INTRODUCTION:

Highly coloured synthetic dye effluents from textile, food, paper and cosmetic industries have been released into receiving water and have contaminated the water resources¹. The effluents of these industries and their disposal into receiving water causes damage to the environment as they may significantly affect photosynthetic activity in aquatic life due to reduced light penetration and may also be toxic to some aquatic life due to presence of metals, chlorides, etc., in them². Textile and other dye effluents are produced at relatively high temperature (50-60°C), even after passing through cooling steps or heat exchangers for temperature reduction³. Thus dyeing industry effluents constitute one of the most problematic wastewaters to be treated not only for their high chemical and biological oxygen demands, suspended solids and content in toxic compounds but also for colour, which is the first contaminant to be recognized by human eye^4-8.

Treatment of these effluents presents several problems mainly due to the toxicity and recalcitrance of dye stuffs. Innovative technologies, such as bioremediation, are needed as alternatives to conventional methods to find inexpensive ways of removing dyes from large volumes of effluents⁹. Many micro organisms are able to remove dye from their environment and their uptake properties have been investigated for wastewater treatment purposes^10-12. If the problem of dye toxicity to the growing cell is overcome by the use of dye-resistant organisms the continually self-replenishing system can be left to run continuously for extended period^13-16. Even though physico-chemical methods are effective in dye removal, problems such as overall cost, regeneration, secondary pollutants, limited versatility, interactions with other waste water constituents and residual sludge generation limit their usage¹⁷.

As an alternative, biological treatments are relatively inexpensive way to remove dyes from wastewater¹⁸. Bioaccumulation is defined as the accumulation of pollutants by actively growing cells by metabolism and temperature-independent and metabolism-dependent mechanism steps. Most descriptions of the dye bioaccumulation begin with or build upon a one-compartment model with a first order uptake and the elimination kinetics¹⁹.

The present paper reports the application and potential of the microorganism for treating synthetic dye under optimal conditions and to study their stability analysis.

MATERIALS AND METHODS:

Dye solution:

The dye stock solution was prepared by dissolving the powdered Reactive Red C2G 29 dye in distilled water to a final concentration of 1000 mg/L and appropriate dilutions of the stock solution were added to the media.

Microorganism and growth conditions:

The microorganism Penicillium chrysogenum MTCC 6477 used for this study was obtained from MTCC (Microbial Test Culture Collection and Gene Bank, Chandigarh) and was maintained in the Czapek’s Yeast Extract Agar medium (Sucrose 30.0 g, yeast extract 5.0 g, agar 15.0 g, K₂HPO₄ 1.0 g, NaNO₃ 30.0 g, KCl 5.0 g, MgSO₄. 7H₂O 5.0 g, FeSO₄.7H₂O 0.1 g in a litre of distilled water and were subcultured for every 30 days. It was grown in the above said medium constituents in the absence of agar. To check the decolourisation ability of the microorganism some trail experiments were performed in the presence and absence of carbon and nitrogen sources in the Czapek’s medium along with the Reactive Red C2G 29 dye.

In order to produce more resistant and efficient strain, adaptation of the cells to the progressively higher concentrations of the dye was performed. Adapted mycelial cells were obtained during the serial subcultures in growth medium supplemented with different concentrations of dye changing between 50 and 500 mg/L at a constant sucrose concentration varied for each experimental set. The culture grown in the medium containing the dye at the lowest level was transferred to the next medium supplemented with a higher concentrations of dye and thus, acclimatized to higher concentrations of dye at same sucrose concentration. Acclimation increases the inhibitory concentration level compared to the unacclimated mixed culture²⁰. When the adapted culture reached to its exponential growth phase, 1.5 mL of the culture medium was transferred to the next culture medium. The adaptation studies were repeated two times for the dye and sucrose concentration and were carried out at 37˚C containing 100 mL of the growth medium.

Bioaccumulation Experiment:

The parameters of sucrose and dye concentrations, affecting the growth and dye bioaccumulation properties of adapted growing mycelial cells were studied. After the sterilization of growth medium including molasses in varying concentrations from 5 to 15 g/L and 1.0 g/L (NH₄)₂SO₄and 1.0 g/L KH₂PO₄, a defined quantity of sterilized dye solution was added. An aliquot of (1%, v/v) of an adapted preculture harvested from the exponential growth phase was transferred to the fresh media (100 mL) supplemented with dye in varying concentrations from 50 mg/Lto 500 mg/Lat a constant sucrose concentrations. The cultivation was carried out at 37˚C in incubator for 5 days. Samples were taken at predetermined time intervals for the analysis of microorganisms and residual dye concentration in the culture media. Before analysis the samples were centrifuged at 4000 rpm for 3 min and the supernatant fraction was analyzed for residual dye.

Bioaccumulation yield is defined as the ratio of accumulated concentration of dye at the end of microbial growth to the initial dye concentration.

