A STUDY ON BIODEGRADATION OF ROOT NODULATING RHIZOBIUM SP Dr. Bharat Jinturkar,
Head Department of Botany and Principal, Late K.G. Kataria College Daund, Pune, Maharashtra, India
Abstract:- Material color contamination is an issue of ecological concern. . In spite of the fact that organisms have been broadly investigated for their color corrupting capacities, little work has been accounted for utilizing root modulating microorganisms. In the current investigation, an endeavor has been made to utilize non-pathogenic, nitrogen fixing soil microbes for biodegradation of material color, Direct Blue 71 (DB71). The 11 secludes of Rhizobium demonstrated potential to decolorize the triazo color DB71. Of these, separate TUR2 (Gen Bank increase no. JF820124) demonstrated the most noteworthy (95%) capacity to decolorize the color. Over expression of oxidative and reductive enzymes in the presence of the dye indicated their role in degradation which was confirmed using analytical techniques like HPLC, FTIR and GC-MS. Phytotoxicity studies have shown these dye metabolites to be non-toxic. Results of this study indicate potential use of Rhizobium sp. in biodegradation of textile dye, and suggest further investigation towards developing a dual technology of bio fertilization coupled with bioremediation.
Keywords:- Bio fertilizer, Bioremediation, Pollution, Root modulating bacteria, Soil restoration.
1. INTRODUCTION
Colors are generally utilized in different ventures, for example, material, tanning, paper, food, hair shading and makeup.
Despite the fact that common colorants, for example, bacterial shades are arising as better other option, manufactured colors are still in wealth in different enterprises. Azo colors are a significant class of colors which are sweet-smelling mixes with at least one (-N=N-) gatherings and speak to about 70% of the general engineered colors delivered.
Nonetheless, during preparing, a lot of colors are legitimately lost into the water contingent upon the color application class. The misfortunes can shift from 2% when utilizing fundamental colors to practically half with certain responsive colors. More often than not, particularly in non-industrial nations, this debased water isn't just arranged straightforwardly in amphibian bodies after essential treatment yet additionally utilized in horticultural fields.
Notwithstanding their impact on the photosynthetic movement in amphibian life brought about by diminished light infiltration, colors likewise have an effect as far as the compound oxygen interest.
Studies have revealed that azo dyes cause toxic, genotoxic, mutagenic and carcinogenic effects to the aquatic organism as well as animals. Physical and chemical methods like adsorption, coagulation, flocculation, filtration, oxidation and electrochemical methods
can be used for removal of dyes from polluted waters7. Nakkeeran et al. have recently demonstrated zinc oxide (ZnO) nano-composite for effective dye removal from synthetic and textile industrial wastewater. However, physical methods create secondary pollution problems resulting from the accumulation and concentration of dyes. Though chemical processes destroy the dye, they are quite expensive.
Microbial degradation of dyes is a cost effective and ecofriendly method compared to both physical and chemical methods. Various fungi and algae have been used in dye degradation. However, adsorption plays a major role in both the cases, thus leaving the dye in the environment. White rot fungi are mainly studied for dye degradation, but they require nitrogen limiting conditions and longer time periods for decolourization, thus limiting them to laboratory levels.
Hence, features like fast growth and easy adaptability without any disposal problems make bacteria a preferred system for dye degradation.
Recently, Saini and co-workers have reported high decolourization potential of two bacterial isolates, Alcaligenes faecalis and Bacillus flexus against Azo dyes and recommended their application for bioremediation of textile effluents. Rhizobium is a common soil bacterium with agricultural importance as a bio fertilizer. They form a symbiotic
association with the roots of leguminous crops leading to formation of nodules, and thereby improve the soil fertility through nitrogen fixation. They are ecofriendly and non-pathogenic. Hence, its biomass, if disposed in the soil, will be an additional advantage unlike other pathogenic isolates used for dye decolourization.
Thus, Rhizobium can be used for biodegradation of textile dyes as well as restoration of soil quality. Considering these factors, the present study was carried out to screen Rhizobium sp. for possible use in biodegradation of triazo dye Direct Blue 71, most commonly used in textile industry.
2. MATERIALS AND METHODS 2.1 Dye and Chemicals
Textile dye DB71 (Dye content 80%) was a generous gift from local industry, Avani Dye Chem. Pvt. Ltd. Mumbai, India. The chemical 2, 2’ w-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid) was obtained from Sigma (USA). Nicotinamide Adenine Dinucleotide was obtained from Sisco Research Laboratory, India. The laboratory media were obtained from Himedia, Mumbai, India. All other chemicals used in our study were of analytical grade.
