3.3. ATP-Based Assay of Total Bacterial Activity
We also measured tATP levels in the soils of the different treatment groups on day-1 and day-50 (
Table 4). The soil tATP level is a quantitative indicator of the number of living cells [
23]. These concentrations of tATP are expressed in terms of microbial equivalents (MEs)/g soil, as representative of the total number of viable microbes and assuming that all free ATP degraded quickly, and that detected ATP was only from living or recently decreased cells [
23,
24]. On day-1 (1 day after inoculation), soils inoculated with
Caballeronia sp. EK had the highest tATP level (mean: 740,168 MEs/g soil), followed by
Methylobacterium sp. PS. (692,184 MEs/g of soil). The controls had much lower levels of tATP (423,601 MEs/g soil), thus confirming that there were significantly more living microbes in the pots inoculated with PSB. However, the control pots still had living cells because of the addition of the organic supplement. We also measured soil tATP levels on day-50. The mean value was greatest in soils that received
Caballeronia sp. EK inoculations (879,597 MEs/g soil), and this level was 18.9% greater than in soils that received
Methylobacterium sp. PS inoculations (713,415 MEs/g soil) and 45.2% greater than the controls (482,273 MEs/g soil). Notably, from day-1 to day-50, the tATP level increased in all three groups. PSB strains had a positive effect on the growth of
Lycopersicon esculentum L. in the acid sulfate soils, and it can be considered as the result explaining the role of microorganisms in promoting vegetation. Our results confirmed that the
Caballeronia sp. EK strain provided the greatest increase in the number of soil microbes.
3.4. Germination and Leaf Growth
We measured the percentage of germination of
Lycopersicon esculentum L. seeds on day-52 in the different treatment groups (
Table 5). The results indicated that soils inoculated with
Caballeronia sp. EK had 80% germination, much greater than soil inoculated with
Methylobacterium sp. PS (57.5%) and the control soil (20%).
We measured the mean number of leaves of
Lycopersicon esculentum L. plants in the different groups every 2 to 4 days beginning on day-25 (
Table 6). These measurements did not include the cotyledons, and the values were from the average of 40 plants per pot. Within-group analysis indicated the control group had 8.7 leaves on day-25, and this number declined to 4.7 leaves on day-47. Over time, these plants had fewer leaves, but leaf size increased. The
Methylobacterium sp. PS group had 18.3 leaves on day-25 (2.1-fold more than the control), and this number stayed at 17.3 leaves as the size of the leaves increased, and then declined after day-40 even though leaf size continued to increase. The
Caballeronia sp. EK group had 10.3 leaves on day-25, but the number and size of these leaves increased, and the maximum number was 17.3 leaves from day-40 to day-52. These results indicated that soil inoculated with these two PSB strains led to increased germination and development of more leaves in tomato plants. Therefore, the PSB analyzed here may have potential for use as biofertilizers in the restoration of acid sulfate soils.
Many species of
Methylobacterium occur in the rhizomes of plants, and they can secrete indole-3-acetic acid (IAA), a promoter of plant growth [
25]. Many species of
Caballeronia and some closely related genera can produce antibiotics or fix atmospheric nitrogen [
26]. This may be why the two PSB strains used in this study promoted plant growth. Most chemical fertilizers are designed to provide nitrogen, because soils normally have abundant carbon and phosphorus [
20]. However, inoculation of PSB can solubilize the inorganic phosphorus in soils, and provide a nearly permanent source of phosphorus to plants if these microbes persist [
16,
17]. The soils described here that were treated with PSB led to significantly increased germination and improved plant growth. These results suggest it may be possible to achieve efficient vegetation of acid sulfate soils by promotion of phosphorus absorption. The three major essential elements for plant growth are carbon, nitrogen, and phosphorus. Carbon is typically abundant, and occurs as carbon dioxide in the air. Nitrogen in the form of chemical salts is often readily available due to the activities of nitrogen fixing microbes in the soil [
17]. However, most phosphorus exists in a form that cannot be absorbed by plants. Although less phosphorus than carbon and nitrogen is required for growth, phosphorus is a limiting factor for plant growth in most environments. Soil microbes are often related to plant growth. As such, the phosphate solubilizing activity of microorganisms in the rhizosphere plays an important role in plant growth. Organic acids in PSB solubilize phosphorus, in which the hydroxyl and carboxyl groups chelate the soil phosphorus, converting it into water-soluble forms [
26]. These chelated forms of phosphorus can be taken up by plants, indicating that inoculation of PSBs may help to facilitate restoration of vegetation.
