research article indirect manganese removal by ... · research article indirect manganese removal...

Research ArticleIndirect Manganese Removal by Stenotrophomonas sp andLysinibacillus sp Isolated from Brazilian Mine Water

Nataacutelia Rocha Barboza1 Soraya Sander Amorim1

Pricila Almeida Santos1 Flaacutevia Donaacuteria Reis2 Mocircnica Mendes Cordeiro3

Renata Guerra-Saacute1 and Versiane Albis Leatildeo2

1Laboratorio de Bioquımica e Biologia Molecular Departamento de Ciencias Biologicas (DECBI) andInstituto de Ciencias Exatas e Biologica (ICEB) Universidade Federal de Ouro Preto 35400-000 Ouro Preto MG Brazil2Laboratorio de BioampHidrometalurgia Departamento de Engenharia Metalurgica e de Materiais (DEMET)Escola de Minas Universidade Federal de Ouro Preto 35400-000 Ouro Preto MG Brazil3Faculdade Unileste 35160-215 Ipatinga MG Brazil

Correspondence should be addressed to Renata Guerra-Sa rguerrasagmailcom

Received 6 August 2015 Revised 9 November 2015 Accepted 11 November 2015

Academic Editor Xiaofeng Fan

Copyright copy 2015 Natalia Rocha Barboza et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Manganese is a contaminant in the wastewaters produced by Brazilian mining operations and the removal of the metal isnotoriously difficult because of the high stability of the Mn(II) ion in aqueous solutions To explore a biological approach forremoving excessive amounts of aqueousMn(II) we investigated the potential ofMn(II) oxidation by both consortium and bacterialisolates from a Brazilian manganese mine A bacterial consortium was able to remove 997 of the Mn(II) A phylogenetic analysisof isolates demonstrated that the predominant microorganisms were members of Stenotrophomonas Bacillus and Lysinibacillusgenera Mn(II) removal rates between 585 and 709 were observed for Bacillus sp and Stenotrophomonas sp while theLysinibacillus isolate 13P removes 827 The catalytic oxidation of Mn(II) mediated by multicopper oxidase was not properlydetected however in all of the experiments a significant increase in the pH of the culture medium was detected No aggregatesinside the cells grown for a week were found by electronic microscopy Nevertheless an energy-dispersive X-ray spectroscopy ofthe isolates revealed the presence of manganese in Stenotrophomonas sp and Lysinibacillus sp grown in K medium These resultssuggest that members of Stenotrophomonas and Lysinibacillus genera were able to remove Mn(II) by a nonenzymatic pathway

1 Introduction

Manganese is a common contaminant in many mine watersgroundwater and freshwaters worldwide [1ndash8] Brazil is oneof the largest producers of manganese ore and the stateof Minas Gerais has the largest reserves of this metal [9]Unfortunately mining activities may result in the dissolutionof manganese-containing minerals and lead to the contami-nation of freshwater with the element Because its solubilityis high manganese concentrations of up to 100mgsdotLminus1 can befound in some Brazilian mine waters and this concentrationis much greater than the limit that was established by theBrazilian legislation for industrial effluents (ie 10mgsdotLminus1)[10]

Typically Mn(II) ions are chemically removed fromeffluents by oxidation to MnO

2

adsorption or precipitationas a carbonate [7 11ndash14] However chemical processes areoften resource intensive because multiple steps are requiredFurthermore such processesmay present low efficiencies andproduce secondary pollution such as toxic byproducts [15ndash17]

Manganese removal using biological processes could bean alternative to chemical routes The role of microbial activ-ity in the remediation of manganese-contaminated watershas been frequently documented [1 2 5 18ndash20] In additionto biosorption the bacteria-mediated precipitation of MnO

2

has been proposed Manganese-oxidizing microorganismsare widespread in nature and are phylogenetically diverse

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 925972 14 pageshttpdxdoiorg1011552015925972

2 BioMed Research International

[16 21ndash23] Although several bacteria promote manganesebioremediation only few species have been extensively stud-ied (ie Pseudomonas Bacillus Leptothrix and Pedomicro-bium) [5 24ndash31] Furthermore few studies have identifiedor tested the ability of local bacterial strains to remove theelement despite high Mn(II) concentrations in some minewaters and groundwater in tropical countries Therefore thegoals of the current study are to (i) enrich a bacterial con-sortium from mine waters with high Mn(II) concentrationsand (ii) isolate and identify the bacterial strains from theenrichment while assessing their manganese removal abilityThis knowledge would be useful for the design of new moreefficient strategies for treatingmanganese-laden wastewaters

2 Material and Methods

21 Sample Sites A water sample was collected from a man-ganese mine in the Iron Quadrangle region (Minas GeraisBrazil) The mine water was stored in plastic containers atroom temperature until laboratory processing A sample ofthe mine water was selected and analyzed for metal contentby inductively coupled plasma optical emission spectrometry(ICP-OES Varian 725)

22 Enrichment A subsample comprising one liter of minewater was filtered through a 022 120583m membrane (MillexMillipore) The membrane was transferred to Falcon tubeswith 10mL of K medium (015 gsdotLminus1 MnSO

4

sdotH2

O 2 gsdotLminus1peptone 05 gsdotLminus1 yeast extract and 10mM HEPES bufferpH 75) Cultures were incubated at 30∘C on an orbitalshaker (150minminus1) for 24 h Subsequently the cultures weretransferred to 250-mL flasks containing 90mL of K mediumand incubated as described above The obtained consortiumwas called CL throughout this study After 24 h 50 (wv)glycerol was added and aliquots of 1mL were stored inEppendorf tubes at minus80∘C until the experiments were con-ducted

23 Isolation Procedures Pure strains were isolated fromthe enriched CL consortium by plating on 15 (wv) agar-solidified K medium The agar plates were amended with50mgsdotLminus1 Mn(II) ion after autoclave sterilization The cul-tures were incubated at 30∘C until colonies formed andorthe culture medium containingMn(II) turned brownishThecolonies which had changed color andor changed themediacolor were selected and bacterial isolates were inoculatedinto K medium without Mn(II) The isolates were preservedat minus80∘C in 50 (wv) glycerol

24 Manganese Removal Experiments Batchwise manganeseremoval experiments at 50mgsdotLminus1 Mn(II) ion concentra-tions in a 2-week-long period were performed with theCL consortium These experiments were accomplished in asmall-scale 3-L bioreactor (BioFlo 110 FermentorBioreactorNew Brunswick Scientific) at a one-liter final volume Priorto the bioreactor experiment 1mL of the CL consortiumpreserved at minus80∘C was cultured in 10mL of K medium(at 30∘C) on an orbital shaker (150minminus1) for 24 h (ie a

preinoculation period) Subsequently the preinoculum wastransferred to 250-mL flasks containing 90mL of K mediumand incubated under the same conditions as describedabove Thereafter 100mL of the culture (approximately 01optical density at 600 nm) was transferred to a bioreactorcontaining 900mL of K medium at pH 75 in the presenceof 50mgsdotLminus1 Mn(II) and maintained at 30∘C under constantstirring (150minminus1) for twoweeks Aliquots were periodicallycollected for manganese concentration pH and bacterialgrowth measurements (by counting cells in a Neubauerchamber) The experiments were performed in duplicateConcurrently a similar bioreactor was operated as a controlin which the bacterial growth was inhibited by a Nipagin(014 wv)-Nipazol (01 wv) addition

Manganese removal by the isolates was performed inflasks at final volumes of 100mL The preinoculum wasprepared as described above for the CL consortium Sub-sequently 10-mL aliquots of the preinoculum were trans-ferred to 250-mL flasks containing 90mL of K medium thatwas supplemented with 50mgsdotLminus1 Mn(II) at an initial pHof 75 The flasks were maintained at 30∘C on an orbitalshaker (150minminus1) for seven days Aliquots were collectedperiodically and the same analyses that were performedfor the consortium CL were performed for the isolates(ie manganese concentration pH and bacterial growthmeasurements using the optical density method in a Hitachi2800A spectrophotometer) In the control flasks under thesame conditions bacterial growth was inhibited by addingNipagin (014 wv)-Nipazol (01 wv) The experimentswere performed in triplicate

For manganese analysis approximately 4-mL aliquots ofculture were centrifuged for 15min at 14681timesg and filteredthrough a 022-120583m membrane The filtrate was collecteddiluted 10 or 25 times in distilled water and acidified withHCl (1 1) solution The dilutions were analyzed by ICP-OESThe amount of manganese that was removed was measuredby the decay of the element in the culture medium

We also evaluated the culture growth on solid K mediacontaining different Mn(II) ion concentrations (140mgsdotLminus1300mgsdotLminus1 600mgsdotLminus1 and 1200mgsdotLminus1) Plates were incu-bated at 30∘C for two weeks after which dark brown-ish colonies or the browning of the culture medium wasobserved

25 Mn(II) Oxidation Assays Abiotic manganese oxidationwas confirmed in control experiments that were performedat different pH values (from 75 to 90 in 05 steps) Sam-ples of 01mL of the K medium that were amended with50mgsdotLminus1 Mn(II) and submitted to the same conditions ofthe experiments with the inoculum were mixed with 05mLof 004 Leucoberbelin blue I (LBB Sigma-Aldrich USA) in45mM acetic acid As a negative control K media without50mgsdotLminus1 Mn(II) or with manganese carbonate (MnCO

3

)solutions were also tested as described above For a positivecontrol K medium with manganese oxide (MnO

2

) was used

26 Mn(II) Oxidation by Cell-Free Filtrate The cell-freefiltrates were prepared by growing the isolates on K medium

BioMed Research International 3

for four days The cultures were then centrifuged at 7155timesgfor 20min the supernatant was filtered through a 022120583mpore size membrane filter and the resulting solution wasnamed the cell-free filtrate The cell-free filtrate was split intwo flasks one of which received proteinase K (100 120583gsdotmLminus1Promega) These flasks (with proteinase K) were incubatedat 37∘C for three hours before the addition of Mn(II)Subsequently approximately 30mgsdotLminus1 Mn(II) was added toall of the flasks (ie with orwithout proteinaseK)whichweremaintained at 37∘C and 150minminus1 for three days Sampleswere collected at 0 h 12 h 24 h 36 h 48 h 60 h and 72 hoursand the manganese concentrations were measured by ICP-OES to determine the removal efficiencies The presence ofoxidized manganese species was monitored by the additionof Leucoberbelin blue dye (LBB Sigma-Aldrich USA) to thesamples

27 Multicopper Oxidase Activity To determine whethermulticopper oxidases were involved in manganese oxidationby the isolates the strains were grown in agar-solidified Kmedium (15 wv) that was supplemented with 50mgsdotLminus1Mn(II) and 221015840-azino-bis(3-ethylbenzthiazoline-6-sulfonicacid) (ABTS Sigma-Aldrich USA) The Petri dishes wereincubated at 30∘C for up to 30 days and the multicopperoxidase activity was determined by the development of a bluecolor in the culture medium [32]

