## 4 Discussion  
In this study, we attempted to elucidate the blackening mechanisms of lotus tubers by analyzing their chemical and microbial characteristics. Based on the linear relationship between the damage index and phosphate concentration in the lotus cultivation fields, along with SEM-EDS/ICP-OES analyses, we found that P and Fe were concentrated in the black spots of lotus tubers (Figures 2 and 3), suggesting that the black spots were caused by the formation of P-Fe compounds. To date, viviariate [Fe\({}_{3}\)(PO\({}_{4}\))\({}_{2}\)\(\cdot\)8H\({}_{2}\)O], as an iron phosphate mineral, has been frequently found in sediments and organic soils under reducing (anaerobic) conditions [25; 26]. Vivianite is stably present in rice paddy soil and forms under reducing conditions when PO\({}_{4}\)\({}^{3-}\) and Fe\({}^{2+}\) concentrations are sufficient and S\({}^{2-}\) concentrations are low [26; 27]. In addition, the ORP of lotus cultivation soil (\(-170\) mV) is known to be similar to that of rice paddy soils (from \(-112\) to \(-187\) mV) [24], and the soil has been used for isolating sources of Fe(III)-reducing bacteria [28]. Vivianite is originally  
Figure 4: Relative abundance rate of predominant operational taxonomic units (OTUs) based on 16S rRNA gene sequences. a colorless mineral, but when exposed to air, the iron is oxidized, and vivianite exhibits several colors, such as blue-green and black [29]. [PERSON] et al. confirmed the presence of vivianite minerals in rice roots, accounting for 25-29% of the total root P [30]. In addition, [PERSON] reported that a 0.1 g P\({}_{2}\)O\({}_{5}\)/kg plant-available P level in the plowing layer of the soil is an important condition for the detection of vivianite in rice roots [26]. Thus, assuming that the black spots of lotus tubers are vivianite, all lotus cultivation fields in this study have >0.1 g P\({}_{2}\)O\({}_{5}\)/kg plant-available P levels (Table 1), indicating that the environments are suitable for the formation of vivianite. Using 16S rRNA gene analysis, we found that DIRB and other reducing bacteria were predominantly present in the black spots of lotus tubers (Figure 4). It has been reported that several organic carbon-oxidizing and Fe(III)-reducing DIRB can induce the precipitation of minerals such as vivianite, siderite, apatite, and magnetite [25; 31; 32]. A recent study revealed that vivianite is formed by the reduction of Fe(III), using activated sludge under anaerobic conditions, and _Rhodoferax_ sp. and _Dechloromonas_ sp. involved in the iron cycles were predominant in the sludge after vivianite formation [33]. In addition, batch experiments using _Geobacter sulfurreducens_ of the family _Geobacteraceae_ for P recovery from sewage have also revealed that sufficient PO\({}_{4}\)\({}^{3-}\) promotes cell growth, leading to higher Fe\({}^{3+}\) reduction and vivianite yield [34]. Thus, it is possible that _Rhodocyclaceae_ OTU001, _Rhodoferax_ OTU002, and _Geobacteraceae_ OTU003, which are predominant DIRBs in the black spots of lotus tubers, can form or induce iron phosphate minerals such as vivianite by reducing Fe\({}^{3+}\) in lotus-cultivation soils. The most predominant OTU001 is closely related to _G. toluolica_ of DIRB, which can degrade toluene, ethylbenzene, phenol, p-cresol, m-cresol, benzaldehyde, and p-hydroxybenzoate [20]. It has been reported that phenolic compounds are abundant in lotus tubers [35]. Therefore, it is likely that _Rhodocyclaceae_ OTU001 utilizes aromatic compounds, such as phenol leached from damaged parts of lotus tubers by _H. diversa_, and reduces Fe(III). Recently, vivianite formation mechanisms using microbial reactions via extracellular electron transfer have been proposed [29; 36]. First, under anaerobic conditions, P (mainly HPO\({}_{4}\)\({}^{2-}\) and PO\({}_{4}\)\({}^{3-}\)) is adsorbed on the surface of Fe(III) oxides or coprecipitated with Fe\({}^{3+}\) in the liquid phase to form FePO\({}_{4}\).