Percent yield=x100 (1)

Two control Erlenmeyer flasks were prepared. First control medium contained molasses without any dye to examine the growth of the mycelium. Second control medium contained both dye and molasses without inoculum to observe any reactions of the media with the dye.

Analytical methods:

During the incubation period, a 4 mL sample was taken at fixed time intervals from each flask for the analysis of microorganism and residual dye concentrations in the culture media. The samples were centrifuged to precipitate the suspended biomass at 12,000 rpm for 10 min. The concentration of the dye in the supernatant fraction was analyzed by determining the absorbance at 540 nm. Absorbance measurements were done by using a UV-VIS Spectrophotometer (ELICO-SL156). Dye free molasses medium is used as the blank.

The centrifuged cells were washed and resuspended in distilled water and the suspension was diluted to 4 ml again. All the experiments were carried out at least two times. The values used in calculations were mostly the arithmetic average of the experimental data.

Application of Monod Model:

The relationship between the specific growth rate (µ), the maximum specific growth rate (µ_m) and the limiting substance (sucrose) concentration (S) in the absence of inhibitory substance often assumes the form of saturation kinetics (K_S) and can be described by the Monod equation ²¹.

(2)

When sucrose is used as the main substrate in the medium in the presence of higher concentrations (C₀) of inhibitory substances such as dyes, microbial growth becomes inhibited, and specific growth rate depends on inhibitor concentration. The following competitive inhibition model describing dye component inhibition was selected for assessing the dynamic behaviour of mycelial cells.

(3)

The value of inhibition constant of dye (K_I) could also be estimated by non-linear regression techniques using the data on the specific growth rate obtained at different initial dye concentrations at a constant sucrose concentration changed for each experimental set assuming S=S₀ at the beginning of the exponential growth.

Traditional description of the model starts with the differential equation for the change in concentration in the source (C_s) and in the organism (C) with time and expressed as a function of uptake rate constant (k_u), elimination rate constant (k_e) is given by the Eq. 4 ²² .

(4)

Integration of Eq.3 results in the familiar monoexponential model.

(5)

This model predicts a monotonic increase in concentration until a steady state equilibrium concentration is reached (dC/dt=0). The stability of the equilibrium depends on the relationship between the elimination constant (k_e) and time lag (T). If 0 < k_eT < e^-1, the model predicts a monotonic damping as the concentration approaches equilibrium. However, if e^-1 < k_eT < π/2, the concentration will approach equilibrium through a series of exponentially damped oscillations ²³.

Continuous system for stability criteria:

A 5.5 L volume column reactor was sterilized by UV radiations for 5 min and packed with 100 mm average diameter laterate pebbles which were acid sterilized using 1.0 N H₂SO₄, washed with distilled water. It was then aseptically transferred into the reactor. 100 ml of the inoculum, before starting their exponential phase was inoculated into the reactor with several divisions. The growth medium (molasses media) was added into the column up to its surface of the laterate pebbles. It was incubated aseptically for a day. Then the suspended broth was replaced with fresh media. This was repeated till a constant biomass was achieved over the surface of the laterate pebbles. Now, the dye with enriched media was then passed into the reactor through peristaltic pump and the outlet sample was analyzed with a regular time interval of 4 h. The process was continued till the saturation point was attained.

RESULTS AND DISCUSSION:

Effect of carbon and nitrogen source on dye decolourisation mechanism:

The growth of the fungus in the decolourisation medium containing all the constituents of Czapek’s medium in a litre of distilled water but the absence of nitrogen source; no mycelial biomass production and dye decolourisation/removal were observed. Similarly the above trial was performed in the presence of nitrogen sources and other nutrients from the Czapek’s medium but in the absence of carbon source; no microbial growth and dye decolourisation/removal were observed. The results indicates (data not shown), the fungus could not utilise the dye as carbon and nitrogen source in the absence of any other carbon and nitrogen sources and thus the chemical structure of the dye was not bio degraded by actively growing mycelia of P. chrysogenum.

Effect of initial sucrose concentration on the specific growth rate and percent yield of Penicillium chrysogenum at a constant concentration of Reactive Red C2G 29 at pH 5:

The growth behaviour of Penicillium chrysogenum was investigated at pH 5.0 at increasing sucrose concentration in the presence of a constant dye concentration. The initial sucrose concentration was changed from 5 g/L to 15 g/L and the initial dye concentration was kept constant between 50 mg/L to 500 mg/L for each experimental set up at pH 5.0. The variation of microbial growth rate (µ) with initial sucrose concentration at constant dye concentration level (Fig. 1), we observed that the rise in initial sucrose concentration up to 15 g/L both in the absence and in the presence of constant dye concentration at changing level increased the specific growth rate of Penicillium chrysogenum. Higher specific growth rates were observed in the media without the dye.