28 DNA Extraction and DGGE-PCR Analysis of CL Consor-tium A 100-mL aliquot of the CL consortium from theman-ganese removal experiment was centrifuged at 7155timesg for20min The pellets were washed three times with sterile PBS(818 gsdotLminus1 NaCl 198 gsdotLminus1 NaHPO

4

sdot7H2

O and 036 gsdotLminus1NaHPO

4

sdotH2

O pH 72) Then the pellets were resuspendedin 2mL of sterile PBS transferred to Eppendorf tubes andcentrifuged at 14681timesg for five minutes DNA was extractedfrom the pellets using a Wizard Genomic kit (Promega)following the manufacturerrsquos protocol and stored at 4∘C priorto use For the DNAg extraction from isolates samples werecultured in the absence of Mn(II) ions overnight at 30∘C onan orbital shaker (150minminus1)Then theDNAg extractionwasperformed as described above

The bacterial communities in consortia from the man-ganese removal experiment were analyzed by denaturing gra-dient gel electrophoresis (DGGE) The DGGE primers thatwere used were 968F1392R [33] 357F518R and 357907R(968F 51015840-AACGCAAGAACCTTAC-31015840 1392R 51015840-ACG-GGCGGTGTGTAC-31015840 357F 51015840-CCTACGGGAGGCAGC-AG-31015840 518R 51015840-ATTACCGCGGCTGCTGG-31015840 907R 51015840-CCGTCAATTCMTTTRAGTTT-31015840) [34] The primers 968Fand 357F contained a CG clamp 51015840-CGCCCGGGGCGC-GCCCCGGGCGGGGCGGGGGCACGGGGGG-31015840 that wasattached to the 51015840 end PCR amplifications were performedin 50-120583L reaction mixtures containing 1x Taq Buffer 15mMMgCl

2

02mMdNTPmix 02mMprimersmix 25U of TaqDNA polymerase (Thermo Scientific Taq DNA PolymeraseFermentas) and 10 ng of DNA template

For primers 968F1392R a PCR program was performedwith an initial denaturation step (94∘C 5min) followed by 35

cycles of denaturation (94∘C 45 s) annealing (63∘C 1min)and extension (72∘C 2min) and a single final extension step(72∘C 20min) For primers 357F518R and 357F907R thePCR thermocycling regime was performed with an initialdenaturation step (94∘C 5min) followed by 35 cycles ofdenaturation (94∘C 1min) annealing (49∘C 1min) andextension (72∘C 1min) and a single final extension step(72∘C 20min) The PCR products were visualized by 12agarose gel electrophoresis

Subsequently 20120583L of PCR product was subjected toDGGE analysis as described by Muyzer et al [35] followingthe manufacturerrsquos protocol for the CBS System ScientificEPS-30 II model DGGE-2401 For the DGGE analysis of theCL consortium a denaturing gradient ranging from 20 to60 (primers pairs 968F1392R and 357F907R) or 40 to 60(primers pair 357F518R) was used (a 100 denaturant wasdefined as 7M urea and 40 formamide) Electrophoresiswas performed in a TAEbuffer at 60∘Cand 100 volts for 155 hThe gels were stained with ethidium bromide and visualizedunder UV light

29 16S rRNA Gene Amplification Sequencing and Phyloge-netic Analyses DNAg was extracted from the isolated purecultures PCR was performed to amplify a region of the 16SrRNA gene fromDNAg extracts using primers 968F1392R asdescribed above (DNA Extraction and DGGE-PCR Analysisof CL Consortium) The PCR products were purified usingstandard protocols [36] and sequenced on a 3500 GeneticAnalyzer (Applied Biosystems) The quality of the 16S rRNAgene sequences was checked using the multiple alignmentCLUSTALW software package [37] A phylogenetic analysiswas performed using the neighbor-joining method and theJones-Taylor-Thornton model [38] The tree topologies wereevaluated by performing a bootstrap analysis with 1000replicates Phylogenetic analyses were conducted using theFigtree 14 software

210 Nucleotide Sequence Accession Numbers The partialsequence of the 16S rRNA gene sequence of Bacillus spLysinibacillus and Stenotrophomonas was deposited in theGenBank under the accession number from KT9629041 toKT9629061 and from KT9706811 to KT9706971

211 Scanning Electron Microscopy (SEM) TransmissionElectron Microscopy (TEM)Energy-Dispersive X-Ray Spec-troscopy (EDX) Analyses The isolates with Mn(II) removalactivity were cultured in liquid K medium containing50mgsdotLminus1 Mn(II) for 7 days as previously described Sub-sequently the suspensions were centrifuged and preparedfor SEMTEM analyses SEMEDX assays were performedusing a JSM-6360LV and FEG-Quanta 200 FEI while aTEMEDX analysis was performed with a Tecnai G2-12-SpiritBiotwin FEI-120 kV and a Tecnai G2-20-SuperTwinFEI-200 kV Experiments and analyses involving electronmicroscopy were performed in the Center of Microscopy atthe Universidade Federal de Minas Gerais Belo HorizonteMG Brazil


0

5

10

15

20

25

30

35

40

45

50

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

pH

66

64

62

60

68

70

72

74

76

78

82

80

Days0 2 5 7 9 12 14

Mn(II) ion concentrationpH

Bioreactor with 50mgmiddotLminus1 Mn(II) ion

(a)

05

1015202530354045505560

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

pH

66

64

62

60

68

70

72

74

76

78

80

Days0 2 5 7 9 12 14


Control

(b)

Bacterial growthtimes10

8

50

40

30

20

10

0

Cell

s num

bers

50mgmiddotLminus1 Mn(II)

1 2 3 4 5 6 7 8 9 10 11 12 13 140

Days

Bioreactor

(c)

Figure 1 Growth and pH variation during Mn(II) ion removal by CL consortium in a 2-week period The CL consortium was inoculated inK medium and was maintained in a bioreactor at 30∘C and under constant stirring (150minminus1)

3 Results

31 Manganese Removal by the Bacterial Consortium Theanalyses of the mine water under study showed manganeseas one of the major constituents (140mgsdotLminus1) at pH 65 [14]Using the K medium the CL consortium that was enrichedfrom this mine water was tested for its Mn(II) removalability as described in Section 2 In these experiments theinitial metal concentration was reduced to 50mgsdotLminus1 tominimize the manganese removal by air oxidation which iscatalyzed at higher pH values [39] The amount of Mn(II)that was removed by the consortium and the profiles ofboth pH and bacterial growth during the experiment are

shown in Figure 1 The concentration of Mn(II) continuouslydecreased over time in the presence of the CL consortiumwith a removal efficiency of 997 and the manganeseresidual concentrationwas 02mgsdotLminus1 whereas the pH variedbetween 736 (initially) and 786 at the end of the experiment(Figure 1(a))

Unlike the inoculated tests the pH values did not increasein the control experiment and remained around 74 whereasno significant manganese removal (344 after 14 days)was observed (Figure 1(b)) This outcome suggests that thepH increase that is shown in Figure 1(a) was promoted bybacterial growth


BioC

L50

mgmiddot

Lminus1

Mn(

II)-1

BioC

L50

mgmiddot

Lminus1

Mn(

II)-2

BioC

L50

mgmiddot

Lminus1

Mn(

II)-1

BioC

L50

mgmiddot

Lminus1

Mn(

II)-1

BioC

L50

mgmiddot

Lminus1

Mn(

II)-2

BioC

L50

mgmiddot

Lminus1

Mn(

II)-2

968F-CG1392R 357F-CG907R 357F-CG518R

Figure 2 CL consortium molecular characterization DGGE anal-ysis of the bacterial 16S rRNA gene as amplified from the totalcommunity DNA of the CL consortium

The bacterial counts peaked (40 times 108 cellsmL) withintwo days when the Mn(II) removal started (Figures 1(a) and1(c)) The bacterial population was stable until the seventhday and thereafter a slight decrease in cell counts wasobserved reaching 25 times 108 cellsmL on the 12th day At thistime we observed greater manganese removal (Figure 1(a))

32 Phylogenetic Analysis of the Isolates from the CL Consor-tium At the end of the 14-day Mn-removal experiment themicroorganisms were recovered by centrifugation and thecollected pellets were used for DNAg extraction and DGGE-PCR analysis The DGGE profile suggests low microbialdiversity because even when using several sets of primers toamplify different regions of the 16S rRNA few ampliconswereobtained (Figure 2)

Bacterial species identification after sequencing of the 16SrDNA fragments from the DGGE gel was not conclusive andan isolation procedure was used to determine the diversityof the CL consortium which resulted in 20 isolates (datanot shown) To identify the genera of the Mn(II)-oxidizingbacteria that were isolated in this study we sequenced the968F1392R 16S rRNA amplicons and used them for thephylogenetic study As shown in Figure 3 15 isolates belongedto the Firmicutes phylum with another five belonging tothe Gammaproteobacteria phylum Among the Firmicutesisolates 12 clustered with species from the Bacillus cereusgroup and the other three isolates were phylogenetically

closer to the genus Lysinibacillus (4P 6P and 13P) The fiveisolates belonging to the phylumGammaproteobacteria wereclustered with genus Stenotrophomonas We also performed abiochemical test using the Bactray I II and III system to iden-tify the species (data not shown)The results corroborated thephylogenetic analyses

33 Manganese Removal by the Isolates Initially two rep-resentatives of each identified genus (Bacillus Stenotroph-omonas and Lysinibacillus) were chosen for growth in solidK medium containing different Mn(II) ion concentrations(140mgsdotLminus1 300mgsdotLminus1 600mgsdotLminus1 and 1200mgsdotLminus1) todetermine whether these genera were capable of Mn(II)removal as indicated by the color of the colony andor ofthe medium These experiments also showed the toleranceof isolates to manganese The six isolates were able to becultured in the presence of 1200mgsdotLminus1 Mn(II) (Figure S1 inSupplementary Material available online at httpdxdoiorg1011552015925972) however growth at this concentra-tion was impaired Moreover colonies of Lysinibacillus spisolates (6P and 13P) showed a brownish color after one weekof growth in 140mgsdotLminus1 300mgsdotLminus1 and 600mgsdotLminus1Mn(II)suggesting the capacity of Mn(II) oxidation to either Mn(III)or Mn(IV) whereas the remaining isolates only made theculture medium color darker (Figure S1)

