation at 600 nm.

Turbidity = OD _{(before centrifugation)} - OD_{(after centrifugation)}

2.5. Decolorization under Different Culture Conditions

Decolorization under different culture conditions was studied by changing carbon source, nitrogen source and dye concentration. In effect, different decolorization efficiency was obtained at different conditions. The effect of aeration was examined under three culture conditions, namely, static (no shaking), agitated (aerobic) and anaerobic. In the case of aerobic decolorization batch tests using the three isolates (E1, E2 and E3), the culture flasks were shaken on a rotary shaker running at 110 rpm/min. In the anaerobic batch test, flasks containing decolorizing medium were sealed with rubber septa and incubated under anaerobic condition and in the static condition flasks were placed in the incubator directly.

2.6. Decolorization by Active and Inactive Cells

After the dye was decolorized in an anaerobic batch test by the isolate E1, the decolorized medium was autoclaved, centrifuged at 8000 rpm for 10 min, incubated with the dye and their decolorization activity was monitored by UV-Vis spectrophtometry.

2.7. Isolation of Bacterial Strains from the Continuous Reactor

Isolation was made from the continuous reactor system that was acclimatized for a month with alkalophilic consortia. Serial dilutions (10^-1to 10^-6) of the samples collected from both anaerobic and aerobic reactors were inoculated into a mineral salt agar medium (MSM) by the spread plate technique. Isolates were inoculated into a synthetic dye media containing 10 mg/L and incubated at 30°C for 5 days. The isolates were tested for their decolorization efficiency under anaerobic batch conditions and the strain that achieved the best decolorization efficiency was selected for further study.

2.8. Analysis
2.8.1. Color Measurement

Color in the influent and effluent samples of the anaerobic/aerobic reactors and color in the anaerobic batch treatment with the selected isolate, E1, was measured with Shimadzu-1800 UV-Vis spectrophotometer at the maximum visible absorbance wavelength of the dye. In both cases samples were centrifuged at 8000 rpm for 10 min and absorbance values of supernatants were recorded for color measurements. In order to measure the real color removal, the absorbance values of the treated media at λ max was calibrated with the control samples containing no dye (Awoke, 2008).

2.8.2. Biodegradation Assay Via TLC

After complete decolorization by the efficient decolorizer isolate (E1) which was selected from the isolates, the decolorized medium was centrifuged at 8000 g for 10 min. And the supernatant extracted with chloroform after alkalization to pH 8 to extract the biotransformed products. Then the extracted product was evaporated in a rotary evaporator. The concentrated extract was dissolved in 1 ml chloroform and used for thin layer chromatography (TLC). The mobile phase for the organic and the aqueous extracts was petroleum ether: chloroform: methanol (4:1:1). The bands of decolorization metabolite were observed under UV light.

2.8.3. HPLC Analysis of Decolorization Metabolites

To determine the dye fragments produced upon decolorization in a continuous reactor system using microbial consortia the treated samples were used directly for HPLC analysis. HPLC analysis was carried out On a Ceccil model Adept CE 4900 chromatograph equipped with a Cecil model CE 4200 UV detector, a column model CE 4601, and a lichrosorb C-18 column with a 4.6 mm inside diameter and 25 cm height. In the continuous reactor, sampling was done before the anaerobic treatment, after anaerobic treatment and aerobic treatments and centrifuged at 8000 g for 10 min, clarified by 0.45 filters, and analyzed with HPLC. In the batch test, the decolorized media under anaerobic condition with the efficient isolate were centrifuged, pH adjusted to 7.5, autoclaved, supplemented with glucose to a final concentration of 0.1% and inoculated with three isolate aerobically that are capable of decolorizing under aerobic, incapable of decolorizing under aerobic and the efficient isolate that is capable of decolorizing under anaerobic condition. Samples before autoclaving (after centrifugation) and incubation with the three isolates were centrifuged at 6000 rpm for 10 min, clarified by 0.45 filters, and analyzed with HPLC. In the analysis of dye fragments, a mobile phase composed of a phosphate buffer solution (0.7 g/L Na₂HPO_4,0.58g/L NH₄H₂PO₄) and methanol with a flow rate of 1 ml min^-1 were used. The elutes were monitored by the UV absorption at 220 nm and 217 nm. To determine the dye fragments produced upon decolorization, the treated samples were used directly for HPLC analysis.

2.8.4. COD, Total Nitrogen and NH₄⁺ Measurements

In the continuous reactor, samples before the anaerobic treatment, after anaerobic treatment and after aerobic treatment were taken and analyzed for chemical oxygen demand (COD), Total nitrogen, ammonia and nitrate. Chemical Oxygen Demand, Total Nitrogen and NH₄⁺measurement were done according to the standard methods of HACH instruction manual and instruments.

The percent removal efficiency of Chemical Oxygen Demand (COD) and Total Nitrogen (TN) was calculated as:

% Removal efficiency = Ci - Cf/CiX100,

Where, Ci = the initial concentration of the feed Cf = final concentration after treatment.

