Bioremediation of acid fast red dye by Streptomyces globosus under static and shake conditions

Two different azo dyes known as acid fast red (AFR) and Congo red (CR) were examined for their decolorization by five strains of actinomycetes (Streptomyces globosus, Streptomyces alanosinicus, Streptomyces ruber, Streptomyces gancidicus, and Nocardiopsis aegyptia) under shake and static conditions. Streptomyces globosus decolorized AFR by 81.6% under static condition while 70.2% dye removal was achieved under shake conditions. Application of Plackett-Burman statistical design revealed that the main factors that affected biosorption capacity were the starch concentration and the inoculum size. Under static conditions, increasing the inoculum size and decreasing starch concentration increased the biosorption % up to 1.14 fold with time reduction, while increasing both the inoculum size and starch concentration under shake conditions increased the biosorption % up to 1.09 fold only. A trial for the use of potato peel for more economic biomass production of S. globous was carried out and (2 g/50 ml) and dried potato peel had the optimum concentration for maximum biomass production (0.3 g/50 ml) which led to considerable biosorption capacity (89.4%). Electron microscopy studies confirmed the dye removal.


INTRODUCTION
Azo dyes are widely used in industries, such as textiles, paper, plastics and leather, industries, etc, for the coloration of products.The effluents emanating from these industries often contain high concentrations of dye wastes.The release of colored wastewaters represents a serious environmental problem and a public health concern (Dos Santos et al., 2007;El Ahwany, 2008).
Dyes are generally believed to be toxic and carcinogenic or prepared from other known carcinogens.The discharge of these dye stuffs from industries into rivers and lakes results in a reduced dissolved oxygen concentration causing anoxic conditions, which subsequently affect aerobic organisms (Chander and Arora, 2007;Vijayaraghavan and Yun , 2008).
Adsorption and precipitation methods, chemical degradation or photo degradation are financially and often also *Corresponding author.E-mail : nermeen_ok@yahoo.co.uk;Tel.0106620217.
Abbreviation: AFR,Acid fast red; CR, Congo red.methodologically demanding, time-consuming and mostly not very effective.As a viable alternative, biological processes have received increasing interest owing to their cost effectiveness and environmental friendliness (Mabrouk and Yousef, 2007).
Actinomycetes strains have been identified which decolorize effluents containing different types of reactive dyes.Adsorption of anthraquinone, phthalocyanine and azo dyes to the cells of some of the strains resulted in the decolorization of the effluents, but no degradation of the dyes was observed (Aksu, 2005).
There are large numbers of reports on the optimization of carbon and nitrogen source by the classical method of medium optimization that changes one independent variable, while fixing other variables at definite levels.Optimizing all the affecting parameters by statistical experimental design, such as Plackett-Burman methodology can eliminate the limitations of single-factor optimization process collectively (Urvish and Akshaya, 2010).
Potato peels contain considerable amounts of carbohydrate which stimulate the cells to express many hydrolytic enzymes .In addition it contains appreciable   (Mabrouk and El-Ahwany, 2008).This work attempted to formulate a suitable media using Plackett-Burman statistical design to increase biosorption of acid fast red by Streptomyces globosus under static and shake conditions and evaluated its biosorption potentiality by growing it on economic sources.

Actinomycetes strains and dye
Five actinomycetes strains were used in the investigation.These were Streptomyces globosus, Streptomyces alanosinicus, Streptomyces ruber, Streptomyces gancidicus, and Nocardiopsis aegyptia, which were isolated from Burollus Lake and the Mediterranean Sea sediments and was kindly provided by Dr. Gehan Abou Elela (Associate professor of marine microbiology, National Institute of Oceanography and Fisheries, Alexandria -Egypt).Two dyes were used in this work; acid fast red and Congo Red (Figure 1) which were obtained from the Asma Company, Kafr El-Dawar city, near Alexandria, Egypt.Dye stock solutions were prepared in water and autoclaved for 15 min at 121°C.

Culture medium and screening test
Actinomycetes strains were cultivated on starch-nitrate medium which had the following constituents: starch, 20 (g/l); KNO3, 1(g/l); K2HPO4, 0.5 (g/l); MgSO4.7H2O,0.5 (g/l); FeSO4, 0.05(g/l); and agar, 15 (g/l) (in case of solid maintenance medium).All constituents were dissolved in the Lake or sea water (pH 7).The media broths were inoculated with 1 ml of seed cultures and incubated at 30°C for six days.A screening test for the biosorption percentage of all the strains was applied for both dyes under shake and static conditions.

Measurement of decolorized dye
After centrifugation of cultures broths, cell pellets were collected from six days culture, washed twice in sterile water and 0.1 g of washed pellets was suspended in dye solution (50 mg/ml), at both static and shake conditions.All tests and their replicates were incubated at 30°C for 2 h, during which the cells were colored due to the uptake of the dye.The mixtures were centrifuged and the residual dye colors in the supernatants were measured according to the dye wave lengths (Table 1).
Biosorption percentage was calculated as follows: (C0 − Ce ) x 100 Biosorption % = C0 C0 = Initial absorbance reading before decolorization and Ce = final absorbance reading after decolorization.The concentrations of the residual dyes in the supernatants were determined by using a standard curve.All results are the mean of replicates (El Ahwany, 2008).