The analysis of the manganese concentration in liquidmedium is shown in Figure 4 The Bacillus isolates (1Gand 10G) showed no statistical differences in manganeseremoval capacities (Figures 4(b) and 4(c)) The 1G isolatesremoved 2781mgsdotLminus1 of the Mn(II) (585) whereas the10G isolates removed 3083mgsdotLminus1 of the Mn(II) (630)Among the Stenotrophomonas sp isolates (7P and 8P) thesame was observed (Figures 4(d) and 4(e)) The 7P isolateremoved 3478mgsdotLminus1 of the Mn(II) (709) from culturemedium and the 8P isolate removed 3212mgsdotLminus1 of theMn(II) (664) For Lysinibacillus sp isolates there was adifference in themanganese removal ability 6P removed only2817mgsdotLminus1 of the Mn(II) (6558) whereas the 13P isolateremoved 3676mgsdotLminus1 of Mn(II) during the experiments(827) In the control test (Figure 4(a)) no manganeseremoval within seven days of the experiments was observed

The solution pH increased to approximately 83 in theexperiments with Bacillus sp isolates (1G and 10G Fig-ures 4(b) and 4(c)) and Stenotrophomonas sp isolate 8P(Figure 4(e)) whereas it reached 85 for Stenotrophomonassp isolate 7P (Figure 4(d)) In the experiments with Lysini-bacillus sp isolates (6P and 13P) the pH increased only to 81(Figures 4(f) and 4(g))

Similar to the experiment with the CL consortium themedium pH did not increase in the control experiment(Figure 4(a)) which indicates that the pH increase in thebiotic experiments was promoted by bacterial growth (Fig-ures 1(a) and 4)

34 AbioticManganeseOxidation in LiquidMedium Theroleof pH in manganese oxidation was analyzed by submitting Kmedium that was supplemented with 50mgsdotLminus1 at differentpH values under similar conditions to those that were used in


100

100100

100

100

100100

100

100100

100

100

100

100100

100100100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100 100 100

100

100

100

100

100

100

100 1

00

100

100

100

100

100

100

100

100

100

100

100100

100

100

100

100

100

100

100

100

100

100

100

100

100100

100

100

100

100

100

100

100

100

10010

0

100100

100

100

100

100

100 100

100 100

100 100

100

100

100

100

100

100

100

100

100

100

100

100

T ptyseos LMG 7888 (NR_11

62831)

T ptyseos ATCC 33301

(NR_0253421)S

terrae

R-32768T

(AM4035892)

S ter

raeR-32

768

(NR_0

425691)

S acid

aminiphilaCCUG 5

4933

(GU9455351)

S acidaminiphila

AMX 19

(NR_02

51041

)

Bacillus sp PL-26 (AF3263691)lowast


S maltophilia IA

M12423

(NR_0415771)

P geniculataATCC19374

(NR_0247081)

S maltophiliaR

551-3

(NR_0748751)

S nitritireducensCCUG54932

(GU9455371)

B an

thra

cis(N

R_0744531

)

6G

9G

1G

12G

10G

8G

B cereus G9842 (CP0011861)

B cereus ATCC14579 (NR_0745401)

B thuringiensis Bt407 (NR_1025061)

B thuringiensis CT-43 (CP0019071)

B thuringiensis HD-771 (CP0037521)

5G

B weihenstephanensisKBAB4 (NR_0749261)

B cereus Rock1-3 (CM0007281)

7G

3GB thuringiensisMC28 (CP

0036871)

B toyonensisBCT-7112 (N

R_1217611)

2G4GB weihenstephanensisDSM

11821

(NR_0246971)

B thuringiensisATCC10792

(NZ_CM

0007531)

B mycoides

273

(NR_0368801)

B th

urin

gien

sisBM

B171

(CP0019031

)B

thur

ingi

ensis

IAM12077

(NR_0434031

)

B ce

reus

ATCC

14579

(NC_0047221

)

B cy

toto

xicu

sNVH

391

-98

(NR_0749141

)

B fu

nicu

lusN

AF001

(NR_0286241

)

B ab

yssa

lisSC

SIO15042

(NR_1096711

)

Bacillus sp MB-11 (AF3263601)lowast

A globiformis (AB0898411)lowast

A gandavensis R 5812 (NR_0254751)

A luteolus CF-25 (NR_0253621)

Rhodococcus sp Ld17 (FJ9669421)lowast

R opacus DSM 43205 (NR_0261861)

R opacus B4 (NR_0746321)

P putida F1 (NR_0747391)

P putida GB-1 (CP0009261) lowast

P putida MnB1 (U709771) lowast

P aeruginosa PAO1 (NR_0748281)

V soli GH9-3 (NR_0438111)

V paradoxus EPS (NR_0746461)

V paradoxus S110 (NR_0746541)

L discophora SP-6 (L339741) lowast

L cholodnii SP-6 (NR_0746231)

L discophora SS-1 (NR_0259161) lowast

X campestrisATCC33913 (NR_0749361)

S koreensis

(AB1668851)

S plymuthica IC1270

(AY55133

21)

K variicola At-22 (NR_0747291

)

K oxytoca KCTC 1686 (NR_102

9821)

K pneumoniae DSM 30104 (NR_1176

851)

P polymyxa DSM 36 (NR_1177301)P polymyxa SC2 (NR_1028031)

P polymyxa DSM 36 (NR_1177291)

P manganicum ATCC 33121 (NR_10484

11)lowast

R denitrificans OCh 114 (NR_1029091)

R litoralis Och 149 (NR_0275931)Roseobacter sp AzwK-3b (DQ22301

71)lowast

L boronitolerans NBRC 103108(NR_1142071

)

L boronitolerans 10

a (NR_04

12761)

L paki

stanensis

NCCP-54(N

R_1131661)

Lysinibacill

us sp R18

(KJ4997861

)

Lysin

ibacil

lussp

POUD (J

Q0077241)

Lysin

ibacil

lussp

BAB-

4156

(KJ7941141

)

L boronitolerans LCD9

(KJ7252361

)

L fusifo

rmis DSM 28

98(NR_04

20721)

L fusiformis NBRC 1

5717

(NR_1126281)

L parviboronicapiens NBRC 10314

4 (NR_114213

1)L fusiformis NBRC157

17 (NR_1125691

)

L sphaericus NBRC 15095 (NR_11262

71)L sphaericus DSM 28 (NR_0420731

)

S malt

ophil

iaLM

G958-

T (X959231)

S m

altop

hilia

K279a

(NC_0109431

)

P pi

ctoru

mLM

G981

(NR_0419571

)

P hi

bisc

icola

ATCC

19867

(NR_0247091

)

S m

alto

phili

a AT

CC 19861

(NR_0408041

)

Sten

otro

phom

onas

sp 4

_O_7

(EF5405161

)

S n

itriti

redu

cens

L2(N

R_0253051

)

5P

7P

8P 12P

10

P

4P

13P 6P

1P

S hu

mi R-32729

(NR_0425681

)

607

887

514

685

999

995

551

605

873

563

51

668

999992

801

644

764

998 944815

968

993

661

84

583

594

664

527

961

835

577

S rhizophila e-p10 (AJ29

34631)

Figure 3 Phylogenetic relationship between the partial 16S rRNA gene sequences from CL consortium isolates and their closest GenBanksequences with the 16S rRNA gene from previously reported known Mn(II)-oxidizing bacterial strains (labeled with lowast) The GenBankaccession numbers of these sequences are shown in brackets Bootstrap values ge 50 with 1000 replicates are indicated at the branch points

experiments ofmanganese removal by the isolates Upon LBBaddition to 01-mL K medium samples the MnIVO

2

-bearingtube in Figure 5 developed a blue color because of a redoxreaction with the dye whereas the flask containing MnIICO

3

remained transparent in the presence of LBB Figure 5 also

indicated that the K medium did not contain species thatwould produce a blue color after mixing with such reagentAbiotic Mn(II) oxidation can be inferred from the blue colorthat developed when the Mn(II) solutions were mixed withLBB at increasing pH values that is Mn(II) oxidation was


Control

Days0 2 5 7

pH

05

10152025303540455055

666870727476788082848688


Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(a)

pH

70

72

74

76

78

80

82

84Isolate 1G Bacillus sp

Days0 2 5 7


Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

0

5

10

15

20

25

30

35

40

45

50

(b)pH

70

72

74

76

78

80

82


Days0 2 5 7


Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

0

5

10

15

20

25

30

35

40

45

50

(c)pH

70

72

74

76

78

80

82

86

88

84

Isolate 7P Stenotrophomonas sp

Days0 2 5 7


05

10152025303540455055

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(d)

pH

70

72

74

76

78

80

82

86

88

84


Days0 2 5 7


05

10152025303540455055

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(e)

pH

706866

727476788082

8688

84

Isolate 6P Lysinibacillus sp

Days0 2 5 7


05

10152025303540455055

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(f)

Figure 4 Continued


pH

706866

727476788082

8688

84


Days0 2 5 7


0

5

10

15

20

25

30

35

40

45

50

55

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(g)

Figure 4 Time course of Mn(II) removal and the pH level of six isolates from the CL consortium The cells were grown in K medium forone week The concentration of Mn(II) was determined according to procedures that were described in Section 2

catalyzed by an increase of 05 units of pH as indicatedby blue color development (Figure 5) Thus pH played animportant role in manganese oxidation promoted by CLconsortium and the isolates

To assess whether bacterial growth would result inthe production of extracellular proteins that could oxidizeMn(II) we conducted Mn(II) oxidation assays with cell-free filtrates in either the presence or absence of proteaseK We did not observe Mn(II) removal by any cell-freefiltrates from isolates 10G (Figure 6(a)) 8P (Figure 6(b)) and13P (Figure 6(c)) in either presence or absence of protease(Figure 6(a)) Similar results were also observed with the cell-free filtrates from isolates 1G 7P and 6P (data not shown)These results indicate that no extracellular proteins wereinvolved in manganese oxidation by the isolates that wereselected herein

35 Multicopper Oxidase Activity Manganese oxidation bymany bacteria occurs through an enzymatic pathway withmulticopper oxidase enzymes (MCO) [29 40 41] Thus thecontribution of MCO to manganese oxidation by isolates 1G10G 7P 8P 6P and 13P was investigated using the ABTSsubstrate [31 32] As a positive outcome a blue color woulddevelop in the culturemediumAs shown in Figure S2 we didnot observe the development of a blue color in any enzymaticassays with isolates 10G 8P and 13P although the color of themedium darkened in the presence of Mn(II) ions The samewas observed for isolates 1G 7P and 6P (data not shown)

36 SEMEDX and TEMEDX Analyses Isolates 1G 10G 7P8P 6P and 13P were cultured in laboratory shaker flasks forseven days and selected for micromorphological responsesto the presence of Mn(II) ions SEM and TEM analysesrevealed no aggregates within the cells after seven days of

culturing (Figure 7) However when isolate 13P was grownin the presence of Mn(II) ions we observed an exopolymerout of the cell that contained manganese as revealed byEDX spectra (Figure 7(x)) The EDX spectra (Figure 7(n))also revealed the presence of manganese on the surface ofisolate 8P Furthermore we could observe spores in cultureof Bacillus sp (Figure 7(d)) as expected as Bacillus sppform spores and therefore survive harsh conditions [42] Inaddition a spherical spore was observed in the culture ofLysinibacillus sp (Figure 7(t))This spore is a defining featureof this bacterium [43]

