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Identification of Ureolytic Bacteria for Concrete Formation and Antifungal Activity from the Soil with Long-Term Application of Urea Fertilizer

Ureolytic bacteria in 12 isolates, namely TCU1-TCU12, obtained from the soil with long-term application of urea fertilizer were screened and characterized. Urea degrading activity of all isolates was ranging from 2.4 to 22.9 mM. The ureolysis profiles of the isolates corresponded to fluctuations in pH value. Urease inhibitors, including acetohydroxamic acid and phenyl phosphorodiamidate, were applied to prove urea degrading activity. Moreover, the antifungal activity was verified by the TCU4 and TCU5 strains. The 16S rRNA gene sequence analysis revealed that among the 12 isolates, eight were Bacillus aryabhattai, three were Bacillus megaterium, and one was Bacillus subtilis. The random amplified polymorphic DNA was used to detect the genetic relationship among the 12 isolates. In addition, next-generation sequencing indicated that Proteobacteria was the most abundant phylum at 47.1% in the soil with long-term application of urea fertilizer. Bacillus species occupied 0.12%. Consequently, these ureolytic bacteria had the potential to be applied in the fields of concrete structure and medicine.


Introduction
Urease, a metalloenzyme, is widely found in various organisms, including bacteria, fungi, and plants. One mole of urea and two mole water can be hydrolyzed into one mole of ammonia carbonate by urease. This process further leads to an increase in pH because of ammonification [1]. Much attention has been focused on the calcium carbonate precipitation using ureolytic bacteria due to ureolytic activity [2][3][4][5]. They are used to prevent brick deterioration through water absorption by biocalcification, which is also known as microbiologically induced calcite precipitation (MICP) [6]. In addition to ureolytic activity, ureases have various biological properties, such as insecticidal activity and the activation of blood platelets [7]. The antifungal activity of urease is also demonstrated in recombinant Helicobacter pylori urease, embryo-specific soybean urease, and jackbean major urease [8]. The National Center for Biotechnology Information (NCBI) GenBank contains information on various bacteria with urease enzymes; however, few studies have been conducted to identify bacteria with a high ureolytic activity from agricultural soil. In the present study, we evaluated the biochemical and genetic characteristics of indigenous ureolytic bacteria in soil with long-term application of urea fertilizer. The genetic relationship of ureolytic bacteria with urea degrading activity, pH value, and urease inhibitor effects was established. Subsequently, the unculturable method of NGS was employed to estimate the isolates in the microbial community structure of the soil.

Screening and cultivation of ureolytic bacteria
The serially diluted soil sample was plated on the modified M9 minimal agar medium (w/v, 0.6% Na 2 HPO 4 , 0.3% KH 2 PO 4 , 0.05% NaCl, 0.1% Urea, 0.2% Glucose, 0.002% phenol red, 2 mM MgSO 4 , 0.1 mM CaCl 2 , and pH value was adjusted to 6.2). Urea was used as the sole nitrogen source for selectively screening ureolytic bacteria, which are capable of degrading urea. Phenol red was used as a pH indicator to confirm the ureolytic bacteria. Colonies were purified and inoculated into the modified M9 minimal medium broth for further analyzing urea degrading activity and DNA.

Analyses of urea degrading and antifungal activity of ureolytic bacteria
Ureolytic bacteria were inoculated into the modified M9 minimal medium broth at 37°C for the time course analysis. To measure the urea degrading activity of ureolytic bacteria, 1 mL of assay broth was harvested and centrifugated. The optical density of the suspension was measured using a spectrophotometer at 570 nm [9]. To explore the effect of urease inhibitors on ureolytic bacteria, acetohydroxamic acid, and phenyl phosphorodiamidate were used to estimate urea degrading activity. To detect the antifungal activity of ureolytic bacteria, Trichoderma harzianum BCRC31867 and Penicillium varians BCRC33128 were used to perform a dual culture assay. Paper discs 6 mm in diameter with the culture broth of ureolytic bacteria were placed surrounding the fungal block of pathogenic fungi. The plates were cultured at 28°C and the growth inhibitors of the pathogenic fungi were observed.

Identification of ureolytic bacteria
Ureolytic bacteria were cultured in a broth medium for the chromosomal DNA extraction. To identify isolates, a set of primers for amplifying 16S rRNA gene (27F, 5ʹ-AGAGTTTGGATCMTGGCTCAG-3ʹ, and 907R, 5ʹ-CCGTCAATTCCTTTRAGTTT-3ʹ) was designed to be used in polymerase chain reaction (PCR). The PCR products were extracted and purified for DNA sequencing. The sequence data were aligned and compared with the data of the NCBI GenBank (http://www.ncbi.nlm.nih.gov/), using a BLAST tool. The phylogenetic tree was constructed using the neighbor-joining method by using MEGA software with 1000 bootstrap replicates [10].

