BIOREMEDIATION OF COPPER CONTAMINATED SOIL USING RHIZOSPHERE AND ROOT NODULE BACTERIA

Abstract

Increased industrialization and urbanization have led to heavy metal contamination of the soil. It is the matter of great concern in recent times as it poses threat to all life forms. The existing physical and chemical remediation approaches for these toxic substances are too expensive and eco-unfriendly. Hence, the focus has been shifted towards more feasible, eco-friendly and cost-effective remediation approaches. Biological remediation is one such naturally driven technology with the potential to overcome the drawbacks of other physical and chemical treatment methods. It is well known that the metal contaminated site harbours natural metaltolerant microbial population that are capable of accumulating and immobilizing the metals from the contaminated environment and restrict their availability to the other living beings. In recent times, endophytic bacteria too are gaining an alarming attention in bioremediation of heavy metal contaminated soils. Keeping this in view, in the present study an attempt has been made to investigate the bioremediation and phytoremediation potential of copper-tolerant bacteria isolated from rhizosphere soil of Cynodon dactylon L. grown in brass effluent contaminated soil. Likewise, root nodule endophytic bacteria isolated from a leguminous plant (Vigna unguiculata L.) grown in uncontaminated soil was also explored for its copper bioremediation and phytoremediation potential. Copper-tolerant rhizobacteria and root nodule endophytic bacteria showing good plant growth promoting factors (PGPF) and higher copper removal potential were chosen for the construction of phytoextraction system with plants Helianthus annuus L. var. CO4 and Vigna unguiculata L. var. RS Gomati, respectively. The efficiency of phytoextraction system was evaluated through seed germination, plant length, chlorophyll, plant biomass and copper accumulation in the plant tissues. Total six copper-tolerant rhizobacterial colonies were isolated on nutrient media after enrichment in copper (II) sulphate (50 to 600 mg/L). The isolated v rhizobacteria were gram-negative bacilli and belonged to genera Stenotrophomonas and Brevundimonas. 16S rRNA analysis identified the rhizobacteria isolates as Stenotrophomonas acidaminiphila MYS1, Stenotrophomonas acidaminiphila MYS2, Stenotrophomonas maltophilia MYS3, Stenotrophomonas acidaminiphila MYS4, Stenotrophomonas sp. MYS5 and Brevundimonas diminuta MYS6. Likewise, four copper-tolerant root nodule endophytic bacterial colonies were isolated on yeast mannitol agar after enrichment in copper (II) sulphate (10 to 500 mg/L) and were identified as Arthrobacter tumbae MYR1, Bacillus safensis MYR2, Bacillus pumilus MYR3 and Bacillus sp. MYR4. The plant growth promoting factors namely IAA production, siderophore, ACC deaminase and phosphate solubilisation were in the range of 5.4 to 7.8 µg/mL, 0.38 to 1.1 cm, 0.37 to 0.93 and 2.2 to 3.8, respectively in rhizobacteria and 5.7 to 10.2 µg/mL (IAA) and 0.21 to 0.5 (ACC deaminase) in root nodule endophytic bacteria. No siderophore production or phosphate solubilisation was observed in root nodule endophytic bacteria. The copper-tolerant rhizobacteria and root nodule endophytic bacteria showed high copper removal at pH 5 and temperature of 32.5ºC. However, the copper concentration was different and was 250 mg/L for rhizobacteria and 600 mg/L for the root nodule endophytic bacteria. The growth and copper removal studies carried out for each bacteria at the optimized pH, temperature and copper concentration displayed that the rhizobacteria Brevundimonas diminuta MYS6 and Stenotrophomonas acidaminiphila MYS4 showed the highest copper removal in the range of 94 – 95%. Likewise, root nodule endophytic bacteria Bacillus safensis MYR2 and Arthrobacter tumbae MYR1 exhibited maximum copper removal of 84%. Growth curves indicated that the bacteria achieved higher copper removal in the stationary