Proceedings of International Forum for Green Asia 2017
Special issue of Evergreen - Joint Journal of Novel Carbon Resource Sciences & Green Asia StrategyP-22

Synergistic Mn(II)-oxidative Removal from a New Mn(II)-oxidizing Bacterialstrain, Pseudomonas sp. strain SK3 and Refinery Wastewater Deposit
Santisak Kitjanukit1, Kenji Takeda2, Satoshi Asano2, and Naoko Okibe1
1

Department of Earth Resources Engineering, Kyushu University
Hydrometallurgical process development group, Niihama Research Laboratories,
Sumitomo Metal Mining Co., Ltd2AbstractSince divalent manganese (Mn(II)) is stable at wide-range of pH, loads of alkaline agents andoxidants are required for its treatment (oxidative precipitation). The present study is aiming toimprove Mn(II)-oxidative removal efficiency in harsh condition mimicking the actual refinery
wastewater. Addition of an aliquot amount of wastewater deposit (identified as Mn oxide) tostrain SK3 cell suspension dramatically improved Mn(II)-oxidative removal (100 ppm ofMn(II) was completely removed within 24 hours). The results suggest that the combination ofboth chemical and bacterial Mn(II) oxidation may be useful for the industrial wastewater
treatment process1. Introduction
High stability of Mn(II) at a wide range of pH
made it difficult to be removed from wastewater.
Conventional technology for Mn(II) treatment
relies on physiochemical reaction [1] involves
the uses of strong oxidant and abundant of thealkaline compound to oxidize and precipitateMn(II) as Mn oxides. More economical andenvironmental benign process is needed.
Previously, a new Mn(II)-oxidizing bacterium,
Pseudomonas sp. SK3 isolated from refinery
wastewater deposit was demonstrated robustMn(II)-oxidizingactivity(Kitjanukit,unpublished data). Mn(II)-oxidizing ability ofstrain SK3 become deteriorated when subjected
to harsher conditions resulted in sluggish
Mn(II) oxidative removal speed. This study
aims to improve the robustness as well as thespeed of Mn removal.

2.2 Preparation of dried sterile Mn oxide
depositRefinery wastewater deposit was soaked with DI
water and autoclaved (120 ℃, 20 min) for
preventinganyeffectfromindigenous
microorganisms. The
depositwasthenfreeze-dried overnight prior to being use in furtherexperiments.
2.3 Manganese removal test
An aliquot of freeze-dried wastewater deposit(0.5g) was added into SK3 cell suspension inPYG medium (0.025% (w/v) yeast extract 0.025%(w/v) peptone, 1 mM glucose, 2.02 mM (100ppm) MgSO4• 7H2O, 0.068 mM CaCl2• 2H2O).
pH of the medium (7.0) was buffered by 15 mM
PIPES. Samples were routinely withdrawn toanalyze pH, Eh, and dissolve Mn concentration.
The solid parts were collected after theexperiment for scanning electron microscope(SEM) observation. Both cells and the deposit
were fixed (with a mixture of glutaraldehyde andformaldehyde), dehydrated (with ascending
concentration of ethanol), and finally sputter
coated, prior to observe with SEM.

2. Materials and methods
2.1 Characterization of wastewater deposit
An aliquot amount of refinery wastewater deposit
was freeze-dried overnight and digested in aquaregia (HNO3:HCl = 3:1, assisted with microwave
irradiation for 60 min), before analyzing its metalcomposition by ICP-OES. XRD analysis also
conducted for identification of metal phase.

3. Results and Discussion
3.1 Characterization of wastewater deposit
The deposit was found to consist mainly ofcrystalline Mn oxides based on ICP-OES andXRD analysis.

59Proceedings of International Forum for Green Asia 2017
Special issue of Evergreen - Joint Journal of Novel Carbon Resource Sciences & Green Asia Strategy

P-22
the inhibitory effect of high temperature andMgSO4 [2]. Moreover, Mn oxide could havefacilitated chemical Mn removal throughoxidation and adsorption.

3.2 Synergistic Mn(II)-oxidative removal bystrain SK3 and Mn oxide
The presence of both strain SK3 cells and Mnoxide improved Mn removal even under
originallyinhibitory
conditions(hightemperature (35℃) and high MgSO4
concentration (200 mM)) to planktonic SK3cells. No synergistic effect was seen when thetemperature was raised to 40℃ (fig 1).

5. Conclusion
The refinery wastewater deposit wascharacterized as Mn oxides. Addition of it
together with strain SK3 effectively promote
Mn(II)-oxidative removal speed. Since it could
provide a site for biofilm formation which
alleviated the inhibitory factors (ex: hightemperature and MgSO4) and also promoted
chemical Mn(II)-oxidation. Further studies areongoing to access the applicability of strain
SK3 for bioprocess of Mn(II)-containingwastewater treatment.
Acknowledgment
This work was partly supported by Sumitomo
Metal Mining Co., Ltd, Japan. S.K. is grateful forthe financial assistance provided by the Kyushu
University Advanced Graduate Program in Global
Strategy for Green Asia.

Figure 1 Mn removal efficiency in the presence ofstrain SK3 and wastewater deposit.

Reference[1] P. Roccaro, C.B., G. Mancini, F.G.A.
Vagliasindi: Desalination, (2007) Vol. 210,
pp.205-214.

3.3 Biofilm formation on wastewater deposit(Mn oxide) surface
Figure 2 showed desiccated biofilm (resultingfrom dehydration step during SEM samplepreparation) of strain SK3 on wastewater
deposit (Mn oxide) surface. Addition of Mnoxide could have provided SK3 cells with asurface to colonize via biofilm forming. Thismay have enabled cells to be less affected by[2] T. Garrett, M. Bhakoo, Z. Zhibing: Progressin Natural Science, (2008) Vol. 18,
pp.1049-1056
Email: ming@mine.kyushu-u.ac.jp

Figure 2 SEM micrograph of the precipitatescollected at the end of experiment showing strain
SK3 cell attached to Mn oxide surface with biofilm

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