Agricultural aquifer storage recovery and transfer (ASTR) stores excess fresh water for later reuse in irrigation. Moreover, water quality improves because chemical pollutants and pathogens will be removed by degradation and attachment to the aquifer material. The source water may contain the bacterial plant pathogen Ralstonia solanacearum which causes plant infections and high yield losses. We used quantitative microbial risk assessment (QMRA) to investigate the removal of R. solanacearum during ASTR to predict infection risks of potato plants after irrigation with the recovered water. Laboratory experiments analyzed the ASTR treatment by investigating the bacterial die-off in the water phase and the removal by attachment to the aquifer sediment. Die-off in the water phase depends on the residence time and ranged between 1.3 and 2.7 log₁₀ after 10 or 60 days water storage, respectively. A subpopulation of the bacteria persisted for a prolonged time at low concentrations which may pose a risk if the water is recovered too early. However, the natural aquifer sand filtration proofed to be highly effective in removing R. solanacearum by attachment which depends on the distance between injection and abstraction well. The high removal by attachment alone (18 log₁₀ after 1 m) would reduce bacterial concentrations to negligible numbers. Upscaling to longer soil passages is discussed in the paper. Infection risks of potato plants were calculated using a dose-response model and ASTR treatment resulted in negligible infection risks of a single plant, but also when simulating the irrigation of a 5 ha potato field. This is the first QMRA that analyzed an agricultural ASTR and the fate of a plant pathogen focusing on plant health. QMRA is a useful (water) management tool to evaluate the treatment steps of water reclamation technologies with the aim to provide safe irrigation water and reduce risks disseminating plant diseases.

An aquifer storage transfer and recovery (ASTR) system was studied in which tile drainage water (TDW) was injected with relatively high NO₃ (about 14 mg/L) concentrations originating from fertilizers. Here we present the evolution of denitrification kinetics at 6 different depths in the aquifer before, and during ASTR operation. First-order denitrification rate constants increased over time before and during the first days of ASTR operation, likely due to microbial adaptation of the native bacterial community and/or bioaugmentation of the aquifer by denitrifying bacteria present in injected TDW. Push-pull tests were performed in the native aquifer before ASTR operation. Obtained first-order denitrification rate constants were negligible (0.00–0.03 d⁻¹) at the start, but increased to 0.17–0.83 d⁻¹ after a lag-phase of about 6 days. During the first days of ASTR operation in autumn 2019, the arrival of injected TDW was studied at 2.5 m distance from the injection well. First-order denitrification rate constants increased again over time (maximum >1 d⁻¹). Three storage periods without injection were monitored in winter 2019, fall 2020, and spring 2021 during ASTR operation. First-order rate constants ranged between 0.12 and 0.61 d⁻¹. Denitrification coupled to pyrite oxidation occurred at all depths, but other oxidation processes were indicated as well. NO₃ concentration trends resembled Monod kinetics but were fitted also to a first-order decay rate model to facilitate comparison. Rate constants during the storage periods were substantially lower than during injection, probably due to a reduction in the exchange rate between aquifer solid phases and injected water during the stagnant conditions. Denitrification rate constants deviated maximally a factor 5 over time and depth for all in-situ measurement approaches after the lag-phase. The combination of these in-situ approaches enabled to obtain more detailed insights in the evolution of denitrification kinetics during AS(T)R.