phase. The rhizobacteria and root nodule endophytic bacteria adapted biosorption and bioaccumulation as a mechanism for copper removal. Bioaccumulation and biosorption study demonstrated that Cu bioaccumulation in the bacterial cells increased with increase in bacterial cell growth. However, biosorption being a metabolism independent process to accumulate copper even after decrease in bacterial cell growth. SEM and EDX analysis confirmed copper biosorption by the bacterial cell surface. The slimy or mucoid texture of the bacterial colonies and copper biosorption capacity of the copper-tolerant bacteria suggested possibility of EPS secretion. Stenotrophomonas acidaminiphila MYS4 showed 34.4% increased EPS secretion in copper exposed bacterial cells when compared to unexposed control bacterial cells. Likewise, Brevundimonas diminuta MYS6 showed 18.6% increased EPS secretion in copper exposed bacterial cells as compared to the control cells. More abundant production of EPS by copper exposed bacteria when compared to control demonstrated their role as a defence mechanism in Cu stress condition. Brevundimonas diminuta MYS6 showed more EPS production (738.7 mg/L) when compared to Stenotrophomonas acidaminiphila (317.6 mg/L) suggesting the vi potential role of EPS in overall copper removal by Brevundimonas diminuta MYS6. FT-IR spectroscopy revealed that hydroxyl, carboxyl, aliphatic amines, imines, alkenes and alkanes are the main dominant functional chemical groups present on EPS with potential to bind Cu ions. The efficiency of phytoextraction systems constructed with H. annuus L. var. CO4 with rhizobacteria and root nodule endophytic bacteria and V. unguiculata L. var. RS Gomati with root nodule endophytic bacteria assessed via seed germination rate, root and shoot length, chlorophyll content, plant biomass (fresh and dry weight) and Cu uptake showed that phytoextraction system, H. annuus L. var. CO4 with Brevundimonas diminuta MYS6 and V. unguiculata L. var. RS Gomati with Bacillus safensis MYR2 was the most efficient systems. Brevundimonas diminuta MYS6 improved root and shoot length by 1.47 and 1.7 fold, plant fresh weight by 9.9 fold, plant dry weight by 15.8 fold and chlorophyll by 2.1 fold, respectively when compared to un-inoculated control plant. It also demonstrated a markedly higher copper accumulation of 235 µg/g in the plant. Likewise, the phytoextraction system constructed with V. unguiculata L. RS Gomati and Bacillus safensis MYR2 showed an increased seed germination of 87.5%, root and shoot length of 6 cm and 24.7 cm, fresh and dry weight of 8.6 gm and 1.5 gm, chlorophyll of 4.7 mg/g and copper accumulation of 490 µg/g when compared to un-inoculated plants. Further the systems were evaluated for the phytoextraction and phytostabilization ability through translocation factor (TF), bio-concentration factor (BCF) and biological accumulation factor (BAF). TF was more than 1 in H. annuus L. var. CO4 inoculated with Stenotrophomonas acidaminiphila MYS4 and in V. unguiculata L. var. RS Gomati inoculated with Arthrobacter tumbae MYR1 and Arthrobacter tumbae MYR1- Bacillus safensis MYR2 co-inoculated plant, suggesting good Cu phytoextraction capability. Likewise, BCF obtained was less than 1 in all the treatments suggesting poor copper phytostabilization potential of the strains and plant. Results obtained in the present study demonstrated that copper-tolerant rhizobacteria and root nodule endophytic bacteria possess good potential for bioremediation and phytoremediation of copper contaminated soil. The phytoextraction system with V. unguiculata L. var. RS Gomati and root nodule endophytic bacterium, Bacillus safensis MYR2 proved to be competent system for copper phytoremediation from contaminated soil. From the study it can be concluded that root nodule endophytic bacteria proved to more advantageous over the rhizobacteria in terms of copper-tolerance, copper removal, promotion of plant growth and phytoextraction of copper from the contaminated soil.