“Impacts of treated wastewater on soil properties with special reference to the soil microbiome and resistome in irrigated Tunisian agricultural soils”.

Research output: Book/ReportPh.D. thesisResearch

Experts and decision-makers continue to disagree on whether it is appropriate to use treated wastewater (TWW) for crop or agricultural irrigation. Since the 1980s, TWW has been used in northern Africa to irrigate fodder crops, but there haven't been enough studies done to look at how it affects the physiochemical properties of the soil, the composition and structure of bacterial communities, and the soil resistome over the long and short term. Our experiment was conducted on Cebala Borj Touil area in northern Tunisia, an agricultural soil which has been receiving TWW for irrigation for more than 25 years. Our study aimed to investigate the impact of long-term TWW irrigation on soil abiotic and biotic properties, to correlate changes in bacterial communities’ structure and composition with different environmental factors affected with this type of irrigation, and to analyze the communities’ resistance profile towards heavy metals and antibiotics contamination. Furthermore, we proceeded with isolation of multiresistant bacteria strain and characterization of its heavy metal resistant and plant growth promoting traits as a way of reusing it as bioremediation mean in heavy metal polluted agricultural soils. Soils samples analyzed in this study were divided to control samples from plots with no history of TWW irrigation, short-term TWW irrigated plots with 3-4 years of TWW irrigation, and long-term TWW irrigated samples from plots with 20-25 years of TWW irrigation. Our results showed an acidification of soils correlated with TWW irrigation years, in addition to an increase of salinity, soil organic matter, and concentrations of major soil ions. Heavy metal contamination was assessed by concentration measurements in different times TWW irrigated soils. Nine different heavy metals were analyzed, Cu, Cd, Pb, Zn, Al, Fe, Mn, Se, and As which all showed a significant rise in long-term TWW irrigated soils. Pollutioninduced bacterial community tolerance (PICT) was investigated for Cu, Zn, Pb, and tetracycline with H3-Leucine incorporation. Our results showed a significantly higher tolerance index for long-term TWW irrigated bacterial communities compared to control and short-term TWW irrigated communities. To highlight the impact of this irrigation on the bacterial communities’ structure and composition, 16S rRNA gene amplicon sequencing was performed on the same samples as used for PICT detection. Statistical analysis based on phyla and genera relative abundance, alpha and beta-diversity analysis, and environmental factors correlation were performed. Our results showed a significant difference in alpha diversity indexes such as Chao1 and ACE. Surprisingly, short-term TWW irrigated bacterial communities had the higher biodiversity index with a better bacterial richness implication. A turnover in bacterial communities’ composition was proved especially at genus level with a dominant appearance and development of particular genera such as Defluviococcus, Pseudarthrobacter, Nocardioides, and Gaiella in long-term TWW irrigation compared to the rest of treatment, which lead us to consider them as TWW contamination bio-indicator. Bacterial strains aim to develop resistance traits against pollutants like heavy metals as a result of soil's ongoing contamination and diverse pollutants' alteration. In this study, the growth response and antioxidant defense protection of a novel strain isolated from agricultural soil were examined in relation to lead exposure. Surprisingly, adding lead to Bacillus simplex strain 115 cultures at different concentrations (0, 0.2, 1.5, and 3 g L-1) had no impact on the bacterial cultures' ability to grow. Protein carbonylation increased (+221%), malondialdehyde levels increased (+2.3 fold), H2O2 levels increased (+6.2 fold), and lipox activity increased (+59%) all as a result of an oxidative stress brought on by the presence of 3 g L-1 lead. The simultaneous up-regulation of superoxide dismutase (+4 fold) and catalase (+416%) activities revealed innate antioxidant defenses, with Fe-SOD showing a more notable increase than the other isoforms. Bioaccumulation tests revealed intracellular as well as extracellular lead accumulation. Metal sequestration in the cell wall accounts for 88.5% to 98.5% of the total endogenous metal accumulation, making bio-adsorption a particularly effective lead resistance mechanism for Bacillus simplex strain 115. Accordingly, it was proposed that this recently identified isolated microorganism might be practical as a biotechnological tool for agricultural soil lead bioremediation. It was our intention to maximize the value of this study and the benefits of the investigation's findings by using the Bacillus simplex strain 115 as a Plant Growth Promoting Bacteria (PGPB) in a polluted environment. In the third and last section of our investigation, we sought to describe the PGPB characteristics of Bacillus simplex. In hydroponically controlled conditions, this bacterium was introduced to help pea (Pisum sativum L.) growth that had been exposed to lead (Pb) toxicity. In 23-day-old plants, the root nodulation system had sufficiently matured to attest to the interaction between the two organisms. The strain 115 had the ability to biosorb lead in addition to its abilities to solubilize phosphate and produce siderophores at levels of 303.8 g P mL-1 and 58.7 psu, respectively. When strain 115 was used to inoculate Pb-stressed pea, dry weights of the roots and shoots recovered (+70% and +61%, respectively). Similar to this, after bacteria inoculation, Pb-treated plants had higher water and protein contents. In comparison to Pb stress alone, strain 115 reduced the relative Pb toxicity by 39.3%. In addition, plants exposed to Pb showed increased activity of catalase and superoxide dismutase (+56% and +51%, respectively). Strain 115 inoculation resulted in a -38% and a -44% restoration of CAT and SOD, respectively. After the Bacillus strain was introduced into Pb-treated plants, concurrently, oxidant stress indicators (H2O2 and 4-hydroxynonenal), osmo-regulators (proline and glycine betaine), and lipoxygenase activity all significantly lowered. When the data is considered as a whole, it provides some support for the strain 115's ability to promote plant growth while reducing Pb stress.
Original languageEnglish
PublisherDepartment of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen
Number of pages159
Publication statusPublished - 2023

ID: 378825751