Fig. 1. Effect of initial sucrose concentration (S₀) on the specific growth rate of P. chrysogenum at a constant Reactive Red C2G 29 concentration at pH 5.0, (¿) C₀= 0 mg/L, (¢) C₀ = 50 mg/L, (p) C₀ = 100 mg/L, (¯) C₀ = 275 mg/L, (r) C₀ = 500 mg/L.

Fig. 2. Effect of initial sucrose concentration (S₀) on the percent yield of P. chrysogenum at a constant Reactive Red C2G 29 concentration at pH 5.0, (¿) C₀ = 50 mg/L, (¢) C₀ = 100 mg/L, (p) C₀ = 275 mg/L, (●) C₀= 500 mg/L.

The presence of dye in the growth medium caused an increase in the lag and log period and therefore a reduction in the mycelial growth rate in all the three different pH, showing thereby that the dye bioaccumulation is a metabolic activity. This suggests that sucrose plays a vital supportive role for the growth as an enriched carbon source. The increase in growth with increasing sucrose concentration could be due to cell defence mechanism and the presence of sufficient nutrient.

The dye uptake yield was affected significantly (Fig. 2) by initial sucrose concentration when the parameters increased considerably up to 15 g/L sucrose concentration for each constant dye concentration studied. The increase in sucrose concentration brings about an increase in the percent yield of the organism.

Effect of initial Reactive red C2G 29 concentration on the specific growth rate and percent yield of Penicillium chrysogenum at a constant concentration of sucrose at pH 5:

The growth behaviour was further investigated with five different initial dye concentrations of 0 mg/L, 50 mg/L, 100 mg/L, 275 mg/L and 500 mg/L with a constant sucrose concentration of 5 g/L, 10 g/L and 15 g/L at pH 5.0 for each experimental set. We observed that the specific growth rate were maximum at pH 5.0 in the absence of dye (Fig. 3). It is evident that the sucrose concentration has a major role in the growth and decreased the inhibitory effects of dye on the mycelial growth (Fig. 3 and 4). That is in 5 g/L sucrose containing growth medium at pH 5.0 with raising concentration from 0 mg/L to 500 mg/L, the specific growth rate diminished.

Fig. 3. Effect of initial reactive red dye C2G 29 on the specific growth rate of P. chrysogenum at a constant sucrose concentration (S₀) and at pH 5.0, (¿) S₀= 5 g/L, (¢) S₀ = 10 g/L, (p) S₀ = 15 g/L.

Initial dye concentrations affect the efficiency of dye uptake through a combination of factors including toxicity of dye. Due to the inhibition effect of the dye on the growth of the microorganism the dye uptake yield decreased with increase in the initial dye concentration in the medium though the amount of dye accumulated by Penicillium chrysogenum increases with the rise in the dye concentration in the medium at pH 5.0.

Fig. 4. Effect of initial reactive red C2G 29 on the percent yield of P. chrysogenum at a constant sucrose concentration (S₀) and at pH 5.0, (¿) S₀= 5 g/L, (¢) S₀ = 10 g/L, (p) S₀ = 15 g/L.

Stability criteria:

The dye bioaccumulation properties of Penicillium chrysogenum were investigated in a continuous system as a function of its stability criteria using a time lag of 4 h. Eq. 4 is modified to Eq. 6 to describe a continuous bioaccumulation model with a time lag (T).

(6)

The methods those outlined in for establishing stability regions of differential population growth models can then be applied directly to bioaccumulation²³. Thus Eq. (6) can be written as,

(7)

The equilibrium concentration, C* can be evaluated by setting dC(t)/dt=0. The continuous bioaccumulation experiment carried out till the saturation point achieved (248^th h), with equilibrium concentration of 49 mg/L obtained from 196^th h (Fig.5).

Fig. 5. Dynamics of single compartment model exhibiting a monotonic damped oscillation.

The value of k_eT obtained was 0.0612; thereby suggesting that the bioaccumulation follows monotonic damping oscillations. Thus the experiment elucidates that the microorganism used is tolerant to higher concentrations of Reactive Red C2G 29 dye.

CONCLUSION:

In this study, the growth and the dye intake capability of Penicillium chrysogenum was analyzed and the level of dye bioaccumulation was found highly dependent on both initial sucrose and initial dye concentrations thereby showing tolerance to high dye concentrations. The stability criteria studied for the organism using the continuous system further indicated that the organism is tolerant to Reactive Red C2G 29 dye at its higher concentrations over a nominal time period. All the above results suggested that Penicillium chrysogenum has a significant potential for the removal of dye from the contaminated water.

ACKNOWLEDGEMENT:

The authors are indebted to all the colleagues, especially those from the Department of Biotechnology, Madha Engineering College for their valuable suggestions on the manuscript and laboratory work.

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Received on 10.04.2011 Accepted on 21.05.2011

Research J. Engineering and Tech. 2(3): July-Sept. 2011 page 167-171