4 Discussion

As stated we investigated the use of bacteria that were col-lected frommine waters for potentialMn(II) bioremediationThis analysis was accomplished by enriching and isolatingbacterial strains that grow in the presence of high Mn(II)concentrations (up to 1200mgsdotLminus1) Both the consortium andisolated strains removedMn(II) ions from the culture media

Specifically the CL consortium removed 997ofMn(II)which corresponded to a residual metal concentration of02mgsdotLminus1 at the end of the experiment (Figure 1(a)) foran initial concentration in the bioreactor of 45mgsdotLminus1 Thisresidual concentration was lower than the limit that wasestablished by the Brazilian regulations for effluents [10]Moreover the final pH increased to 786 (Figure 1(a)) with theconsortium A similar pH increase during Mn(II) removalby the isolates was also observed (Figure 4) whereas bothnegligible Mn(II) removal and no increase in pH wereobserved in the control experiments (Figure 1(b)) There-fore the change in solution pH contributed to manganeseremoval This result was speculated because pH is one ofthe major factors affecting manganese oxidation [1 7 8


MnCO3

MK + LBB +MK + LBB +MnO2

MK pH 75 +LBB

MK pH 80 +LBB

MK pH 85 +LBB

MK pH 90 +LBB

MK pH 75 +Mn(II) + LBB Mn(II) + LBB Mn(II) + LBB Mn(II) + LBB

MK pH 80 + MK pH 85 + MK pH 90 +

Figure 5 Role of pH in manganese oxidation K medium that was supplied with or without 50mgsdotLminus1Mn(II) at different pH values (75ndash90)wasmaintained at 30∘C and under constant stirring (150minminus1) for seven daysThe samples were collected and 004 LBB reagent was addedto them Manganese oxidation was indicated by the sample color turning blue MK K medium LBB Leucoberbelin blue I reagent MnCO

3

manganese carbonate MnO

2

manganese oxide

14 44] Furthermore XANES spectroscopy analysis of thesolids that formed during the bioreactor experiments withthe CL consortium and in the experiments with the isolatesbelonging to the Bacillus (1G and 10G) and Stenotrophomonas(7P and 8P) genera indicated that Mn(II) was precipitatedas MnO

2

(data not shown) as the pH increased Thereforewe hypothesize herein that bacterial metabolism promotedthe increase of pH which in turn enabled Mn(II) removal bychemical oxidation as will be further discussed

Regarding the strains that were isolated in the currentstudy five were identified as Stenotrophomonas sp (Figure 3)Members of this genus play important ecological and clinicalroles and tolerate high concentrations of metals such as AgCd and Hg [45] Although these species can be found in awide range of environments for example in soil plants wastegas biofilters and sewage [46] we showed for the first timethat strains of this genus have tolerance tomanganese (FigureS1) and promote its removal by the oxidation of Mn(II) ions(Figure 4)

Isolates 4P 6P and 13Pwere identified asLysinibacillus spwhile the other 12 isolates belonged to the B cereus groupsensu lato (Figure 3) Cerrato et al [25] isolated B cereusand Lysinibacillus sp strains from a biofilm in drinking watersystems and found that B cereus and L sphaericus isolateswere capable of some manganese oxidation Other studiesalso related B cereus and L sphaericus with Mn(II) removalby either oxidation or adsorption [47 48] but themechanism

was not discussed by these authors Furthermore in suchworks the strains removed lessMn(II) and neededmore timeto promote the manganese removal than did the strains thatwere isolated in the current study

SEMEDX and TEM images (Figures 7(n) and 7(x))revealed manganese outside the cells (as indicated by anarrow) of isolate 8P (Stenotrophomonas sp) and in anexopolymer-like structure in isolate 13P (Lysinibacillus sp)For the latter the colonies turned darker in the presenceof Mn(II) which may indicate manganese biosorption bythis isolate These observations are consistent with those ofother works which report manganese deposition around theexosporium of Bacillus sp SG-1 and around the sheaths inLysinibacillus sp [15 49] For the latter no manganese wasdetected inside the cells The inability of some manganese-oxidizing microorganisms to accumulate manganese oxidesaround their cells has been ascribed to the lack of exopolymerproduction as for the marine bacteria strain SSW

22

[15] Inthat case the colonies did not turn brown but caused thebrowning of Mn(II)-bearing agar Figure S1 of the currentstudy shows a similar behavior for isolates 1G 10G 7Pand 8P which did not turn the colonies brown It is likelythat these isolates were not able to produce an exopolymerfor manganese deposition making it impossible to findmanganese around the cells

Although few manganese removal studies with B cereusstrains have been published so far the mechanism of


10G10G + protease

12 24 36 48 60 720

Time (hours)

0

5

10

15

20

25

30

35

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

Cell-free filtratefrom isolate 10G Bacillus sp

(a)

8P8P + protease

12 24 36 48 60 720

Time (hours)

0

5

10

15

20

25

30

35

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

Cell-free filtratefrom isolate 8P Stenotrophomonas sp

(b)

13P13P + protease

12 24 36 48 60 720

Time (hours)

0

5

10

15

20

25

30

35

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

Cell-free filtratefrom isolate 13P Lysinibacillus sp

(c)

Figure 6 Cell-free filtrate Mn(II) oxidation The experiments were conducted at 37∘C For certain experiments 100120583gmL protease wasadded to the filtrate to examine the role of proteins inMn(II) oxidationThe samples were collected periodically andmanganese removal wasmeasured by inductively coupled plasma optical emission spectrometryThe presence of manganese oxides was monitored by the addition ofLBB to the samples


SEMStrains

Bacil

luss

p1

G

(a)

SEMStrains + Mn(II)

Bacil

luss

p1

G

(b)

StrainsTEM

Bacil

luss

p1

G

(c)

Strains + Mn(II)TEM

Bacil

luss

p1

G

(d)

SEMStrains

Bacil

luss

p10

G

(e)

SEMStrains + Mn(II)

Bacil

luss

p10

G

(f)

StrainsTEM

Bacil

luss

p10

G

(g)

Strains + Mn(II)TEM

Bacil

luss

p10

G

(h)

SEMStrains

Sten

otro

phom

onas

sp

7P

(i)

SEMStrains + Mn(II)

Sten

otro

phom

onas

sp

7P

(j)

StrainsTEM

Sten

otro

phom

onas

sp

7P

(k)

Strains + Mn(II)TEM

Sten

otro

phom

onas

sp

7P

(l)

SEMStrains

Sten

otro

phom

onas

sp

8P

(m)

SEMStrains + Mn(II)

Sten

otro

phom

onas

sp

8P

(n)

StrainsTEM

Sten

otro

phom

onas

sp

8P

(o)

Strains + Mn(II)TEM

Sten

otro

phom

onas

sp

8P

(p)

SEMStrains

Lysin

ibac

illus

sp

6P

(q)

SEMStrains + Mn(II)

Lysin

ibac

illus

sp

6P

(r)

StrainsTEM

Lysin

ibac

illus

sp

6P

(s)

Strains + Mn(II)TEM

Lysin

ibac

illus

sp

6P

(t)

SEMStrains

Lysin

ibac

illus

sp

13

P

(u)

SEMStrains + Mn(II)

Lysin

ibac

illus

sp

13

P

(v)

StrainsTEM

Lysin

ibac

illus

sp

13

P

(w)

Strains + Mn(II)TEM

Lysin

ibac

illus

sp

13

P

(x)

Figure 7 SEM and TEM images of isolates 1G 10G 7P 8P 6P and 13P that were cultured on K medium that was supplied with or without50mgsdotLminus1 Mn(II) EDX spectra of selected areas from the cell surface

manganese oxidation by Bacillus SG-1 has been extensivelystudied and MCO are involved in this process [50 51]Such a manganese oxidation mechanism was also related toother bacteria such as Pseudomonas putida (GB-1 andMnB1)

and L discophora SS-1 [30 40 41 50 51] In the currentstudy no MCO activity was observed during manganeseremoval by the isolates (Figure S2) Furthermore we alsotested whether some extracellular proteins were involved


in manganese oxidation The results (Figure 6) indicate noparticipation of the proteins in the cell-free filtrates thatwere produced from each isolate in manganese oxidationin the current study because no manganese removal wasobserved with or without proteinase K addition (Figure 6)These results suggest that manganese oxidation by strains ofBacillus Stenotrophomonas and Lysinibacillus did not occurby a direct mechanism

In addition to the enzymatic mechanism involvingMCOthere is also an indirect or nonenzymatic pathway for theremoval of manganese through the metabolism or growthof microorganisms This important mechanism appears tohave been overlooked in many studies addressing biologicalmanganese removal because pH is a key parameter inMn(II) oxidation as previously described Mn(II) oxidationby oxygen is thermodynamically favorable but its kineticsare slow Hydroxyl (OHminus) ions are among the many catalystsof chemical manganese oxidation and the rate increases 10times as the pH is changed from 80 to 88 [52] Manganeseoxyhydroxides are also catalysts of Mn(II) oxidation par-ticularly Mn(OH)

2

When its solubility limit is reached theprecipitated Mn(II)-hydroxide is quickly oxidized to Mn(III)and Mn(IV) [52] Therefore chemical manganese oxidationdoes occur when the pH is increased to values above 80 asdemonstrated by the results in Figure 6

Despite many attempts in buffering the experimentalconditions using HEPES the bacterial growth promotes anincrease in pH values in the current study (Figures 1 and5) Therefore we propose that the biological manganeseoxidation by the isolates and the CL consortium followed anonenzymatic pathway because the final pH attained valuesin which chemical manganese oxidation predominated Thisargument is supported by the fact that we observed Mn(II)oxidation in abiotic experiments (Figure 5) In summary thepresence of manganese-oxidizing bacteria is not required forMn(II) oxidation provided that the bacteria can increase thesolution pH

5 Conclusions

These results extend our knowledge aboutMn(II) removal byoxidation It was demonstrated thatmine bacteria are efficientinMn(II) removal through an indirect pathway by increasingthe pH growth medium The obtained results allow us toconclude the following

(i) The CL consortium that was obtained from the minewater was able to remove 997 of themanganese thatwas present in the growth medium and the residualmanganese concentration was 02mgsdotLminus1

(ii) Three different genera of manganese-removing bac-teria were isolated from the CL consortium Most ofthe isolates belonged to the Bacillus sp genus Thesecond-most-abundant genuswas Stenotrophomonasand bacteria belonging to this genus were related tomanganese oxidation for the first time Three isolateswere identified as Lysinibacillus

(iii) The six studied isolates (two representatives for eachgenus) were able to oxidize manganese and promote

manganese removal range from 585 to 827 inseven days in laboratory-scale reactors