3. Results

3.1. Color Measurement from the Continuous Reactor

From Figure 2a, the maximum absorbance of the dye (λ max = 540 nm) was 0.631. After the anaerobic stage (Figure 2b), the maximum absorbance of the dye was found to be 0.025. The color removal efficiency of the reactor was approximately 96.0%. After the dye was treated in an anaerobic condition, it was pumped into a second stage known as the aerobic reactor. From Figure 4c, the absorbance of the dye after aerobic treatment or the effluent was found to be 0.0. And the color removal efficiency of reactive Red 184 at λ_maxwas 100%. Thus the major color removal was attained at the anaerobic stage than the aerobic stage. After biodegradation of the dye, the absorbance peaks in the visible region disappeared while the absorption peak in the UV range did not diminish, indicating complete decolorization.

Figure 2. Color removal efficiency of a) dye b) anaerobic effluent c) aerobic effluent

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3.2. HPLC Analysis of Biodegradation Products

At 220 nm, HPLC analysis of the parent compound, the dye (Figure 3a) shows the presence of two peaks with retention times 0.62 and 1.35 minute. The peak area of the peak with retention time 0.62 was 47.6 mv. For the second peak with retention time 1.35, the area of the peak was 23.3 mv. At the same wavelength, the dye after anaerobic treatment was analyzed by HPLC and it gives three new peaks with different retention times than the chromatograms of the parent molecule (Figure 3b). The retention times of the peaks were, 0.46, 0.58 and 1.23 minute. The peak areas of the three peaks were 121, 245 and 46 mv respectively. The appearance of these three new peaks with different retention times after anaerobic treatment can be attributed by the cleavage of the azo bond by the consortia into aromatic compounds. It should be noted that due to the unavailability of authentic standards, the chromatographic peaks appearing in samples taken after the anaerobic system could not be identified or quantified. Therefore, it is reasonable to consider that the three peaks contain aromatic amines originating from total reduction of Reactive Red azo dye. Further HPLC analysis of the effluent after aerobic treatment at 220 nm, gives three peaks with retention times, 0.46, 0.58 and 1.25 minute (Figure 3c). The areas of the three peaks were 12, 61, 52 mv respectively. At the end of aerobic phase, the HPLC analysis seems to indicate that the decolorization metabolites produced during the anaerobic phase were removed in the subsequent aerobic phase. Figure 5c shows that when the products produced during the anaerobic stage were metabolized aerobically, a less aromatic compound was formed. This is because the product peak area of the peaks decreased tremendously.

Figure 3. HPLC chromatograms of the a) dye, b) anaerobic effluent c) aerobic effluent at 220nm

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3.3. Thin Layer Chromatography/TLC

The TLC chromatograms under UV light showed that the decolorized sample had two bands indicating that Reactive Red was cleaved into two fragments (data not shown).

3.4. Decolorization of Reactive Red by Active and Inactive Cells

In the study of active verses inactivated cells, half of the decolorized media by the isolate E1 in an anaerobic batch experiment were autoclaved and half of the remaining decolorized media were left without being autoclaved. In the study it is only active (non autoclaved) cells were able to decolorize the dye whereas inactivated cells are unable to do so. This shows that decolorization was primarily taking place by degradation than adsorption.

3.5. COD, Total Nitrogen, and NH₄⁺Measurements

In Table 1, treatment of synthetic dye wastewater with a continuous anaerobic/aerobic process showed that evolution of NH₄⁺in the subsequent aerobic process increases from the anaerobic reactor (25.6 mg/L) to the aerobic reactor (54 mg/L). The pH after the anaerobic treatment decreases to 8.92 and it increases to 9.42 after aerobic treatment (Table 1). As Figure 4 shows, the Chemical Oxygen Demand (COD) and Total Nitrogen (TN) removal efficiency after anaerobic treatment were 66.6% and % 50.2 % respectively. And the overall COD and Total nitrogen removal efficiency after aerobic treatment were 94.26 % and 77.9 % respectively.

Table 1. The ineffluent and effluent parameters of anaerobic/aerobic system

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Tables index

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Figure 4. Comparison of COD and Total Nitrogen Removal in an anaerobic and Overall

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3.6. Isolation of Isolates from the Continuous Reactor

isolate E1, which is isolated from the anaerobic reactor were tested for its decolorization efficiency, as a function of time, with various concentration of dyes ranging from 1 mg/L, 2.5 mg/L and 5 mg/L and it shows a pronounced efficiency in decolorizing reactive Red 184 in 48 h. Similarly, the decolorization efficiency of E2 and E3, which were isolated from the aerobic reactor, were tested under aerobic and anaerobic conditions, respectively. Isolate E2 decolorized the dye within four days of incubation in a shaker (data not shown). When the isolate E3 was incubated with the dye under aerobic condition, its decolorization efficiency under this condition was almost negligible. Unlike isolate E2, it was capable of decolorizing the dye in 48 hours of incubation. In order to test whether the three isolates are able to grow on the decolorized media using it as a sole nitrogen source or not the decolorized medium by isolate E1 under anaerobic condition were centrifuged at 600 g for 10 min and the pH of the supernatant was adjusted to 7.5. And then the supernatant was autoclaved and placed in to a 50 ml flask. Since the cells already utilized the carbon source during decolorization, glucose was separately autoclaved and added to each flask to a final concentration 0.1%. The three flasks were inoculated with the three isolates and incubated under aerobic condition. The sole nitrogen source for the isolates was the dye decolorization metabolite, which mainly consist of aromatic amines. Samples were taken every 12 hours and the cell growth (turbidity) was measured at 600 nm (Figure 5). All the three isolates are able to grow.

Figure 5. Growth profile of three isolates, E1, E2 and E3 on decolorization product of isolate E1 under aerobic condition

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