The
Methylobacterium sp. PS strain examined in the present study is a typical genus of the Methylobacteriaceae family that occurs in plant stems, leaves, flowers, and roots, and usually has a pink carotenoid pigment.
Methylobacterium species utilize C1 compounds as a carbon source, and can also use methanol generated from plant metabolism as a substrate.
Methylobacterium species can reportedly use byproducts from plants and can invade plant seeds (including tomato seeds), thereby promoting the secretion of phytohormones. Previous research reported that plants treated with
Methylobacterium had longer roots than control plants that did not receive this treatment [
14,
25,
27]. Phytohormones play an important role in plant growth and yield. IAA, the first auxin to be isolated, is an important phytohormone that plays an important role in cell growth, division, and differentiation [
25,
27,
28]. Because some bacteria can synthesize IAA, previous researchers suggested that inoculating soils with IAA-producing bacteria could stimulate plant growth. In fact, previous research indicated that the IAA level was greater in soils inoculated with
Methylobacterium sp., as were the amounts of two cytokines (
trans-zeatin riboside and dihydro-zeatin riboside) in tomato plants growing in these soils [
14,
25]. Moreover, the resulting plants had increased levels of IAA and cytokines, and greater growth controls [
14,
25,
29].
The MBF1239 strain of
Methylobacterium produces two types of alkyl quinolones, a class of compounds that have antibiotic properties. Many prescribed quinolone-based antibiotics are effective against Gram-negative bacteria (except
Pseudomonas aeruginosa). Thus, some species in the
Methylobacterium genus can be classified as pathogenic bacteria and others as antibiotic-producing bacteria. It was hypothesized that infection of plants by pathogenic bacteria or nematodes functioned as a stressor that led to the symbiosis of plants with antibiotic-producing bacteria [
26,
30]. Some of these microbes may also perform nitrogen fixation. These nitrogen-fixing bacteria use ATP to reduce atmospheric nitrogen to produce ammonia, and the ammonia is then used for intracellular synthesis of various amino acids, such as glutamine. Some bacteria can fix nitrogen alone, and other bacteria fix nitrogen by forming symbiotic relationships with plant roots [
26,
30]. For example, leguminous bacteria (
Rhizobia) infect the roots of legumes, leading to the formation of root nodules, a symbiotic relationship. In this case,
Rhizobia provide a nitrogen source to the plants, and the plants provide ions and a suitable habitat necessary for bacterial growth. Some species in the genus
Burkholderia perform independent nitrogen fixation, and others in this genus perform symbiotic nitrogen fixation [
31]. Further investigations should clarify how and to what extent inherent molecular properties of secreted hormones by PSB impact plant growth.
The acidic drainage from acidic sulfate soils (produced by the oxidation of pyrite) contains high concentrations of heavy metals, and this is the cause of the damage to farmland and the poor growth of vegetation. Remediation of acidic soils can be achieved by covering up the pyrite with alkaline material to reduce atmospheric exposure, and this intervention reduces acidic drainage and stabilizes the soil. However, this method is not an eco-friendly intervention for the restoration of poor landscapes. For the restoration of acidic soils, priority should be given to introducing vegetation, an eco-friendly intervention that stabilizes slopes and improves landscapes. When a slope is covered with plants, the organic content in the soil continuously increases over time, and this reduces the dissolved oxygen concentration of rainwater that penetrates the embankment layer. This effect reduces pyrite oxidation and thereby reduces the generation of acidic drainage. A general method to suppress acidic drainage is prevention of the oxidation of sulfide minerals by application of a surface coating. However, the materials responsible for acidic drainage still contain some amount of water, so acidic drainage will eventually increase over time, and this underground acidic drainage can move to the surface by capillary action, eventually killing plants. A more appropriate method for the efficient revegetation of acid sulfate soils is the application of biofertilizers using PSB, as described in the present study.