(iv) We did not detect multicopper oxidase activityalthoughmanganese oxidation at pH values of 80 andabove was confirmed We then proposed a nonen-zymatic manganese oxidation pathway comprisingthe modification of the pH growth medium by theisolates

(v) The ability of the isolates to increase the pH growthmedium could be useful in the treatment of minewaters by producing alkalinity and oxidizing man-ganese concomitantly which are key to improvingcommercial applications

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was sponsored by the Conselho Nacional dePesquisa e Desenvolvimento (CNPq) the Fundacao deAmparo a Pesquisa do Estado de Minas Gerais (FAPEMIG)the Financiadora de Estudos e Projetos (FINEP) Vale andthe Universidade Federal de Ouro Preto The authors wouldlike to acknowledge theCenter ofMicroscopy at theUniversi-dade Federal deMinasGerais (httpwwwmicroscopiaufmgbr) for providing the equipment and technical supportfor experiments involving electron microscopy The authorswould especially like to thankKinulpeHonorato-Sampaio forhis technical assistance

References

[1] M S Burger G A Gagnon S S Mercer and G D ShupeldquoManganese removal during bench-scale biofiltrationrdquo WaterResearch vol 42 no 19 pp 4733ndash4742 2008

[2] K L Johnson and P L Younger ldquoRapid manganese removalfrom mine waters using an aerated packed-bed bioreactorrdquoJournal of Environmental Quality vol 34 no 3 pp 987ndash9932005

[3] F Luan C M Santelli C M Hansel and W D BurgosldquoDefining manganese(II) removal processes in passive coalmine drainage treatment systems through laboratory incuba-tion experimentsrdquoApplied Geochemistry vol 27 no 8 pp 1567ndash1578 2012

[4] W D Burgos H Tan C M Santelli and M C HanselldquoImportance of fungi in biological Mn(II) oxidation in lime-stone treatment bedsrdquo in Proceedings of the National Meetingof the American Society of Mining and Reclamation ASMRPittsburgh Pa USA June 2010

[5] C M Santelli D H Pfister D Lazarus L Sun W DBurgos and C M Hansel ldquoPromotion of Mn(II) oxidationand remediation of coal mine drainage in passive treatmentsystems by diverse fungal and bacterial communitiesrdquo Appliedand Environmental Microbiology vol 76 no 14 pp 4871ndash48752010

[6] H Tan G Zhang P J Heaney S M Webb and W DBurgos ldquoCharacterization ofmanganese oxide precipitates from


Appalachian coal mine drainage treatment systemsrdquo AppliedGeochemistry vol 25 no 3 pp 389ndash399 2010

[7] A M Silva F L S Cruz R M F Lima M C Teixeira and VA Leao ldquoManganese and limestone interactions during minewater treatmentrdquo Journal of Hazardous Materials vol 181 no1ndash3 pp 514ndash520 2010

[8] S M Bamforth D A C Manning I Singleton P L Youngerand K L Johnson ldquoManganese removal from mine watersmdashinvestigating the occurrence and importance of manganesecarbonatesrdquo Applied Geochemistry vol 21 no 8 pp 1274ndash12872006

[9] I B D M Ibram Informacoes e AnalIses da Economia MineralBrasileira 2012

[10] CONAMA ldquoRESOLUCAO N∘ 430 13052011rdquo CND Ambi-ente Editor 2011

[11] N D W Clearinghouse Iron and Manganese Removal ANational Drinking Water Clearinghouse Fact Sheet 1988

[12] Z Zainol and M J Nicol ldquoIon-exchange equilibria of Ni2+Co2+ Mn2+ and Mg2+ with iminodiacetic acid chelating resinAmberlite IRC 748rdquo Hydrometallurgy vol 99 no 3-4 pp 175ndash180 2009

[13] D Savova N Petrov M F Yardim et al ldquoThe influence of thetexture and surface properties of carbon adsorbents obtainedfrom biomass products on the adsorption of manganese ionsfrom aqueous solutionrdquo Carbon vol 41 no 10 pp 1897ndash19032003

[14] A M Silva E C Cunha F D R Silva and V A LeaoldquoTreatment of high-manganese mine water with limestone andsodium carbonaterdquo Journal of Cleaner Production vol 29-30pp 11ndash19 2012

[15] H L Ehrlich ldquoMicrobes as geologic agents their role inmineralformationrdquo Geomicrobiology Journal vol 16 no 2 pp 135ndash1531999

[16] A P Das L B Sukla N Pradhan and S Nayak ldquoManganesebiomining a reviewrdquo Bioresource Technology vol 102 no 16 pp7381ndash7387 2011

[17] H Gallard andU von Gunten ldquoChlorination of natural organicmatter Kinetics of chlorination and of THM formationrdquoWaterResearch vol 36 no 1 pp 65ndash74 2002

[18] C K Hope and T R Bott ldquoLaboratorymodelling of manganesebiofiltration using biofilms of Leptothrix discophorardquo WaterResearch vol 38 no 7 pp 1853ndash1861 2004

[19] V A Pacini A M Ingallinella and G Sanguinetti ldquoRemoval ofiron andmanganese using biological roughing up flow filtrationtechnologyrdquo Water Research vol 39 no 18 pp 4463ndash44752005

[20] RMariner D B Johnson andK BHallberg ldquoCharacterisationof an attenuation system for the remediation ofMn(II) contam-inated watersrdquo Hydrometallurgy vol 94 no 1ndash4 pp 100ndash1042008

[21] R Schweisfurth ldquoManganese-oxidizing bacteria I Isolationand identification of various manganese-oxidizing bacteriardquoZeitschrift fur Allgemeine Mikrobiologie vol 13 no 4 pp 341ndash347 1973

[22] K H Nealson and J S Ford ldquoSurface enhancement of bacterialmanganese oxidation implications for aquatic environmentsrdquoGeomicrobiology Journal vol 2 no 1 pp 21ndash37 2009

[23] W C Ghiorse ldquoBiology of iron-and manganese-depositingbacteriardquo Annual Review of Microbiology vol 38 pp 515ndash5501984

[24] X-Z Lin A-G Gao and H-W Chen ldquoIsolation and phylo-genetic analysis of cultivable manganese bacteria in sedimentsfrom the Arctic OceanrdquoActa Ecologica Sinica vol 28 no 12 pp6364ndash6370 2008

[25] J M Cerrato J O Falkinham III A M DietrichW R KnockeC W McKinney and A Pruden ldquoManganese-oxidizing and -reducing microorganisms isolated from biofilms in chlorinateddrinking water systemsrdquo Water Research vol 44 no 13 pp3935ndash3945 2010

[26] C M Hansel C A Zeiner C M Santelli and S M WebbldquoMn(II) oxidation by an ascomycete fungus is linked to super-oxide production during asexual reproductionrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 109 no 31 pp 12621ndash12625 2012

[27] W Yang Z Zhang Z Zhang et al ldquoPopulation structure ofmanganese-oxidizing bacteria in stratified soils and propertiesof manganese oxide aggregates under manganese-complexmedium enrichmentrdquo PLoS ONE vol 8 no 9 Article IDe73778 2013

[28] J Zhang L W Lion Y M Nelson M L Shuler and W CGhiorse ldquoKinetics ofMn(II) oxidation by Leptothrix discophoraSS1rdquo Geochimica et Cosmochimica Acta vol 66 no 5 pp 773ndash781 2002

[29] AV Soldatova C ButterfieldO FOyerinde BM Tebo andTG Spiro ldquoMulticopper oxidase involvement in bothMn(II) andMn(III) oxidation during bacterial formation ofMnO

2

rdquo Journalof Biological Inorganic Chemistry vol 17 no 8 pp 1151ndash11582012

[30] K Geszvain J K McCarthy and B M Tebo ldquoElimination ofmanganese(IIIII) oxidation in Pseudomonas putida GB-1 by adouble knockout of two putative multicopper oxidase genesrdquoApplied and Environmental Microbiology vol 79 no 1 pp 357ndash366 2013

[31] J Su P Bao T Bai et al ldquoCotA a multicopper oxidase fromBacillus pumilus WH4 exhibits manganese-oxidase activityrdquoPLoS ONE vol 8 no 4 Article ID e60573 2013

[32] E S Hartikainen A Hatakka and M A Kahkonen ldquoImpactof cadmium chromium cobalt lithium and manganese to thegrowth of fungi and production of enzymesrdquo Expert Opinion onEnvironmental Biology vol 2 no 3 2013

[33] A T Nielsen W-T Liu C Filipe L Grady Jr S Molin and DA Stahl ldquoIdentification of a novel group of bacteria in sludgefrom a deteriorated biological phosphorus removal reactorrdquoApplied and EnvironmentalMicrobiology vol 65 no 3 pp 1251ndash1258 1999

[34] O Sanchez J M Gasol R Massana J Mas and C Pedros-AlioldquoComparison of different denaturing gradient gel electrophore-sis primer sets for the study of marine bacterioplankton com-munitiesrdquo Applied and Environmental Microbiology vol 73 no18 pp 5962ndash5967 2007

[35] G Muyzer E C De Waal and A G Uitterlinden ldquoProfilingof complex microbial populations by denaturing gradient gelelectrophoresis analysis of polymerase chain reaction-amplifiedgenes coding for 16S rRNArdquo Applied and Environmental Micro-biology vol 59 no 3 pp 695ndash700 1993

[36] J Sambrook TManiatis and E F FritschMolecular Cloning ALaboratoryManual Cold SpringHarbor Laboratory Press NewYork NY USA 2nd edition 1989

[37] A Aiyar ldquoTheuse of CLUSTALWandCLUSTALX formultiplesequence alignmentrdquoMethods inMolecular Biology vol 132 pp221ndash241 2000


[38] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987

[39] I A Katsoyiannis and A I Zouboulis ldquoBiological treatment ofMn(II) and Fe(II) containing groundwater kinetic considera-tions and product characterizationrdquoWater Research vol 38 no7 pp 1922ndash1932 2004

[40] G-J Brouwers J P M de Vrind P L A M Corstjens PCornelis C Baysse and E W de Vrind-de Jong ldquocumA a geneencoding a multicopper oxidase is involved inMn2+ oxidationin Pseudomonas putida GB-1rdquo Applied and EnvironmentalMicrobiology vol 65 no 4 pp 1762ndash1768 1999

[41] G J Brouwers E Vijgenboom P L A M Corstjens J P M DeVrind and E W De Vrind-de Jong ldquoBacterial Mn2+ oxidizingsystems and multicopper oxidases an overview of mechanismsand functionsrdquo Geomicrobiology Journal vol 17 no 1 pp 1ndash242000

[42] T Abee M N Groot M Tempelaars M Zwietering RMoezelaar and M V D Voort ldquoGermination and outgrowthof spores of Bacillus cereus group members diversity and roleof germinant receptorsrdquo Food Microbiology vol 28 no 2 pp199ndash208 2011

