A strategy to supply molecular oxygen for microbial calcium precipitation was developed for the first time. Firstly, a controlled oxygen-releasing tablet (ORT) containing CaO and lactic acid with a suitable ratio of 9:1 was developed. It can provide a stable oxygen supply and maintain pH in the range of 9.5-11.0 for 45 days while contacting with water. In the presence of oxygen, a self-healing bacterium H4 spores germinated more effectively and maintained high metabolic activity. Furthermore, H4 vegetative cells induced 50 % more calcium precipitation than that obtained without oxygen supply. Finally, a binary self-healing system containing bacterial spores and ORT was established. The calcium precipitation experiments showed that H4 in the binary self-healing system precipitated 27.5 mM calcium with oxygen supply after 32 days and dissolved oxygen (DO) concentration of the solution decreased from 15 to 4 mg l, while only 6.9 mM calcium precipitation was obtained without oxygen supply. This work can disclose the effect of oxygen on microbial calcium precipitation and further lay a foundation for the establishment of ternary self-healing system containing bacteria, ORT, and nutrients, which will be promising for the self-healing of cracks deep inside the concrete structure.

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Successful cultivation of black truffle (Tuber melanosporum) requires a long-term investment and the maintenance of the symbiosis throughout its preproductive and productive years. Monitoring the symbiosis over time is challenging, as it requires methods that can detect the belowground proliferation of the fungus associated with its host tree. In this study, we used a chronosequence design to study the expansion pattern of this fungus as the host tree grows. We hypothesize that this expansion can be estimated by monitoring T. melanosporum DNA from soil beneath host trees of different ages (3, 5, 7, 10, 14, and 20 years old) and at different distances from the trunk of the trees (40, 100, and 200 cm). We also wished to evaluate the presences of Tuber brumale and Tuber indicum, potentially problematic truffle species, in these plantations. To detect the mycelium of T. melanosporum in these soils, we extracted DNA and performed polymerase chain reaction (PCR) with Tuber species-specific primers, and to estimate DNA amount, we measured relative band intensities from the amplicons in agarose gels. Both age and distance were related to T. melanosporum DNA quantity, which was more abundant in the oldest age classes, reaching a plateau in 5-7 years. At 40 cm from the tree, there were no differences in T. melanosporum DNA amounts in orchards of different ages, but at 100 and 200 cm, younger orchards had less T. melanosporum DNA. We did not detect DNA from T. brumale or T. indicum in any of our samples.