Optimization experiment
The effect of medium components on dyes biosorption was studied by applying the Plackett-Burman (1946) experimental design.In this experiment, seven factors (medium components) were screened in eight combinations organized according to the Plackett Burman design matrix (Table 3).Increase of the original component level is represented by the (+) sign, while decrease of the original component level is represented by (-) sign.The main effect of each factor was determined using the following equation: Where Exi is the variable main effect, and Mi+ and Mi-are the biosorption % and the wet weight (g/ml) in the trials, where the independent variables were present in high and low concentrations, respectively, and N is the number of trials divided by two.Statistical t-values for equal unpaired samples were calculated using Microsoft Excel to determine the variable significance.From main effect results, an optimized medium was predicted which will give maximum biosorption %.Verification test was done to confirm the validity of the optimized medium.Results of the verification test were recorded after different time intervals.
Biosorption capacity was calculated according to the following equation: Concentration of acid fast red biosorbed by cells (mg/ml) Biosorption capacity = mg biomass

Biomass production using agricultural waste
A trial for using potato peels for more economic biomass production of S. globous was carried out.Optimized media (free of starch and KNO3) with different concentrations (0.75, 1, 2, 3, 4 and 5 g/50 ml) of dried, finely grounded potato peel were prepared.Biomasses were collected after six days incubation, washed twice with sterile distilled water and were subjected to dye solution (50 mg/ml).Results were recorded in the static condition (Mabrouk and El Ahwany, 2008).

Transmission electron microscope (TEM) studies
Fresh samples of S.globosus were fixed using a universal electron microscope fixative as described by McDowell and Trump (1976).Series dehydration steps were followed using ethyl alcohol and propylene oxide.The sample was then embedded in labeled beam capsules and polymerized.Thin sections of cells with adsorbed dye were cut using LKB 2209-180 ultra microtome and stained with a saturated solution of urinyl acetate for half hour and lead acetate for 2 min (McDowell and Trump 1976).The procedure was applied to  the control cells not exposed to dye solution and to dye-exposed cells to observe the location of the dye.Electron micrographs were taken using a transmission electron microscope (JEM-100 CX Joel), at the Electron Microscope Unit, Faculty of Science, Alexandria University, Egypt.

Screening tests
Acid fast red and Congo red were screened for their biosorption % by five actinomycetes strains under static and shake conditions.The results in Table 1 revealed that the highest biosorption percentages were achieved under static conditions.Moreover, S. globous was the most efficient strain that gave the highest biosorption % (81.6) with acid fast red (50mg/l) which was selected for use in the examination of different factors controlling biosorption %.

Plackett-Burman optimimization experiments
Growth factors and their levels (Table 2) which affect acid fast red biosorption by S. globous, were screened using the Plackett-Burman design.The results achieved from the several combinations matrix of Plackett-Burman design (Table 3) revealed that, biosorption % under static conditions was higher than that obtained under shake conditions.Table 4 records the quantitative results of the Plackett-Burman design for S. globosus biomass (g/ml) and its corresponding dye removal (biosorption) (mg/ml).
In spite that the results clarified that acid fast red biosorption capacity under static condition was higher than that under shake condition, it was noticed that, the biomass production was mostly the reverse.Calculation of the biosorption % main effect (under static and shake conditions) and its corresponding t-value (Table 5) revealed that inoculum size and starch concentration were highly significant variables.
The interaction effect between starch concentrations and inoculums sizes was plotted to illustrate their effect on biosorption % under static conditions (Figure 4).The figure showed that, increase in the inoculum size with the low concentrations of starch gave maximum dye biosorp-   tion % while under shake conditions (Figure 5) increase of the inoculum sizes with increased concentrations of starch gave maximum biosorption %.

Verification experiment
To verify the results obtained from the statistical analysis of Plackett-Burman design, a verification tests were performed in duplicates using the predicted optimized media against the basal condition media under static condition (Table 6).At different time intervals, the results obtained revealed that, after only 1 h, biosorption % was elevated to 1.14 fold increase than that in the case of basal condition with time reduction (from 2 h to 1 h) which  indicated the validity of the design while under shacked condition, biosorption % was elevated to only 1.09 fold increase after 2 h of contact (data not shown).These results recommend the application of static condition in AFR biosorption by S.globosus.

Transition electron microscope (TEM) studies
Figure 6 shows the TEM micrographs of the native and dye exposed cells of S. globosus.The control cells (a) appeared with regular cell wall and no dense areas were seen.Cells exposed to acid fast red dye (b) under static conditions showed dense dark areas distributed inside the cell.

