IMPROVEMENT ON BACTERIA-INDUCED CALCIUM
MINERALIZATION ABILITY OF BACILLUS PSEUDOFIRMUS BY AN
INTEGRATED HIGH-THROUGHPUT SCREENING STRATEGY
J.L. Zhang1, X. Deng1，X. Feng2, N. X. Han2, H. M. Jonkers3
1
Institute of Eco-environmental Science, College of Life Science, Shenzhen University, Shenzhen, P.R. China
2
Guangdong Provincial Key Laboratory of Durability for Marine Civil Engineering; Shenzhen Durability Centre for Civil Engineering; College of Civil Engineering, Shenzhen University, Shenzhen, P.R. China
3
Faculty of Civil Engineering and GeoSciences, Delft University of Technology, Delft, The Netherlands
Keywords: biomineralization, Bacillus pseudofirmus, UV-induced Mutation, High throughput screening,
ABSTRACT
The CaCO3-mineralizing bacteria from different taxonomic groups have shown
potential in restoration of construction material such as concretes, cements and stony materials. However, these strains are far from the demand of practical application due to some shortages, including the low mineralizing capacity.
In this work, an integrated high-throughput screening(HTS) strategy was developed in an attempt to screen enhanced CaCO3-producing mutants of Bacillus
pseudofirmus. The isolates, mutagenized by ultraviolet radiation (UV), were cultivated
in the suitable media containing calcium ion by using 96-deep-well microliter plates. The residual calcium in supernatants from micro-cultivation plates was determined by O-Cresolphthalein Complexone method to evaluate the CaCO3-producing activity. On
the other hand, the activity of carbonic anhydrase of the isolates, which is responsible for the formation of CO32-, was also monitored to further evidence the
bacteria-induced calcium mineralization process.
As a result, the mutant strain B388 with the highest calcium mineralizing capability (92.67%, 11.1% higher than that of the original strain) was obtained from about 3000 isolates. It is proved that this novel HTS strategy is a promising procedure for selecting high efficient calcium mineralizing microorganisms.
1. INTRODUCTION
Self-healing of concrete by microorganisms has been proved to be a promising strategy during the recent years [1, 2, 3, 4]. Although protective vehicles were necessary for the effctiveness of bacterial self-healing process [1, 3, 4], the calcium mineralizing activity of the self-healing microorganism itself was also crucial. The aim of this work, therefore, was to improve CaCO3-producing capability of a previously used bacterial
strain in our group.
2. MATERIALS
Strain Bacillus pseudofimus DSM8715 was provided by Dr. H. M. Jonkers(Delft University of Technology, Delft, Netherlands); Calcium standard concentrate,O-cresolphthalein complexone(OCPC) and 8-Hydroxyquinoline were purchased from Sigma-Aldrich (St. Louis, MO);3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) was purchased from Ameresco(Solon,OH) ; 2-Amino-2-methyl-1-propanol(AMP) and L-sodium lactate was purchased from Aladdin(shanghai).
3. METHODS
The UV-mutagenesis of strain DSM 8715
Strain DSM8715 was grown overnight on LB Agar plates containing 1% Na2CO3 at
30℃.After washed twice with 1% Na2CO3, the cells were resuspended in the same
buffer and diluted to about 108 cells/ml which was detected with bacterial microscopic counting method. Then the bacterial suspension were exposed to UV-light for 16 min which resulted in a death rate of 92%. The UV-treated cell suspension was cultivated in the LB broth containing 1% Na2CO3 for 6 h and then gradiently diluted and spread
on LB agar plates. After 48 h cultivation at 30℃, single colonies formed and were respectively transfered onto the LB agar in the wells of 96-well microplates(Corning Inc.; Corning, NY). The inoculated microplates(plate 1) were cultivated for 24 h at 30℃ and then preserved at 4℃ for further tests.
