Abstract

Bio-calcification is a natural phenomenon catalyzed by the urease enzyme, which is produced by a wide range of bacterial strains. The bio-calcification process relies on several physicochemical factors like pH, Ca²⁺, inoculum size (serve as a nucleation site), temperature, etc. In this study, seven urease-producing bacteria (BS-1-BS-7) were isolated from the garden soil of the two locations and the soil collected from a cave located at Sahastradhara, Dehradun Uttarakhand, India. These isolates were tested for urease activity and CaCO₃ precipitation. Among the bacterial isolates, BS-6 produced the maximum ammonium ions and CaCO₃ precipitate (14.6 mg/50 ml) and therefore it was selected for the optimization study. The two important factors – inoculum size (4%, 12%, and 20%; OD value 1.2 at 600 nm) and CaCl₂ concentration (25, 50, and 100 mM) were optimized for bio-calcification process (CaCO₃ precipitation). The optimum inoculum size and CaCl₂ concentration for the CaCO₃ precipitation by the isolate BS-6 were found to be 12% and 50 mM, respectively. Two-way ANOVA (analysis of variance) indicates that both factors, inoculum size and CaCl₂ concentration significantly (p < 0.05) affect the CaCO₃ precipitation. The present study signifies the role of Ca²⁺ and inoculum size in the bio-calcification process. The selected bacterial strain BS-6 was further identified as a novel strain Advenelle sp. AV1 based on the 16S rRNA sequencing. This is the first report that shows Advenelle sp. AV1 as urease producing and bio-calcite forming bacteria.

摘要

生物钙化是一种由脲酶催化的自然现象，它由多种细菌菌株产生。生物钙化过程依赖于几个物理化学因素，如 pH 值、Ca ²⁺、接种物大小（作为成核位点）、温度等。在这项研究中，7 种产脲酶细菌 (BS-1-BS-7)从这两个地点的花园土壤中分离出来，并从位于印度德拉敦北阿坎德邦 Sahastradhara 的一个洞穴中收集到的土壤。测试这些分离物的脲酶活性和CaCO ₃沉淀。在细菌分离物中，BS-6产生最大的铵离子和CaCO ₃沉淀（14.6 毫克/50 毫升），因此它被选择用于优化研究。针对生物钙化过程（CaCO ₃沉淀）优化了两个重要因素——接种物大小（4%、12% 和 20%；OD 值在 60​​0 nm 时为 1.2）和 CaCl ₂浓度（25、50 和 100 mM） . 发现分离株 BS-6 沉淀CaCO ₃的最佳接种量和 CaCl ₂浓度分别为 12% 和 50 mM。双向方差分析（方差分析）表明，接种量和 CaCl ₂浓度这两个因素显着（p  < 0.05）影响 CaCO ₃沉淀。本研究表明 Ca ^2+的作用和生物钙化过程中的接种量。选择的细菌菌株BS-6被进一步鉴定为新菌株Advenelle sp。AV1 基于 16S rRNA 测序。这是第一份显示Advenelle sp 的报告。AV1 作为产生脲酶和形成生物方解石的细菌。