Impact of microbial-induced calcium carbonate precipitation by native Bacillus licheniformis on enzymatic activity in Khuzestan sand dunes

Document Type : Research Paper

Authors

1 Agricultural Sciences and Natural Resources University of Khuzestan

2 Department of Biology, Faculty of Science, Shahid Chamran University of Ahvaz, Ahvaz, Iran. Biotechnology and Biological Science Research Center, Shahid Chamran University of Ahvaz, Ahvaz, Iran

Abstract

Bio and non-bio additives are considered effective approaches for increasing the stabilization of sand dunes, and one of the novel methods to achieve this is Microbial Carbonate Precipitation (MICP). This study aimed to investigate the effect of MICP using indigenous bacteria Bacillus licheniformis on the enzymatic activity of sand dunes. This study evaluated the impact of vinasse (a byproduct of sugarcane industries) as a culture medium, as well as the effect of vinasse pH, the addition of urea and calcium chloride, and incubation time on the activities of soil catalase, dehydrogenase, and urease enzymes.  The results showed that the indigenous isolates of B. licheniformis were capable of generating enzymatic activity similar to or better than that of the indicator bacterium S. pasteurii in the soil. The highest catalase and dehydrogenase activities in soils treated with B. licheniformis were measured at 3.2 KMnO4.g-1.h-1 and 7.609 µg TPF.g-1 dm.16h-1, respectively, while the highest urease activity was observed in the treatment with S. pasteurii, registering at 7.512 µgNH4+.N. g-1.2h-1. In addition, the incubation time and initial pH of vinasse significantly influenced enzymatic activity. This research demonstrated that the use of indigenous bacteria in MICP processes could serve as a sustainable and economical method for improving soil characteristics and enhancing its structure. 

Keywords

Main Subjects

Introduction

Recently, there has been increasing emphasis on ecosystem-based and biological methods for soil conservation, such as stabilizing sand dunes, has led to innovative strategies that leverage soil microbial communities. The microbially induced calcium carbonate precipitation (MICP) method generates carbonate and ammonium ions through the hydrolysis of urea by microbes, resulting in calcium carbonate precipitates in the presence of calcium. Ureolytic bacteria are particularly suitable for biocement technology due to their straightforward processes. Selecting appropriate strains for MICP is crucial, with Sporosarcina pasteurii being the primary focus due to its effectiveness. Moreover, indigenous bacteria offer advantages over non-native strains, as they adapt better to local environments and can reduce costs. This study examines the enzymatic activity of sandy soils treated with vinasse and Bacillus licheniformis in comparison to S. pasteurii.

Material and methods

The sampling area features sandy soil with neutral pH and high electrical conductivity. Three isolates of B. licheniformis were obtained from sandy dunes as indigenous strains, while S. pasteurii served as a control. Bacterial inoculum reached approximately 109 CFU. Collected sands were sterilized and placed in experimental trays, with three types of vinasse applied, differing in pH. Treatments included acidic and neutral vinasses, supplemented with urea and calcium chloride as cementing agents. Sampling occurred at the end of first, third, and fifth weeks of incubation to measure enzymatic activities of catalase, dehydrogenase, and urease. Data analysis used a split-plot design and Duncan's multiple range test at a 5% significance level.

Results and discussion

The highest catalase activity was observed in the combination of B. licheniformis strain 1D2, vinasse from alcohol and yeast mixture, with acidic pH, along with urea and calcium chloride at the third week. The catalase enzyme activity indicates the activity of aerobic microorganisms and soil fertility. Studies show that microbial communities under oxidative stress (higher H2O2 production) have increased catalase concentrations. Some reports indicate increased catalase enzyme activity in soils irrigated with vinasse, leading to improved performance and growth of sugarcane seedlings.

Statistical analysis indicated that bacterial treatments and incubation time significantly affect dehydrogenase enzyme activity in sand dunes. B. licheniformis strain 1D1 and S. pasteurii exhibited the highest activity, respectively. The increase in dehydrogenase activity signifies a rise in microbial population and soil ecosystem dynamics. Dehydrogenases are important as indicators for assessing microbial activity in soil, and their activity has been documented in sandy soils. During the incubation period, dehydrogenase activity showed a downward trend due to the depletion of carbon sources.

The highest urease activity was observed with S. pasteurii and B. licheniformis strain 1S5 in neutral vinasse combined with calcium chloride and urea. Urease activity showed a decreasing trend from the first to the fifth week. S. pasteurii is one of the most common urease-producing bacteria, and urease activity has also been reported in B. licheniformis RC636. In this study, higher urease activity in sand dunes was noted when isolates grown in neutral vinasse, as these isolates prefer a pH higher than acidic. Urease activity was greater in treatments with urea and calcium chloride, leading to the production of ammonia and carbonate and the precipitation of calcium carbonate. The declining trend in enzyme activity was associated with cellular aging and the salinity of the vinasse environment.

Conclusions

The study results indicate that indigenous isolates performed better in tests, and selecting isolates with lower urease activity but a slower release rate promotes more effective bonding between particles (like sand). Field-scale investigations could yield even better results.

Author Contributions

Negin Pirhadi: Data collection, data analysis and interpretation, writing the original draft.

Habib Nadian: project administration, reviewing and editing the manuscript.

Bijan Khalili moghadam: Contribution to experimental design, reviewing and editing the manuscript, supervision,

Hossein Motamedi: Conceptualization, methodology, providing resources, reviewing and editing the manuscript,

 All authors have read and agreed to the published version of the manuscript. All authors contributed equally to the conceptualization of the article and writing of the original and subsequent drafts.

Data Availability Statement

Data available on request from the authors.

Acknowledgements

This work is part of the doctoral dissertation of [Negin Pirhadi]. The authors would like to thank the reviewers and editor for their critical comments that helped to improve the paper. The authors gratefully acknowledge the support and facilities provided by the Department of Soil Science, Faculty of Agriculture, Agricultural Science and Natural Resources University of Khuzestan, Ahvaz, Iran.

The authors would like to thank all participants of the present study.

Ethical considerations

The authors refrained from engaging in data fabrication, falsification, plagiarism, and any form of misconduct.

Conflict of interest

The author does not declare any conflicts of interest.

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Iranian Journal of Soil and Water Research
Volume 56, Issue 8
November 2025
Pages 2285-2299

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