The screening of high-effenciency calcium mineraling mutant isolates
The O-Cresolphthalein Complexone(OCPC) method[6] was applied to evaluate the CaCO3-producing activity of all mutant isolates.The high throughput screening
strategy was introduced in the selection of mutant isolates.Cultures in the above microplate(plate 1) were respectively transfered into the ASWM4 medium(L-sodium lactate(60%) 15ml, yeast extract 1g, NaNO3 2g, MgCl2 0.1g, KH2PO4 0.02g, CAPS
23g, atificial sea water[5] without MgCl
2 and NaHCO3 1L, pH=10.5) in a 96-deep-well
microplate(plate 2) using a multi-channel pipettor and precultivated for 36 h. 10μL culture of each well was then transfered into another 96-deep-well microplate(plate 3) and cultivated for 7 d. The culture from plate 3 was centrifugated (6000×g, 5 min) and 3.75μL supernatant of each well was transfered into coressponding well of another 96-well microplate(plate 4) with 300μL calcium assay reagents(25 mg/L OCPC and 0.5 g/L 8-Hydroxyquinoline in a buffer containing 45g/L AMP) added into each well. The mixtures were gently agitated and further incubated at 30℃ for 10 min. Light absorbance of each well was measured at 575 nm on a 96-well plate spectrometera (SpectraMax 190, Molecular Devices, Corp.). Standard curve was obtained by using Ca2+ at following concentrations(mM): 0, 0.25, 0.5, 1, 2, 3,4 and 8. Calcium mineralizing activity was calculated using the following equation:
R=( C0- C )/C0×100%
R, the calcium mineralizing ratio;
C, the residual calcium concentration after 7-day cultivation; C0, the initial calcium concentration;
4. RESULTS
Considering that the measurement of calcium ion in supernatant might be easy and highly efficient, the reduction of free calcium ions in the medium was determined to indirectly assess the yield of CaCO3 during the experiment. ASWM4 was a newly
designed medium for the calcium mineralizing assay. Artificial sea water and high pH were used in an attempt to mimic the coastal concrete environment. The results showed that strain DSM8715 was able to resist the high salinity and induce the
Figure 1: CaCO3 production by strain DSM8715 and B388.
A, The CaCO3 particles produced by strain DSM8715 in a flask; B, Optical
photomicrograph of the CaCO3 particles produced by strain DSM8715; C, the
calcium mineralizing ratio of strain DSM8715 and B388 after 7-day incubation. formation of CaCO3 particles in ASWM4 liquid medium(Fig. 1, A and B). The calcium
mineralizing ratio of DSM8715 was remarkably higher than that of the control (no bacterial strain) (Fig.1, C).
The established high-throughput screening method was used to select Bacillus
pseudofirmus mutants with high CaCO3-producing activity. Totally 3000 single
colonies were obtained and among them the calcium mineralizing activity of one mutant strain designated as B-388 reached 92.67%, displaying 11.11% increase compared with that of the original strain DSM8715, which was 83.4%. Comparatively, the calcium mineralizing ratio of the medium ASWM4 without bacterial induction was
B
C
A
only 24.39%. It might be due to the high pH value of the medium. CO2 in the air
reacted with OH- in the medium and resulted in the formation of CO32-, which further
reacted with Ca2+ to form insoluble CaCO3.
5. CONCLUSIONS
In summary, a simple and easy HTS strategy was developed for the screening of CaCO3-producing microorganisms after UV-irradiation treatment. The efficiency of
this procedure depended on the high-throughput characteristics of each step in the screening process, especially for bacteria cultivation, culture centrifugation and Ca determination. By using this novel method, more than 3000 strains were evaluated in 3 weeks and one strain (B-388) with 11% improvement of calcium mineralizing activity was achieved, proving that this method is a rapid and reliable way to screen high efficient CaCO3-producing microorganisms.
ACKNOWLEDGEMENTS
Financial support of the National Natural Science Foundation of China for this study (Project No.51120185002 and No.50925829) is gratefully acknowledged.
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