2.2 Culture Isolation
Rhizobium societies were detached from root knobs of Sesbania sesban from different modern regions around Navi Mumbai. The solid and whole root knobs were chosen in the wake of washing it completely under faucet water. They were then submerged for 5-10 s in 95% ethanol (to break the surface pressure and eliminate air rises from the tissue) and surface cleaned by absorbing sodium hypochlorite (2.5% v/v) answer for 2-4 min.
The root knobs were then flushed with refined water multiple times and squashed in a test tube utilizing glass pole keeping up aseptic conditions. The exudates were additionally streaked on Congo Red Yeast extricate Mannitol agar (CRYEMA) and brooded till development showed up at 25±2°C. Lackluster or white settlements were gotten for additional studies177. Distinguishing proof of the separates was completed by 16SrRNA quality arrangement investigation and the groupings were stored with NCBI.
2.3 Dye Decolourization Experiments Screening for the Most Efficient Strain All the 11 isolates were grown till their late log growth phase (24 h) at 25°C in 50 mL Tryptone yeast extract (TY) broth on a rotary shaker (120 rpm). The cultures were then supplemented with 30 mg L-1 DB71 and incubated under static condition. Aliquots were withdrawn at 0 h and every 24 h up to 168 h.
These aliquots were then centrifuged at 10000 rpm for 15 min. The absorbance of the supernatant was measured using UV-Visible spectrophotometer at 585 nm (λmax for DB71) for calculating the percent decolourization. Control samples were also prepared with 30 mg L-1 dye in TY medium without culture and were run in parallel with the assay. Decolourization was expressed in terms of percentage and was calculated as follows:-
% decolourization = [(initial absorbance – final absorbance) / initial absorbance] ×
100.
The isolate with maximum decolourization ability was used for further dye degradation studies. All the experiments were carried out in triplicates and the observations were recorded as mean with standard deviation.
2.4 Decolourization at Static and Shaking Conditions using Rhizobium sp. TUR2
The decolourization ability under static and shaker (120 rpm) condition were also tested for the selected isolate of Rhizobium sp. using 30 mg L-1 of DB71.
The isolate was grown until the exponential growth phase before inoculation. The decolourization assay was done in the same manner as mentioned above. The differences in the % decolourization under both the conditions were recorded. Control samples without culture were used as biotic controls for the assay.
3. RESULTS AND DISCUSSION 3.1 Culture Isolation
A sum of 11 bacterial disengages were gotten from the root knobs of Sesbania sesban (L.) developing around different modern regions present in the zone of our investigation. The S. sesban culture is normally utilized as green compost for soil rebuilding in light of its root nodulating
microbes. Rhizobia nodulating S. sesban has demonstrated a wide hereditary variety with separates having succession comparability to rhizobia having a place with genera Rhizobium, Mesorhizobium, Sinorhizobium and Allorhizobium24. In the current examination, the BLAST results for 11 confines indicated most extreme succession arrangement with the genera Rhizobium.
3.2 Dye Decolourizaton Experiments Screening for the Most Efficient Isolate The decolourization productivity of the apparent multitude of 11 strains was checked by estimating the absorbance after each 24 h of brooding for 168 h. All the strains were discovered to be compelling in decolourization of the color with a normal of over 75% color decolourization. Seclude TUR2 (Gen Bank promotion no. JF820124) was discovered to be generally effective, demonstrating 95% decolourization in 168 h and was utilized for additional color debasement examines.
A few microorganisms have been found to decolorize, change or even totally mineralize azo colors. Numerous bacterial species have indicated the capacity to decolorize a wide scope of material colors.
Elisangela et al. revealed decolourization of DB71 in 48 h and 168 h utilizing Staphylococcus arlettae strain VN-11 and Klebsiella sp. strain VN-31, individually.
Decolourization contemplates utilizing Rhizobium sp. TUR2 Decolourization of DB71 by TUR2 was discovered to be higher (95%) under static condition when contrasted with shaker condition (10%).
A similar trend of enhanced decolourization of suffocated azo dyes, under aerobic conditions by bacteria and fungus, has also been described by Kalme et al. and Eichlerova et al. A study reported that under aerobic conditions azo dyes are resistant to attack by bacteria. Azo dye decolourization is an enzymatic process involving the reduction of azo bond. Under aerobic conditions, the presence of oxygen leads to competition between oxygen and azo compounds for the reduced electron carriers.
