The Use of Purple Non-Sulfur Bacteria as Biofertilizers for Reducing Heavy Metals in Rice Plant and Global Warming

Abstract

Purple non-sulfur bacteria (PNSB) are attractive biofertilizers as they can reduce heavy metals in rice plant and greenhouse gases. A total of 235 PNSBs were isolated from various paddy fields contaminated and un-contaminated by cadmium (Cd) and/or zinc (Zn). Only Rhodopseudomonas palustris TN110 and Rubrivivax gelatinosus TN414 showed great potential as biofertilizers releasing NH4+ via N2 fixationand plant growth promoting substances including 5-aminolevulinic acid (ALA) and indole-3-acetic acid (IAA). In addition, both strains acted as bioremediation agents by reducing heavy metals (Cd and Zn) and greenhouse gases (CH4 and CO2). However, only R. palustris TN110 bioremediated Cd by biosynthesis of cadmium sulfide (CdS) nanoparticles and simultaneously fixed N2. This strain produced uniform CdS nanoparticles under its optimal microaerobic-light conditions (pH 7.5, 30 °C and 3,000 lux). The half maximal inhibitory concentration (IC so) of the produced CdS nanoparticles was 1.76 mM. The produced CdS nanoparticles at ICso up-regulated two genes associated with N2 fixation: Mo-Fe nitrogenase gene (nifH) and V-Fe nitrogenase gene (vnfG) at 2.83- and 2.27-fold changes, respectively. The results of gene expressions agreed with the amounts of NH4 released. Therefore, only R. palustris TN110 was further studied to obtain an optimal mixed carrier to support bacterial cells in solid formulation. Rubber wood ash (RWA), decanter cake (DCC), rice husk ash (RHA), and spent coffee grounds (SCG) were the carriers studied. With the use of D-optimal experimental design, a mixed carrier of RWA, DCC, RHA and SCG had an optimum ratio of 3: 4:2: 1. The optimal mixed carrier contained roughly 10 log CFU/g R. palustris TN110 and is packed in nylon-LLDPE bags using vacuum sealed at 500 W with a bacterial population decrease of only 3 log cycles after storage for 6 months at room temperature (25-3 °C). PNSB biofertilizer(s) (R. palustris TN110, Ru. gelatinosus TN414 and a mixed culture of both strains at 1: 1) were investigated for their phytotoxicity based on rice (Oryza sativa L.) seed germination index and found that all PNSB biofertilizers tested either in solid or liquid form showed no phytotoxicity at their optimal dilutions. All PNSB biofertilizer(s) at their optimal dilutions from liquid and solid forms containing 10 cells/mL for TN110 and 107 cells/mL for TN414 and a mixed culture produced remarkable increases in rice growth on the basis of shoot and root lengths compared with controls (tap water and a mixed carrier with no culture) for both Cd-contaminated and un-contaminated paddy soils. The application of R. palustris TN110 in solid form to Cd-contaminated paddy soil significantly improved rice growth based on the maximum shoot dry weight and root length. Additionally, all PNSB biofertilizer(s) in the solid form was better than the liquid form in cutting down Cd and Zn accumulation in rice shoots and roots in Cd-contaminated paddy soil. R. palustris TN110 was effective in Cd removal whereas Ru. gelatinosus TN414 performed better in decreasing Zn in rice shoot and root. Finally, greenhouse gas emission in rice straw biodegradation model in paddy soil slurry by PNSB biofertilizer(s) was investigated by focusing on light/dark cycles and PNSB biofertilizer(s) formulations. Proliferation between PNSB and methanogens populations under the different light/dark cycles: 0/24, 8/16, 12/12, 16/8 and 24/0 h were evaluated and found that light promoted PNSB growth to compete with methanogens for reducing greenhouse gas emissions. R. palustris TN110 was more effective in reducing CH4 and CO2 emissions compared with other biofertilizer(s). The carrier control dramatically increased total daily gas volume; all PNSB biofertilizer(s) from liquid form under all light/dark cycles throughout 10 day- incubation showed higher total daily gas volume reduction than a solid form. Strain TN110 in liquid form under natural light cycle (12/12 h) at day 10 was able to reduce 72.27% CH+ and 34.38% CO2 emissions. PNSB biofertilizer(s) in the solid form was a suitable form for promoting rice growth and reducing accumulated heavy metals (Cd and Zn) in contaminated soil; however, the liquid form was better for reducing greenhouse gas emissions. Overall discoveries in this research demonstrated that PNSB: R. palustris TN110 and Ru. gelatinosus TN414 are effective as biofertilizers and bioremediation agents for supporting rice cultivation in Cd-contaminated paddy soil. Moreover, the PNSB could alleviate greenhouse gas emissions by competing proliferation with methanogens that are responsible for greenhouse gas production. This thesis revealed that PNSB biofertilizer(s) has a great potential to be biofertilizers and also bioremediation agents for producing safe rice. Therefore, application of the PNSB to paddy fields with contamination with heavy metals or un-contamination would be an eco-friendly practice to produce safe rice along with improving soil quality and reducing global warming for sustainable agriculture.