Presented at 1st International KAMPAI Symposium
6th and 7th November 2017
Sapporo, Hokkaido, JapanIMMOBILIZATION OF LEAD-CONTAMINATED MINEWASTE BY PARARHODOBACTER SP
Mwandira W1, Nakashima K2 , Kawasaki S2, Chirwa M3, Banda K.E.3, and Nyambe I.A.4
1Graduate School of Engineering, Hokkaido University, Japan
2 Faculty of Engineering, Hokkaido University, Japan

3Geology Department, University of Zambia, Zambia

4Integrated Water Resources Management Center, University of Zambia, Zambia

14 November 2017

Presentation outline■ Introduction■ Objectives■ Methodology■ Results and discussion■ Conclusions■ References

2Introduction
Rapid urbanization and industrialization has resulted in extensive exploitation of themineral resource has resulted in environmental pollution by toxic metals (Cd, Cu, Pb,Hg, Cr, and Fe) which pose a threat to the environment and to human health.

Slag

Leaching residue

Ore stock piles

Sources of lead in
the environment
from mining

MiningAir emissions

Waste rockAIR

Smelting

WATER

SOIL

3

Techniques for removing lead from soil
REMEDIATION TECHNIQUE

UNIT

COST ($)

ADDITIONAL FACTORS/EXPENSES

Vitrification

Tonne

75 - 425Long term monitoring

CappingTonne

100 - 500

Transport/excavation/Monitoring

Chemical treatment

Tonne

100 - 500

Recycling of contaminants

Electrokinetic

Tonne

20 - 200

Long term monitoring

Thermal treatment

Tonne

750 - 1200

Long term monitoring

Phytoextraction

Tonne

5 - 40Long term monitoring

Sources: http://www.building.co.uk

Physico-chemical methods take longer and have high costs associated with energy and chemical
consumption in addition to possible emission of secondary pollutants. Therefore there is a the needto develop bioremediation methods. One method is to immobilize heavy metals based on microbiallyinduced calcium carbonate precipitation (MICP): Eco-friendly
 Easily controlled
 Low cost
4

MICP mechanism
MICP involves the hydrolysis of urea into ammonium and carbarmate by ureasecatalysis which results in CaCO3 formation in the presence of Ca ions.
ureaurease

CO(NH2)2 + 2H2O → 2NH3 + HCO3- + H+HCO3- + Ca2+ + OH- → CaCO3↓ + H2O
sandurea

CaCO3

Ca2+

ureaseproducing
bacteria
Pararhodobacter sp. was selected for investigation because it has shown high urease activity and can maintain theenzyme activity for a long time.
5

Objectives
1.Investigate the effects of lead on microbial growth;
2.Determine the effectiveness of lead removal
Pararhodobacter sp. in bioprecipitation experiment;by

3.Determine the effects of varying the injection interval of
the bacteria on unconfined compressive strength (UCS)
for fine and coarse-grained sand;

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METHODOLOGYMaterials
Sand and mine waste
Sand type

Nutrient and cementation solution

Mean diameter (D50)(μm)

ChemicalChemical concentration
(g/L)

1200

Nutrient Broth (g)Toyora sand200

NH4Cl (g)10Leach plant residue

75

NaHCO3 (g)2.12(NH2)2CO (g)30.03

CaCl2 (g)55.49

Misunami sand

Kiln Slag

Other conditions
Parameters>2500

Conditions

Temperature

30oC

Bacteria OD600

1.0Injection intervals

1,2,4,7

Medium3

Bacteria
Pararhodobacter sp.
isolated from
Okinawa, Japan.

ZoBell2216 media
8Effect of lead on microbial growth-viable plate countPararhodobacter sp.
Bacterial Plate culture

Incubate
24 hrs, 30 °C, 160 rpm

Transfer
Colony streak
ZoBell Medium
5 mL)

24-hr Pre-culture(5 mL)

Different
concentrationof leadTransfer1 mL

Main-culture(100 mL)

Serial dilutions and counting
colony forming unit (CFU)
48-hr culture (100 mL)(Number of colonies × Dilution factor)
𝐶𝐶𝐶𝐶𝐶𝐶=𝑚𝑚𝑚𝑚
Volume of liquid culture sample plated(Miles And Misra, 1938)

9Bioprecipitation experiment

Incubate
24 hrs, 30 °C, 160 rpm

Transfer
Colony streak
Pararhodobacter sp.
Bacterial Plate culture

ZoBellMedium
5 mL)

24-hr Pre-culture(5 mL)
Supernatant

Transfer
1 mL

Main culture (100mL)
48 hrs, 30 °C, 160 rpm

ICPE AES

Bacteria culture

XRD

Centrifuge
SEM

Precipitate++CaCl+Urea

2Incubate(6hrs, 30°C, 160 rpm)

PbCl210Conceptual model of experimental setup

Solution

Solidification
Solution

Solution

1 cm 2 cm

Solidification

2 cm

Solidification

2 cm

Bacteria andsolidification
solution

EXPERIMENTAL CONDITIONS
Urea
0.5M

FilterFilterControl Valve
pH and Pb/Zn analysis

Fine sand(170 µm)

6 cm

Coarse sand(1.2 mm)

7 cm

Fine sand(170 µm)