Hence, the reduction of azo bond is inhibited. Dye decolourization rate is greatly influenced by its chemical structure. Highly substituted and high molecular weight dyes are more difficult to treat. It has been reported that the azo
dyes with hydroxyl or amino group are more likely to be degraded than those with methyl, methoxy, sulfo or nitro groups. DB71 is a triazo dye substituted with sulfo group.
3.3 Enzyme Activities during Decolourization of Dye
The role of oxidoreductive enzymes, such as lignin peroxidase, laccase, tyrosinase, azoreductase, NADHDCIP reeducates, riboflavin reductase and aminopyrine N- demethylase in bacterial decolourization and degradation of azo dyes have been well reviewed by Saratale et al. For fungal cells, the major enzymes for biodegradation are lignin peroxidase, laccase and manganese peroxidase in the present study, induction in enzyme activities of lignin peroxidase (2.5 fold) and azoreductase (3 fold) was observed at 72 h.
The intracellular enzyme activity of azoreductase was observed to be higher as compared to its extracellular activity.
Kalyani et al. also reported similar induction of intracellular lignin peroxides and azoreductase during biodegradation of Reactive Red 2 by Pseudomonas sp.
Azoreductase has been reported as the key enzyme responsible for reductive azo bond cleavage in bacterial species.
Increase in azoreductase activities was also observed in Kocuria rosea MTCC 1532 for decolourization of suffocated azo dye methyl orange. The lignolytic enzyme systems have been widely studied in fungi for degradation of polymeric dyes. Recent reports have also demonstrated the role of bacterial lignin peroxides in decolourization of suffocated azo dyes like Direct Red 5B and direct brown MR. The presence of a very low concentration of dye after 72 h incubation could be the reason for decreased enzyme activities thereafter. Thus, it can be presumed that lignin peroxides and azoreductase are the major enzymes responsible in decolourization of DB71 by Rhizobium sp.
TUR2.
3.4 Biodegradation analysis
Decolourization of dyes using bacteria could be due to adsorption of the dye or biodegradation. During adsorption, the cell biomass becomes deeply coloured because of the adsorbed dye whil
e in case of biodegradation, the original
biomass color is retained. UV-Vis spectral analysis (200-800 nm) of the supernatant, at different time intervals, showed a decrease in peak area at 585 nm (λmax for DB71) and disappearance of peak after 120 h (Fig. 1). The biomass, after decolourization, showed no uptake of the
color of DB71 and remained colourless.
Asad et al. stated that, in biodegradation, the major visible peak disappears completely. The above results suggest that probably the decolourization is a result of biodegradation of DB71.
Fig. 1 - UV-Vis spectra of DB71 at 0h and after decolourization (120 h) The HPLC chromatogram of the DB71
gave a single peak at retention time (RT) 1.679 min, whereas the biodegraded products of DB71 extracted after 120 h was observed as two major peaks and one minor peak at RT 3.062 min, 3.470 min and 2.342 min, respectively. These observed peaks confirmed the degradation of DB71. Kalme et al. obtained five peaks in HPLC analysis during biodegradation of Direct Blue 6.
3.5 Phytotoxicity studies
In India, the emanating delivered from material businesses is as yet utilized in farming, particularly in the regions that are situated in the region of these ventures. Despite the fact that natural medicines are compelling, now and again a few mixes may get changed into more poisonous finished results. Subsequently, it is imperative to investigate the idea of these finished results. The phytotoxicity study uncovered a lower germination rate in the seeds of P. mungo (70%) and T.
aestivum (half) on introduction to DB71 contrasted with those presented to the corrupted color metabolites (90%).
The mean plumule and revolutionary length in DB71 treated plants were additionally discovered to be lower than that got for the metabolite treated plants. These outcomes show the non-harmful nature of biodegraded results of DB71 by Rhizobium sp. TUR2.
A comparative decrease in germination
rate, plumule and revolutionary length was gotten for responsive blue 8 (RB8) treated seeds contrasted with the debased color metabolites.
4. CONCLUSION
The current examination showed the color corrupting capacities of Rhizobium sp.
TUR2. It was seen that TUR2 could biodegrade the azo color DB71. Two proteins viz azoreductase and lignin peroxidase assumed part in biodegradation of the color. The biodegraded items were discovered to be non harmful in nature. The huge part of this Rhizobium sp. In color debasement, as announced in our examination, gives another road into examinations identified with the capability of utilizing these understand bio fertilizers for biodegradation of material colors.
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