Potato peel as an economic source
Chemical analysis of potato peels (Table 7) revealed that, carbohydrates constituted the highest % of its dried materials (64.47%).The protein was 13.52% while all other components were minors.Different potato peel concentrations were tested as an economic source for S. globosus biomass production for AFR biosorptionl process.Figure 7 shows that, the use of 2 g of dried potato peel was the optimum concentration for biomass production under static conditions (0.3 g/50ml), and led to maximum dye removal (89.4%), while more increase in potato peel concentrations gave lower biomass production, and less biosorption efficiency.

DISCUSSION
Azo compounds constitute the largest and the most diverse group of synthetic dyes and are widely used in a number of industries (Pandey et a., 2007;El Ahwany 2008).Azo dyes can still be removed from wastewater by the microbial biomass via the process of biosorption.Biosorption by actinomycetes strains is also becoming a promising azo dyes removal process from wastewaters (Aksu, 2005;Abou-Elela, 2006).This study provides evidence that metabolizing cells of S. globosus biomass are capable of removing color from the solutions of the two tested azo dyes; Congo red and acid fast red.Acid fast red was removed with the highest decolonization percentage (82%) under static condition.
The results proved that static conditions are the most     t-value significant at the 1% level = 3.70; t-value significant at the 5% level = 2.45; t-value significant at the 10% level = 1.9; t-value significant at the 20% level = 1.37 powerful tool in removing azo dyes.These results agreed with that of El Ahwany (2008), in the study on decolonization of fast red acid by metabolizing cells of Oenococcus oeni ML34 where dye removal was also under static conditions.Sani et al. (1998) disagrees with the results of this study.Decolonization rates for all the dyes in static condition were found to be less than the shake culture and also were dependent on biomass concentration.
Increase in the inoculums size led to an increase in biosorption capacity.These results agreed with that of Mohana et al. (2008) and Mabrouk and Yusef (2008); they used response surface methodology for optimization of medium for decolonization of textile dye direct black 22 by a novel bacterial consortium.Their results revealed that decolonization values above 80% were observed when high concentration of glucose and inoculum size was applied.Statistical analysis also revealed that low starch addition (10g/l) had a significant effect.The results of this study, also agreed with that of Abedin (2008); addition of low concentration of starch (5 g/l) gave crystal violet decolonization of 96% by F.solani while, previous results indicated that the availability of a supplementary carbon source seems to be necessary for faster growth and decolorization (El-Sersy, 2001;Mohana et al., 2008).
The results revealed that, high concentration of K 2 HPO 4 and low concentration of KNO 3 increased the availability of dye removal by S.globosus, but Abedin (2008) reported that, Malachite Green removal by F. solani increased by increasing the concentration of NaNO 3 and K 2 HPO 4 .In addition, increasing K 2 HPO 4 and decreasing MgSO 4 concentrations in this study enhanced dye decolourization.On the contrary, increasing K 2 HPO 4 and MgSO 4 concentrations led to a positive effect on AFR decolorization by B.subtilius (Mabrouk and Yusef , 2008).
The verification test revealed that, after only 1 h contact of S. globosus biomass with dye solution, a higher decolorization of 90% was achieved.Longer contact time revealed low decolorization which may be due to desorption of dye (Acemioglu et al., 2010).
Potato peels are agro-industrial by-products that could have good biotechnological potential application.Nevertheless such waste was not tested extensively in previous studies (Mabrouk and El Ahwany, 2008).Therefore, it was used for economic studies as a carbon and nitrogen source replacement for starch and KNO 3 .
It is worthy to mention that a powerful removal of AFR (89.4%) by S. globosus biomass (0.3 g wet weight/ ml) was achieved after 1 h which may be promising for using in industrial bioremediation.

Figure 1 .
Figure 1.Chemical structure of acid fast red (AFR) and Congo red (CR) azo dyes.

Figure 2 .
Figure 2. The main effects of different factors affecting acid fast red biosorption (%) by S. globosus under static conditions.

Figure3.
Figure3.The main effects of different factors affecting acid fast red biosorption (%) by S. globosus under shake conditions.

Figure 6 .
Figure 6.Transmission electron micrographs showing (a) S. globosus cells in absence of dye and (b) S. globosus cells in dye solution after 1 h contact.

Figure 7 .
Figure 7. Effect of different concentrations of potato peel on growth of S. globosus and the equivalent AFR biosorption (%).

Table 1 .
Screening of actinomycetes strains and their biosorption % for acid fast red and Congo red.

Table 2 .
Screening for growth factors affecting acid fast red biosorption by S. globosus and their levels in the Plackett-Burman experiment.

Table 3 .
The applied Plakett-Burman experimental design for the seven culture variables with its biosorption (%).

Table 4 .
Quantitative results of the Plackett-Burman design for S. globosus biomass and dye biosorption.

Table 5 .
Statistical analysis of the Plackett-Burman experimental design results.

Table . 6
. Verification experiment for AFR biosorption (%) by S. globosus cells which were grown on basal versus optimized medium under static condition.Results were obtained under static condition. 2an optimum medium formula was predicted according to the results obtained from the Plackett-Burman experiment. 1

Table 7 .
Proximate chemical composition of potato peel.