Bioremediation of copper contaminated soil by using biogas slurry and zeoilte

Abstract

Copper (Cu) is an important and essential element for the plants; its deficiency or excess negatively affects the plant growth. In Cu contaminated soil, plant growth promoting bacteria can improve the plant growth and development. Biogas slurry (BGS) is the residual waste material from anaerobic digestion of manures to biogas and zeolite is an aluminoslicates mineral. A series of experiments under controlled and natural environment were conducted to evaluate the hypothesis that whether the noxious effects of Cu on plant and bacterial community may be reduced by using diverse bacterial strains, BGS and zeolite. Bacterial strains, BGS and zeolite may stabilize Cu in soil and ease Cu stress on plants. Soya bean (Glycine max L.) was exposed to various levels of Cu (0, 100, 150, 200, 250 and 300 mg Cu L-1solution) to rate its effect on germination and growth of four individual varieties. Copper toxicity reduced seed germination and seedling growth of soya bean.Soya bean cultivar Ajmeri-2014 was found tolerant to Cu stress, whereas Rawal-1 was sensitive to Cu stress. The sensitive cultivar (Rawal-1) of soya bean was further used in successive experiments. The different levels of each BGS (2, 3, 4, and 5%) and zeolite (0.2, 0.4, 0.6, and 0.8%) were used to check their effect on growth of delicatesoya bean varieties exposed to different levels of Cu (0, 100, 150, 200, 300 mg Cu kg-1 soil). Biogas slurry applied at @ 4% and 5% considerablyimproved the growth of seedlings, dry and fresh weight of soya bean. Likewise, zeolite at the rate of 0.4% increased seedling growth, fresh and dry weight of soya bean under Cu stress. Forty bacterial strains were isolated through enrichment technique. The minimum inhibitory concentration (MIC) of these strains oscillatebetween 200-900 mg Cu L-1solution on Luria Bertani (LB) media patri plates and from 40, 15 strains displayed tolerance to 800 mg Cu L-1solution in LB broth which wasassessed for soya bean growth promoting activity under various Cu intensities. Cu stress hindered the soya bean growth but Cu tolerant bacteria (CuTB)considerably increased growth of soya beanunder normal and Cu contaminated soils. Due to better performance, CuTB strains viz., HM, F-2 and PIN were found the most operative in enhancingbiomass and soya beangrowth in normal and Cu polluted soil. Selected CuTB, BGS and zeolite based on preceding experiments were further evaluatedalone and in combination for increasing soya bean productivity, physiology, antioxidants and reducing Cu uptake in Cu contaminated soil (0, 150, 300 mg Cu kg-1soil) in natural environment. Development and growth, physiology and yield of soya bean were strictly affected by the externaluse of Cu, however, working of soya bean enhancedby the application of diversetype of bacteria, BGS and zeolite under normal and Cu-polluted conditions. Through the application of BGS+HM strain maximum rise in growth, physiology and yield of soya bean were recorded compared to other zeolite+HM. This treatment combination improved the relative water content, whereas reduction in electrolyte leakage by use of CuTB+BGS and CuTB+zeolite confirms their progressive role in alleviation of Cu strain on soya bean. Beside these, activities/levels of osmolytes and antioxidants,external application of Cu expressivelyenhanced. Zeolite+CuTB also helped out in mitigation of stress. However, the combined application of different bacteria, BGS and zeolite significantly increased osmolytes accumulation especially BGS and HM strain in soya bean, but the activities of catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR), greatly differed to Cu stress. Generally, the application of different bacteria, BGS and zeolite decreased the CAT, APX, GR,malondialdehyde (MDA) and superoxide dismutase (SOD) activities. It was relveaed that Cu buildup in plant partsand soil through itshigh level usage in soil, however, CuTB, BGS and zeolite provocatively reduced its available part in soil and soya beanuptake of Cu. Copper accumulation in soya bean decreased throughuse of BGS with HM and F-2. CuTB strains had positive effect for indole acetic acid, phosphorous solubilization, ACC-deamianse, exopolysacharide, catalase and oxidase activities. 16S rRNA gene based identification had shown their connection with genera Bacillus. Under normal and Cu stressed conditions, the concentration of organic acid was high in rhizosphere, because the stressed plant released these acids to tolerate Cu stress but addition of bacterial strains, BGS and zeolite was helpful under stress in controlling organic acid release in plant rhizosphere and chelating Cu. In short, the use of BGS along with HM strain was more helpful in chelation of Cu and improvement of plant growth under normal and stressed conditions as compared to control.