Random amplified polymorphic DNA
The genetic relationship among ureolytic bacteria was further determined using a random amplified polymorphic DNA (RAPD) amplification technique. The RAPD amplification was conducted with 10 different 10-mer random primers. The PCR included the following steps: an initial denaturation of 5 min at 94°C, 40 cycles for 1 min at 94°C, 30 s at 37°C, and 3 min at 72°C with a final extension of 5 min at 72°C. The DNA bands were recorded as binary data and analyzed using NTSYS-PC software by using the Unweighted Pair Group Method with Arithmetic Mean method.

Metagenomic analysis of 16S rRNA gene
To comprehensively examine the soil microbial community structure, metagenomics sequencing was conducted using the next-generation sequencing (NGS) method. The procedure included the amplification of the 16S rRNA gene of the microbial soil DNA. The primer set, 517F: 5ʹ- TTGCCAGCAGCCGCGGTAA-3ʹ and 926R: 5ʹ-CCCCGTCAATTYYTTTRAGTTT-3ʹ, was designed for targeting the hypervariable V3-V4 region of the 16S rRNA gene. The triplicate PCR amplicons were pooled and purified through gel extraction for metagenomic sequencing. The library was constructed using a TruSeq DNA Sample Preparation kit (Illumina, San Diego, CA, USA) and quantified using a GeneRead Library Quant Kit (Qiagen, Valencia, CA, USA). The sequences of 2 × 300 bp paired-end reads were generated using a Miseq Reagent kit V3 (Illumina) for the Miseq sequencer (Illumina).

Results and discussion
To characterize ureolytic bacteria, the details of the urea degrading activity, cell growth, and pH value were monitored during 96 h of incubation, and the results are shown in Fig. 1. The ammonia released during urea degradation in the liquid medium ranged between 2.4 and 22.9 mM with variations in all isolates. The maximal ammonia release was recorded in the TCU2 isolate followed by the TC11 isolate. However, the minimal urea degrading activity was observed in TCU1 and TCU4-TCU8 isolates. During ureolysis, an increase in pH value was accompanied by urea degrading activity because of ammonification [1,11]. Therefore, the time courses of pH value were also determined from an initial pH value of 6.0-6.3 after 96 h incubation. As expected, the pH change observed in all the isolates were in agreement with the degree of ammonia released. The maximal pH increase was observed in the TCU2 strain from 6.3 to 7.4 after 96 h of incubation and associated with the high urea degrading activity. In addition, the cell density of the TCU2 strain was higher than that of the others. The minimal ammonia release was consistent with the unchanged pH value in the TCU1 and TCU4-TCU8 isolates. Furthermore, the growth of these strains was slightly increased. The application of urease inhibitors can reduce the ammonia loss and confirm urease activity. The effect of two urease inhibitors, acetohydroxamic acid and phenyl phosphorodiamidate acting as a metal chelator and competitive inhibitor, respectively, on the maximal urea degrading activity of the TCU2 isolate was explored during 96 h of incubation (Fig. 2). Ureolysis of TCU2 strain was markedly suppressed by both acetohydroxamic acid and phenyl phosphorodiamidate. A high concentration of urea inhibitors did not cause pH fluctuations during the ureolysis process. However, after 72 h of cultivation, ammonia release was slightly increased by adding 0.01% urea inhibitors. The pH profiles also corresponded to urea degrading activity. H. pylori urease showed antifungal activity [12]. To detect the antifungal activity of ureolytic bacteria, a dual culture assay was performed using two pathogenic fungi, T. harzianum BCRC31867 and P. varians BCRC33128 (Fig. 3). Consequently, only TCU4 and TCU5 isolates were effectively antagonistic against the T. harzianum BCRC31867 and P. varians BCRC33128 fungi. Furthermore, other isolates had no effect on the growth inhibition of the two fungi. To identify ureolytic bacteria, 16S rRNA gene sequence and phylogeny analyses were performed (Fig. 4A). These ureolytic bacteria belonged to the Bacillus genus using the BLAST program, which were consistent with previous studies [2][3][4][5]7]. Phylogenetic analysis identified eight isolates of B.