[43] J L Jurat-Fuentes and T A Jackson ldquoBacterial entomopatho-gensrdquo in Insect Pathology chapter 8 pp 265ndash349 Elsevier 2ndedition 2012

[44] M Divekar Isolation and characterization of a manganeseoxidizing bacterium from the Mediterranean marine spongeSuberites domuncula [Dissertation] Fachbereich Biologie derJohannes Gutenberg-Universitat 2010

[45] D Pages J Rose S Conrod et al ldquoHeavy metal tolerance inStenotrophomonas maltophiliardquo PLoS ONE vol 3 no 2 ArticleID e1539 2008

[46] R P Ryan S Monchy M Cardinale et al ldquoThe versatilityand adaptation of bacteria from the genus StenotrophomonasrdquoNature Reviews Microbiology vol 7 no 7 pp 514ndash525 2009

[47] H Abu Hasan S R Sheikh Abdullah N Tan Kofli and S KKamarudin ldquoEffectivemicrobes for simultaneous bio-oxidationof ammonia and manganese in biological aerated filter systemrdquoBioresource Technology vol 124 pp 355ndash363 2012

[48] I Douterelo S Husband and J B Boxall ldquoThe bacteriologicalcomposition of biomass recovered by flushing an operationaldrinking water distribution systemrdquoWater Research vol 54 pp100ndash114 2014

[49] D Emerson and W C Ghiorse ldquoIsolation cultural mainte-nance and taxonomy of a sheath-forming strain of Leptothrixdiscophora and characterization of manganese-oxidizing activ-ity associated with the sheathrdquo Applied and EnvironmentalMicrobiology vol 58 no 12 pp 4001ndash4010 1992

[50] G J Dick J W Torpey T J Beveridge and B M Tebo ldquoDirectidentification of a bacterial manganese(II) oxidase the multi-copper oxidase MnxG from spores of several different marineBacillus speciesrdquo Applied and Environmental Microbiology vol74 no 5 pp 1527ndash1534 2008

[51] L E Mayhew E D Swanner A P Martin and A S TempletonldquoPhylogenetic relationships and functional genes distributionof a gene (mnxG) encoding a putative manganese-oxidizingenzyme in Bacillus speciesrdquo Applied and Environmental Micro-biology vol 74 no 23 pp 7265ndash7271 2008

[52] J J Morgan ldquoKinetics of reaction between O2

and Mn(II)species in aqueous solutionsrdquo Geochimica et CosmochimicaActa vol 69 no 1 pp 35ndash48 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


[16 21ndash23] Although several bacteria promote manganesebioremediation only few species have been extensively stud-ied (ie Pseudomonas Bacillus Leptothrix and Pedomicro-bium) [5 24ndash31] Furthermore few studies have identifiedor tested the ability of local bacterial strains to remove theelement despite high Mn(II) concentrations in some minewaters and groundwater in tropical countries Therefore thegoals of the current study are to (i) enrich a bacterial con-sortium from mine waters with high Mn(II) concentrationsand (ii) isolate and identify the bacterial strains from theenrichment while assessing their manganese removal abilityThis knowledge would be useful for the design of new moreefficient strategies for treatingmanganese-laden wastewaters

2 Material and Methods

21 Sample Sites A water sample was collected from a man-ganese mine in the Iron Quadrangle region (Minas GeraisBrazil) The mine water was stored in plastic containers atroom temperature until laboratory processing A sample ofthe mine water was selected and analyzed for metal contentby inductively coupled plasma optical emission spectrometry(ICP-OES Varian 725)

22 Enrichment A subsample comprising one liter of minewater was filtered through a 022 120583m membrane (MillexMillipore) The membrane was transferred to Falcon tubeswith 10mL of K medium (015 gsdotLminus1 MnSO

4

sdotH2

O 2 gsdotLminus1peptone 05 gsdotLminus1 yeast extract and 10mM HEPES bufferpH 75) Cultures were incubated at 30∘C on an orbitalshaker (150minminus1) for 24 h Subsequently the cultures weretransferred to 250-mL flasks containing 90mL of K mediumand incubated as described above The obtained consortiumwas called CL throughout this study After 24 h 50 (wv)glycerol was added and aliquots of 1mL were stored inEppendorf tubes at minus80∘C until the experiments were con-ducted

23 Isolation Procedures Pure strains were isolated fromthe enriched CL consortium by plating on 15 (wv) agar-solidified K medium The agar plates were amended with50mgsdotLminus1 Mn(II) ion after autoclave sterilization The cul-tures were incubated at 30∘C until colonies formed andorthe culture medium containingMn(II) turned brownishThecolonies which had changed color andor changed themediacolor were selected and bacterial isolates were inoculatedinto K medium without Mn(II) The isolates were preservedat minus80∘C in 50 (wv) glycerol

24 Manganese Removal Experiments Batchwise manganeseremoval experiments at 50mgsdotLminus1 Mn(II) ion concentra-tions in a 2-week-long period were performed with theCL consortium These experiments were accomplished in asmall-scale 3-L bioreactor (BioFlo 110 FermentorBioreactorNew Brunswick Scientific) at a one-liter final volume Priorto the bioreactor experiment 1mL of the CL consortiumpreserved at minus80∘C was cultured in 10mL of K medium(at 30∘C) on an orbital shaker (150minminus1) for 24 h (ie a

preinoculation period) Subsequently the preinoculum wastransferred to 250-mL flasks containing 90mL of K mediumand incubated under the same conditions as describedabove Thereafter 100mL of the culture (approximately 01optical density at 600 nm) was transferred to a bioreactorcontaining 900mL of K medium at pH 75 in the presenceof 50mgsdotLminus1 Mn(II) and maintained at 30∘C under constantstirring (150minminus1) for twoweeks Aliquots were periodicallycollected for manganese concentration pH and bacterialgrowth measurements (by counting cells in a Neubauerchamber) The experiments were performed in duplicateConcurrently a similar bioreactor was operated as a controlin which the bacterial growth was inhibited by a Nipagin(014 wv)-Nipazol (01 wv) addition

Manganese removal by the isolates was performed inflasks at final volumes of 100mL The preinoculum wasprepared as described above for the CL consortium Sub-sequently 10-mL aliquots of the preinoculum were trans-ferred to 250-mL flasks containing 90mL of K medium thatwas supplemented with 50mgsdotLminus1 Mn(II) at an initial pHof 75 The flasks were maintained at 30∘C on an orbitalshaker (150minminus1) for seven days Aliquots were collectedperiodically and the same analyses that were performedfor the consortium CL were performed for the isolates(ie manganese concentration pH and bacterial growthmeasurements using the optical density method in a Hitachi2800A spectrophotometer) In the control flasks under thesame conditions bacterial growth was inhibited by addingNipagin (014 wv)-Nipazol (01 wv) The experimentswere performed in triplicate

For manganese analysis approximately 4-mL aliquots ofculture were centrifuged for 15min at 14681timesg and filteredthrough a 022-120583m membrane The filtrate was collecteddiluted 10 or 25 times in distilled water and acidified withHCl (1 1) solution The dilutions were analyzed by ICP-OESThe amount of manganese that was removed was measuredby the decay of the element in the culture medium

We also evaluated the culture growth on solid K mediacontaining different Mn(II) ion concentrations (140mgsdotLminus1300mgsdotLminus1 600mgsdotLminus1 and 1200mgsdotLminus1) Plates were incu-bated at 30∘C for two weeks after which dark brown-ish colonies or the browning of the culture medium wasobserved

25 Mn(II) Oxidation Assays Abiotic manganese oxidationwas confirmed in control experiments that were performedat different pH values (from 75 to 90 in 05 steps) Sam-ples of 01mL of the K medium that were amended with50mgsdotLminus1 Mn(II) and submitted to the same conditions ofthe experiments with the inoculum were mixed with 05mLof 004 Leucoberbelin blue I (LBB Sigma-Aldrich USA) in45mM acetic acid As a negative control K media without50mgsdotLminus1 Mn(II) or with manganese carbonate (MnCO

3

)solutions were also tested as described above For a positivecontrol K medium with manganese oxide (MnO

2

) was used

26 Mn(II) Oxidation by Cell-Free Filtrate The cell-freefiltrates were prepared by growing the isolates on K medium





4

sdot7H2


4

sdotH2



2









0

5

10

15

20

25

30

35

40

45

50

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

pH

66

64

62

60

68

70

72

74

76

78

82

80

Days0 2 5 7 9 12 14



(a)

05

1015202530354045505560

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

pH

66

64

62

60

68

70

72

74

76

78

80

Days0 2 5 7 9 12 14


Control

(b)


8

50

40

30

20

10

0

Cell

s num

bers


1 2 3 4 5 6 7 8 9 10 11 12 13 140

Days

Bioreactor

(c)


3 Results





BioC

L50

mgmiddot

Lminus1

Mn(

II)-1

BioC

L50

mgmiddot

Lminus1

Mn(

II)-2

BioC

L50

mgmiddot

Lminus1

Mn(

II)-1

BioC

L50

mgmiddot

Lminus1

Mn(

II)-1

BioC

L50

mgmiddot

Lminus1

Mn(

II)-2

BioC

L50

mgmiddot

Lminus1

Mn(

II)-2

968F-CG1392R 357F-CG907R 357F-CG518R












100

100100

100

100

100100

100

100100

100

100

100

100100

100100100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100 100 100

100

100

100

100

100

100

100 1

00

100

100

100

100

100

100

100

100

100

100

100100

100

100

100

100

100

100

100

100

100

100

100

100

100100

100

100

100

100

100

100

100

100

10010

0

100100

100

100

100

100

100 100

100 100

100 100

100

100

100

100

100

100

100

100

100

100

100

100


62831)


(NR_0253421)S

terrae

R-32768T

(AM4035892)

S ter

raeR-32

768

(NR_0

425691)

S acid

aminiphilaCCUG 5

4933

(GU9455351)

S acidaminiphila

AMX 19

(NR_02

51041

)



S maltophilia IA

M12423

(NR_0415771)


(NR_0247081)

S maltophiliaR

551-3

(NR_0748751)


(GU9455371)

B an

thra

cis(N

R_0744531

)

6G

9G

1G

12G

10G

8G

B cereus G9842 (CP0011861)





5G



7G


0036871)


R_1217611)


11821

(NR_0246971)


(NZ_CM

0007531)

B mycoides

273

(NR_0368801)

B th

urin

gien

sisBM

B171

(CP0019031

)B

thur

ingi

ensis

IAM12077

(NR_0434031

)

B ce

reus

ATCC

14579

(NC_0047221

)

B cy

toto

xicu

sNVH

391

-98

(NR_0749141

)

B fu

nicu

lusN

AF001

(NR_0286241

)

B ab

yssa

lisSC

SIO15042

(NR_1096711

)






R opacus DSM 43205 (NR_0261861)






V soli GH9-3 (NR_0438111)







S koreensis

(AB1668851)

S plymuthica IC1270

(AY55133

21)


)