7 cm

Coarse sand(1.2 mm)

pH and Pb/Zn analysis

0.5M

Nutrient Broth

3 g/L

Sodium Hydrogen
Carbonate

2 g/L

Ammonium
Chloride

10 g/L

Temperature30oCExperimental
Time14 Days

Bacteria OD60

1.0

Fine sand (Toyorasand)Course sand(Misunami sand)

FilterControl Valve

Calcium Chloride

Control Valve
pH and Pb/Zn analysis

Injection intervals

170µm
1.2mm
1,2,4,711Waste types at Kabwe Mine Site
Leach plant residue(LPR)

Kiln slag

12RESULTS AND DISCUSSION

Effect of lead on microbial growth and urease
Colonies of Pararhodobacter in lead
10.0

ControlPb = 0.01mM

Pb = 0.05mM

Pb = 0.25mM

Log cell/mL

8.0
6.0
4.0
2.00.00mM

0.01mM 0.05mM 0.25mM 0.5mM
Lead concentrations

The bacterium can be used for bioremediation, as the effect of lead on thebacteria is negligible for the concentration evaluated.
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Lead bioprecipitation - ICP-AES

Removal percentage (%)

100

 Leadoccurred
withpercentage of 100%.

80

removal toxic Pb2+ ions from soluble form toinsoluble forms hence detoxifying thetoxic lead ions60

 Comparison between this study andprevious studies involving ureolytic
bacteria:
Rhodobacter
spharoides(90.31%)(Li et al., 2016); Sporosarcina
pasteurii (100%) (Mugwar and Harbottle,2016); Enterobacter cloacae (68.1%)(Kang etal., 2015); and Terrabacter tumescens(100%)(Li et al., 2015).

40
200a0.5

15

Lead concentration (mM)
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Lead bioprecipitation – SEM and XRD

Precipitate formation
due to MICP

SEM image precipitate

XRD of precipitate SEM-framboidal aggregates were identified as vaterite, whereas spherical and rhombohedral shaped
precipitates were identified as calcite.
 XRD - confirm calcium carbonate formation induced by Pararhodobacter. The free lead ions in solutionare immobilized in the matrix of calcium carbonate or formation of PbCO3. (C = Calcite V = Vaterite; L= Lead Oxide (PbO)).
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Estimated UCS and optimal injection interval

The more the bacterial injection increased, the more the increase in UCS. This is due to the numberof nucleation sites available for microbial precipitation.
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Sand solidification samples

Pictorial images of the results of all syringe tests after 14 days while varying the bacterial injection
interval to (a) one (b) two (c) four and (d) seven times. Left, fine sand; center, coarse sand; right, mixtureof course and fine sand.
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Conclusions
1. Lead has negligible effect on microbial growth and urease activity ofPararhodobacter and therefore, Pararhodobacter sp. can be used for
remediation purposes.
2. Pararhodobacter was effective in complete removal of lead by changing
toxic Pb2+ ions from soluble form to other forms hence detoxifying thetoxic lead ions ;3. These results will facilitate the possible bioremediation of lead in bothfine and coarse materials as an eco-friendly and sustainable method ofheavy metal remediation.

19References1.

Mugwar, A.J., Harbottle, M.J., 2016. Toxicity effects on metal sequestration by microbially-induced carbonate
precipitation. J. Hazard. Mater. 314, 237–248. doi:10.1016/j.jhazmat.2016.04.039

2.

Li, X., Peng, W., Jia, Y., Lu, L., Fan, W., 2016. Bioremediation of lead contaminated soil with Rhodobacter
sphaeroides. Chemosphere 156, 228–235. doi:10.1016/j.chemosphere.2016.04.098

3.

Li, M., Cheng, X., Guo, H., Yang, Z., 2015. Biomineralization of Carbonate by Terrabacter Tumescens for Heavy
MetalRemovalandBiogrouting
Applications.
J.
Environ.
Eng.
C4015005–C4015005.
doi:10.1061/(ASCE)EE.1943-7870.0000970.

4.

Kang, C.H., Oh, S.J., Shin, Y., Han, S.H., Nam, I.H., So, J.S., 2015. Bioremediation of lead by ureolytic bacteria
isolated from soil at abandoned metal mines in South Korea. Ecol. Eng. 74, 402–407.
doi:10.1016/j.ecoleng.2014.10.009

5.

Lorens, R.B., 1981. Strontium, cadmium, manganese, and cobalt distribution coefficients in calcite as a functionof calcite precipitation rate. Geochim. Cos- mochim. Acta 5, 553–561.

6.

https://wwwbrr.cr.usgs.gov/projects/SW_inorganic/index.html, accessed 17th August 2017.

7.

http://www.osatelegraph.org/current-events/the-fruitvale-and-the-nationwide-lead-crisis, accessed 17th August
2017.

8.

A. Miles, S. Misra, and J. Irwin, (1938). The estimation of the bactericidal power of the blood,” J. Hyg. (Lond), pp.
732–749.

9.

Mwandira, W., Nakashima, K. and Kawasaki, S. “Bioremediation of lead-contaminated mine waste byPararhodobacter sp. based on the microbially induced calcium carbonate precipitation technique and its effects
on strength of coarse and fine grained sand”, Ecological Engineering, Vol 109, 2017, pp. 57-64.

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Thank you for your attention

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