Irrigation with surface water carrying plant pathogens poses a risk for agriculture. Managed aquifer recharge enhances fresh water availability while simultaneously it may reduce the risk of plant diseases by removal of pathogens during aquifer passage. We compared the transport of three plant pathogenic bacteria with Escherichia coli WR1 as reference strain in saturated laboratory column experiments filled with quartz sand, or sandy aquifer sediments. E. coli showed the highest removal, followed by Pectobacterium carotovorum, Dickeya solani and Ralstonia solanacearum. Bacterial and non-reactive tracer breakthrough curves were fitted with Hydrus-1D and compared with colloid filtration theory (CFT). Bacterial attachment to fine and medium aquifer sand under anoxic conditions was highest with attachment rates of max. k _att1 = 765 day ^-1 and 355 day ^-1, respectively. Attachment was the least to quartz sand under oxic conditions (k _att1 = 61 day ^-1). In CFT, sticking efficiencies were higher in aquifer than in quartz sand but there was no differentiation between fine and medium aquifer sand. Overall removal ranged between < 6.8 log ₁₀ m ⁻¹ in quartz and up to 40 log ₁₀ m ⁻¹ in fine aquifer sand. Oxygenation of the anoxic aquifer sediments for two weeks with oxic influent water decreased the removal. The results highlight the potential of natural sand filtration to sufficiently remove plant pathogenic bacteria during aquifer storage.

Managed aquifer recharge (MAR) can provide irrigation water and overcome water scarcity in agriculture. Removal of potentially present plant pathogens during MAR is essential to prevent crop diseases. We studied the die-off of three plant pathogenic bacteria in water microcosms with natural or filtered tile drainage water (TDW) at 10 and 25◦C and with natural anoxic aquifer water (AW) at 10◦C from a MAR site. These bacteria were: Ralstonia solanacearum (bacterial wilt), and the soft rot Pectobacteriaceae (SRP) Dickeya solani and Pectobacterium carotovorum sp. carotovorum (soft rot, blackleg). They are present in surface waters and cause destructive crop diseases worldwide which have been linked to contaminated irrigation water. Nevertheless, little is known about the survival of the SRP in aqueous environments and no study has investigated the persistence of R. solanacearum under natural anoxic conditions. We found that all bacteria were undetectable in 0.1 mL samples within 19 days under oxic conditions in natural TDW at 10◦C, using viable cell counting, corresponding to 3-log₁₀ reduction by die-off. The SRP were no longer detected within 6 days at 25◦C, whereas R. solanacearum was detectable for 25 days. Whereas in anoxic natural aquifer water at 10◦C, the bacterial concentrations declined slower and the detection limit was reached within 56 days. Finally, we modelled the inactivation curves with a modified Weibull model that can simulate different curve shapes such as shoulder phenomena in the beginning and long tails reflecting persistent bacterial populations. The nonlinear model was shown to be a reliable tool to predict the die-off of the analysed plant pathogenic bacteria, suggesting its further application to other pathogenic microorganisms in the context of microbial risk assessment.

Many cyanobacteria produce extracellular polymeric substances (EPS) mainly composed of heteropolysaccharides with unique characteristics that make them suitable for biotechnological applications. However, manipulation/optimization of EPS biosynthesis/characteristics is hindered by a poor understanding of the production pathways and the differences between bacterial species. In this work, genes putatively related to different pathways of cyanobacterial EPS polymerization, assembly, and export were targeted for deletion or truncation in the unicellular Synechocystis sp. PCC 6803. No evident phenotypic changes were observed for some mutants in genes occurring in multiple copies in Synechocystis genome, namely ∆wzy (∆sll0737), ∆wzx (∆sll5049), ∆kpsM (∆slr2107), and ∆kpsM∆wzy (∆slr2107∆sll0737), strongly suggesting functional redundancy. In contrast, Δwzc (Δsll0923) and Δwzb (Δslr0328) influenced both the amount and composition of the EPS, establishing that Wzc participates in the production of capsular (CPS) and released (RPS) polysaccharides, and Wzb affects RPS production. The structure of Wzb was solved (2.28 Å), revealing structural differences relative to other phosphatases involved in EPS production and suggesting a different substrate recognition mechanism. In addition, Wzc showed the ATPase and autokinase activities typical of bacterial tyrosine kinases. Most importantly, Wzb was able to dephosphorylate Wzc in vitro, suggesting that tyrosine phosphorylation/dephosphorylation plays a role in cyanobacterial EPS production.