9821)


851)




11)lowast



71)lowast


)

L boronitolerans 10

a (NR_04

12761)

L paki

stanensis

NCCP-54(N

R_1131661)

Lysinibacill

us sp R18

(KJ4997861

)

Lysin

ibacil

lussp

POUD (J

Q0077241)

Lysin

ibacil

lussp

BAB-

4156

(KJ7941141

)


(KJ7252361

)

L fusifo

rmis DSM 28

98(NR_04

20721)

L fusiformis NBRC 1

5717

(NR_1126281)


4 (NR_114213


17 (NR_1125691

)



)

S malt

ophil

iaLM

G958-

T (X959231)

S m

altop

hilia

K279a

(NC_0109431

)

P pi

ctoru

mLM

G981

(NR_0419571

)

P hi

bisc

icola

ATCC

19867

(NR_0247091

)

S m

alto

phili

a AT

CC 19861

(NR_0408041

)

Sten

otro

phom

onas

sp 4

_O_7

(EF5405161

)

S n

itriti

redu

cens

L2(N

R_0253051

)

5P

7P

8P 12P

10

P

4P

13P 6P

1P

S hu

mi R-32729

(NR_0425681

)

607

887

514

685

999

995

551

605

873

563

51

668

999992

801

644

764

998 944815

968

993

661

84

583

594

664

527

961

835

577


34631)



2


3




Control

Days0 2 5 7

pH

05

10152025303540455055

666870727476788082848688


Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(a)

pH

70

72

74

76

78

80

82


Days0 2 5 7


Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

0

5

10

15

20

25

30

35

40

45

50

(b)pH

70

72

74

76

78

80

82


Days0 2 5 7


Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

0

5

10

15

20

25

30

35

40

45

50

(c)pH

70

72

74

76

78

80

82

86

88

84


Days0 2 5 7


05

10152025303540455055

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(d)

pH

70

72

74

76

78

80

82

86

88

84


Days0 2 5 7


05

10152025303540455055

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(e)

pH

706866

727476788082

8688

84


Days0 2 5 7


05

10152025303540455055

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(f)

Figure 4 Continued


pH

706866

727476788082

8688

84


Days0 2 5 7


0

5

10

15

20

25

30

35

40

45

50

55

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(g)







4 Discussion




MnCO3


MK pH 75 +LBB

MK pH 80 +LBB

MK pH 85 +LBB

MK pH 90 +LBB


MK pH 80 + MK pH 85 + MK pH 90 +


3


2

manganese oxide


2






22




10G10G + protease

12 24 36 48 60 720

Time (hours)

0

5

10

15

20

25

30

35

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)


(a)

8P8P + protease

12 24 36 48 60 720

Time (hours)

0

5

10

15

20

25

30

35

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)


(b)

13P13P + protease

12 24 36 48 60 720

Time (hours)

0

5

10

15

20

25

30

35

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)


(c)



SEMStrains

Bacil

luss

p1

G

(a)

SEMStrains + Mn(II)

Bacil

luss

p1

G

(b)

StrainsTEM

Bacil

luss

p1

G

(c)

Strains + Mn(II)TEM

Bacil

luss

p1

G

(d)

SEMStrains

Bacil

luss

p10

G

(e)

SEMStrains + Mn(II)

Bacil

luss

p10

G

(f)

StrainsTEM

Bacil

luss

p10

G

(g)

Strains + Mn(II)TEM

Bacil

luss

p10

G

(h)

SEMStrains

Sten

otro

phom

onas

sp

7P

(i)

SEMStrains + Mn(II)

Sten

otro

phom

onas

sp

7P

(j)

StrainsTEM

Sten

otro

phom

onas

sp

7P

(k)

Strains + Mn(II)TEM

Sten

otro

phom

onas

sp

7P

(l)

SEMStrains

Sten

otro

phom

onas

sp

8P

(m)

SEMStrains + Mn(II)

Sten

otro

phom

onas

sp

8P

(n)

StrainsTEM

Sten

otro

phom

onas

sp

8P

(o)

Strains + Mn(II)TEM

Sten

otro

phom

onas

sp

8P

(p)

SEMStrains

Lysin

ibac

illus

sp

6P

(q)

SEMStrains + Mn(II)

Lysin

ibac

illus

sp

6P

(r)

StrainsTEM

Lysin

ibac

illus

sp

6P

(s)

Strains + Mn(II)TEM

Lysin

ibac

illus

sp

6P

(t)

SEMStrains

Lysin

ibac

illus

sp

13

P

(u)

SEMStrains + Mn(II)

Lysin

ibac

illus

sp

13

P

(v)

StrainsTEM

Lysin

ibac

illus

sp

13

P

(w)

Strains + Mn(II)TEM

Lysin

ibac

illus

sp

13

P

(x)







2



5 Conclusions










Acknowledgments


References
































2


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





4

sdot7H2


4

sdotH2



2









0

5

10

15

20

25

30

35

40

45

50

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

pH

66

64

62

60

68

70

72

74

76

78

82

80

Days0 2 5 7 9 12 14



(a)

05

1015202530354045505560

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

pH

66

64

62

60

68

70

72

74

76

78

80

Days0 2 5 7 9 12 14


Control

(b)


8

50

40

30

20

10

0

Cell

s num

bers


1 2 3 4 5 6 7 8 9 10 11 12 13 140

Days

Bioreactor

(c)


3 Results





BioC

L50

mgmiddot

Lminus1

Mn(

II)-1

BioC

L50

mgmiddot

Lminus1

Mn(

II)-2

BioC

L50

mgmiddot

Lminus1

Mn(

II)-1

BioC

L50

mgmiddot

Lminus1

Mn(

II)-1

BioC

L50

mgmiddot

Lminus1

Mn(

II)-2

BioC

L50

mgmiddot

Lminus1

Mn(

II)-2

968F-CG1392R 357F-CG907R 357F-CG518R












100

100100

100

100

100100

100

100100

100

100

100

100100

100100100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100 100 100

100

100

100

100

100

100

100 1

00

100

100

100

100

100

100

100

100

100

100

100100

100

100

100

100

100

100

100

100

100

100

100

100

100100

100

100

100

100

100

100

100

100

10010

0

100100

100

100

100

100

100 100

100 100

100 100

100

100

100

100

100

100

100

100

100

100

100

100


62831)


(NR_0253421)S

terrae

R-32768T

(AM4035892)

S ter

raeR-32

768

(NR_0

425691)

S acid

aminiphilaCCUG 5

4933

(GU9455351)

S acidaminiphila

AMX 19

(NR_02

51041

)



S maltophilia IA

M12423

(NR_0415771)


(NR_0247081)

S maltophiliaR

551-3

(NR_0748751)


(GU9455371)

B an

thra

cis(N

R_0744531

)

6G

9G

1G

12G

10G

8G

B cereus G9842 (CP0011861)





5G



7G


0036871)


R_1217611)


11821

(NR_0246971)


(NZ_CM

0007531)

B mycoides

273

(NR_0368801)

B th

urin

gien

sisBM

B171

(CP0019031

)B

thur

ingi

ensis

IAM12077

(NR_0434031

)

B ce

reus

ATCC

14579

(NC_0047221

)

B cy

toto

xicu

sNVH

391

-98

(NR_0749141

)

B fu

nicu

lusN

AF001

(NR_0286241

)

B ab

yssa

lisSC

SIO15042

(NR_1096711

)






R opacus DSM 43205 (NR_0261861)






V soli GH9-3 (NR_0438111)







S koreensis

(AB1668851)

S plymuthica IC1270

(AY55133

21)


)


9821)


851)




11)lowast



71)lowast


)

L boronitolerans 10

a (NR_04

12761)

L paki

stanensis

NCCP-54(N

R_1131661)

Lysinibacill

us sp R18

(KJ4997861

)

Lysin

ibacil

lussp

POUD (J

Q0077241)

Lysin

ibacil

lussp

BAB-

4156

(KJ7941141

)


(KJ7252361

)

L fusifo

rmis DSM 28

98(NR_04

20721)

L fusiformis NBRC 1

5717

(NR_1126281)


4 (NR_114213


17 (NR_1125691

)



)

S malt

ophil

iaLM

G958-

T (X959231)

S m

altop

hilia

K279a

(NC_0109431

)

P pi

ctoru

mLM

G981

(NR_0419571

)

P hi

bisc

icola

ATCC

19867

(NR_0247091

)

S m

alto

phili

a AT

CC 19861

(NR_0408041

)

Sten

otro

phom

onas

sp 4

_O_7

(EF5405161

)

S n

itriti

redu

cens

L2(N

R_0253051

)

5P

7P

8P 12P

10

P

4P

13P 6P

1P

S hu

mi R-32729

(NR_0425681

)

607

887

514

685

999

995

551

605

873

563

51

668

999992

801

644

764

998 944815

968

993

661

84

583

594

664

527

961

835

577


34631)



2


3




Control

Days0 2 5 7

pH

05

10152025303540455055

666870727476788082848688


Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(a)

pH

70

72

74

76

78

80

82


Days0 2 5 7


Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

0

5

10

15

20

25

30

35

40

45

50

(b)pH

70

72

74

76

78

80

82


Days0 2 5 7


Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

0

5

10

15

20

25

30

35

40

45

50

(c)pH

70

72

74

76

78

80

82

86

88

84


Days0 2 5 7


05

10152025303540455055

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(d)

pH

70

72

74

76

78

80

82

86

88

84


Days0 2 5 7


05

10152025303540455055

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(e)

pH

706866

727476788082

8688

84


Days0 2 5 7


05

10152025303540455055

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(f)

Figure 4 Continued


pH

706866

727476788082

8688

84


Days0 2 5 7


0

5

10

15

20

25

30

35

40

45

50

55

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(g)







4 Discussion




MnCO3


MK pH 75 +LBB

MK pH 80 +LBB

MK pH 85 +LBB

MK pH 90 +LBB


MK pH 80 + MK pH 85 + MK pH 90 +


3


2

manganese oxide


2






22




10G10G + protease

12 24 36 48 60 720

Time (hours)

0

5

10

15

20

25

30

35

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)


(a)

8P8P + protease

12 24 36 48 60 720

Time (hours)

0

5

10

15

20

25

30

35

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)


(b)

13P13P + protease

12 24 36 48 60 720

Time (hours)

0

5

10

15

20

25

30

35

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)


(c)



SEMStrains

Bacil

luss

p1

G

(a)

SEMStrains + Mn(II)

Bacil

luss

p1

G

(b)

StrainsTEM

Bacil

luss

p1

G

(c)

Strains + Mn(II)TEM

Bacil

luss

p1

G

(d)

SEMStrains

Bacil

luss

p10

G

(e)

SEMStrains + Mn(II)

Bacil

luss

p10

G

(f)

StrainsTEM

Bacil

luss

p10

G

(g)

Strains + Mn(II)TEM

Bacil

luss

p10

G

(h)

SEMStrains

Sten

otro

phom

onas

sp

7P

(i)

SEMStrains + Mn(II)

Sten

otro

phom

onas

sp

7P

(j)

StrainsTEM

Sten

otro

phom

onas

sp

7P

(k)

Strains + Mn(II)TEM

Sten

otro

phom

onas

sp

7P

(l)

SEMStrains

Sten

otro

phom

onas

sp

8P

(m)

SEMStrains + Mn(II)

Sten

otro

phom

onas

sp

8P

(n)

StrainsTEM

Sten

otro

phom

onas

sp

8P

(o)

Strains + Mn(II)TEM

Sten

otro

phom

onas

sp

8P

(p)

SEMStrains

Lysin

ibac

illus

sp

6P

(q)

SEMStrains + Mn(II)

Lysin

ibac

illus

sp

6P

(r)

StrainsTEM

Lysin

ibac

illus

sp

6P

(s)

Strains + Mn(II)TEM

Lysin

ibac

illus

sp

6P

(t)

SEMStrains

Lysin

ibac

illus

sp

13

P

(u)

SEMStrains + Mn(II)

Lysin

ibac

illus

sp

13

P

(v)

StrainsTEM

Lysin

ibac

illus

sp

13

P

(w)

Strains + Mn(II)TEM

Lysin

ibac

illus

sp

13

P

(x)







2



5 Conclusions










Acknowledgments


References
































2


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

5

10

15

20

25

30

35

40

45

50

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

pH

66

64

62

60

68

70

72

74

76

78

82

80

Days0 2 5 7 9 12 14



(a)

05

1015202530354045505560

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

pH

66

64

62

60

68

70

72

74

76

78

80

Days0 2 5 7 9 12 14


Control

(b)


8

50

40

30

20

10

0

Cell

s num

bers


1 2 3 4 5 6 7 8 9 10 11 12 13 140

Days

Bioreactor

(c)


3 Results





BioC

L50

mgmiddot

Lminus1

Mn(

II)-1

BioC

L50

mgmiddot

Lminus1

Mn(

II)-2

BioC

L50

mgmiddot

Lminus1

Mn(

II)-1

BioC

L50

mgmiddot

Lminus1

Mn(

II)-1

BioC

L50

mgmiddot

Lminus1

Mn(

II)-2

BioC

L50

mgmiddot

Lminus1

Mn(

II)-2

968F-CG1392R 357F-CG907R 357F-CG518R












100

100100

100

100

100100

100

100100

100

100

100

100100

100100100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100 100 100

100

100

100

100

100

100

100 1

00

100

100

100

100

100

100

100

100

100

100

100100

100

100

100

100

100

100

100

100

100

100

100

100

100100

100

100

100

100

100

100

100

100

10010

0

100100

100

100

100

100

100 100

100 100

100 100

100

100

100

100

100

100

100

100

100

100

100

100


62831)


(NR_0253421)S

terrae

R-32768T

(AM4035892)

S ter

raeR-32

768

(NR_0

425691)

S acid

aminiphilaCCUG 5

4933

(GU9455351)

S acidaminiphila

AMX 19

(NR_02

51041

)



S maltophilia IA

M12423

(NR_0415771)


(NR_0247081)

S maltophiliaR

551-3

(NR_0748751)


(GU9455371)

B an

thra

cis(N

R_0744531

)

6G

9G

1G

12G

10G

8G

B cereus G9842 (CP0011861)





5G



7G


0036871)


R_1217611)


11821

(NR_0246971)


(NZ_CM

0007531)

B mycoides

273

(NR_0368801)

B th

urin

gien

sisBM

B171

(CP0019031

)B

thur

ingi

ensis

IAM12077

(NR_0434031

)

B ce

reus

ATCC

14579

(NC_0047221

)

B cy

toto

xicu

sNVH

391

-98

(NR_0749141

)

B fu

nicu

lusN

AF001

(NR_0286241

)

B ab

yssa

lisSC

SIO15042

(NR_1096711

)






R opacus DSM 43205 (NR_0261861)






V soli GH9-3 (NR_0438111)







S koreensis

(AB1668851)

S plymuthica IC1270

(AY55133

21)


)


9821)


851)




11)lowast



71)lowast


)

L boronitolerans 10

a (NR_04

12761)

L paki

stanensis

NCCP-54(N

R_1131661)

Lysinibacill

us sp R18

(KJ4997861

)

Lysin

ibacil

lussp

POUD (J

Q0077241)

Lysin

ibacil

lussp

BAB-

4156

(KJ7941141

)


(KJ7252361

)

L fusifo

rmis DSM 28

98(NR_04

20721)

L fusiformis NBRC 1

5717

(NR_1126281)


4 (NR_114213


17 (NR_1125691

)



)

S malt

ophil

iaLM

G958-

T (X959231)

S m

altop

hilia

K279a

(NC_0109431

)

P pi

ctoru

mLM

G981

(NR_0419571

)

P hi

bisc

icola

ATCC

19867

(NR_0247091

)

S m

alto

phili

a AT

CC 19861

(NR_0408041

)

Sten

otro

phom

onas

sp 4

_O_7

(EF5405161

)

S n

itriti

redu

cens

L2(N

R_0253051

)

5P

7P

8P 12P

10

P

4P

13P 6P

1P

S hu

mi R-32729

(NR_0425681

)

607

887

514

685

999

995

551

605

873

563

51

668

999992

801

644

764

998 944815

968

993

661

84

583

594

664

527

961

835

577


34631)



2


3




Control

Days0 2 5 7

pH

05

10152025303540455055

666870727476788082848688


Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(a)

pH

70

72

74

76

78

80

82


Days0 2 5 7


Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

0

5

10

15

20

25

30

35

40

45

50

(b)pH

70

72

74

76

78

80

82


Days0 2 5 7


Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

0

5

10

15

20

25

30

35

40

45

50

(c)pH

70

72

74

76

78

80

82

86

88

84


Days0 2 5 7


05

10152025303540455055

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(d)

pH

70

72

74

76

78

80

82

86

88

84


Days0 2 5 7


05

10152025303540455055

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(e)

pH

706866

727476788082

8688

84


Days0 2 5 7


05

10152025303540455055

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(f)

Figure 4 Continued


pH

706866

727476788082

8688

84


Days0 2 5 7


0

5

10

15

20

25

30

35

40

45

50

55

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(g)







4 Discussion




MnCO3


MK pH 75 +LBB

MK pH 80 +LBB

MK pH 85 +LBB

MK pH 90 +LBB


MK pH 80 + MK pH 85 + MK pH 90 +


3


2

manganese oxide


2






22




10G10G + protease

12 24 36 48 60 720

Time (hours)

0

5

10

15

20

25

30

35

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)


(a)

8P8P + protease

12 24 36 48 60 720

Time (hours)

0

5

10

15

20

25

30

35

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)


(b)

13P13P + protease

12 24 36 48 60 720

Time (hours)

0

5

10

15

20

25

30

35

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)


(c)



SEMStrains

Bacil

luss

p1

G

(a)

SEMStrains + Mn(II)

Bacil

luss

p1

G

(b)

StrainsTEM

Bacil

luss

p1

G

(c)

Strains + Mn(II)TEM

Bacil

luss

p1

G

(d)

SEMStrains

Bacil

luss

p10

G

(e)

SEMStrains + Mn(II)

Bacil

luss

p10

G

(f)

StrainsTEM

Bacil

luss

p10

G

(g)

Strains + Mn(II)TEM

Bacil

luss

p10

G

(h)

SEMStrains

Sten

otro

phom

onas

sp

7P

(i)

SEMStrains + Mn(II)

Sten

otro

phom

onas

sp

7P

(j)

StrainsTEM

Sten

otro

phom

onas

sp

7P

(k)

Strains + Mn(II)TEM

Sten

otro

phom

onas

sp

7P

(l)

SEMStrains

Sten

otro

phom

onas

sp

8P

(m)

SEMStrains + Mn(II)

Sten

otro

phom

onas

sp

8P

(n)

StrainsTEM

Sten

otro

phom

onas

sp

8P

(o)

Strains + Mn(II)TEM

Sten

otro

phom

onas

sp

8P

(p)

SEMStrains

Lysin

ibac

illus

sp

6P

(q)

SEMStrains + Mn(II)

Lysin

ibac

illus

sp

6P

(r)

StrainsTEM

Lysin

ibac

illus

sp

6P

(s)

Strains + Mn(II)TEM

Lysin

ibac

illus

sp

6P

(t)

SEMStrains

Lysin

ibac

illus

sp

13

P

(u)

SEMStrains + Mn(II)

Lysin

ibac

illus

sp

13

P

(v)

StrainsTEM

Lysin

ibac

illus

sp

13

P

(w)

Strains + Mn(II)TEM

Lysin

ibac

illus

sp

13

P

(x)







2



5 Conclusions










Acknowledgments


References
































2


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


BioC

L50

mgmiddot

Lminus1

Mn(

II)-1

BioC

L50

mgmiddot

Lminus1

Mn(

II)-2

BioC

L50

mgmiddot

Lminus1

Mn(

II)-1

BioC

L50

mgmiddot

Lminus1

Mn(

II)-1

BioC

L50

mgmiddot

Lminus1

Mn(

II)-2

BioC

L50

mgmiddot

Lminus1

Mn(

II)-2

968F-CG1392R 357F-CG907R 357F-CG518R












100

100100

100

100

100100

100

100100

100

100

100

100100

100100100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100 100 100

100

100

100

100

100

100

100 1

00

100

100

100

100

100

100

100

100

100

100

100100

100

100

100

100

100

100

100

100

100

100

100

100

100100

100

100

100

100

100

100

100

100

10010

0

100100

100

100

100

100

100 100

100 100

100 100

100

100

100

100

100

100

100

100

100

100

100

100


62831)


(NR_0253421)S

terrae

R-32768T

(AM4035892)

S ter

raeR-32

768

(NR_0

425691)

S acid

aminiphilaCCUG 5

4933

(GU9455351)

S acidaminiphila

AMX 19

(NR_02

51041

)



S maltophilia IA

M12423

(NR_0415771)


(NR_0247081)

S maltophiliaR

551-3

(NR_0748751)


(GU9455371)

B an

thra

cis(N

R_0744531

)

6G

9G

1G

12G

10G

8G

B cereus G9842 (CP0011861)





5G



7G


0036871)


R_1217611)


11821

(NR_0246971)


(NZ_CM

0007531)

B mycoides

273

(NR_0368801)

B th

urin

gien

sisBM

B171

(CP0019031

)B

thur

ingi

ensis

IAM12077

(NR_0434031

)

B ce

reus

ATCC

14579

(NC_0047221

)

B cy

toto

xicu

sNVH

391

-98

(NR_0749141

)

B fu

nicu

lusN

AF001

(NR_0286241

)

B ab

yssa

lisSC

SIO15042

(NR_1096711

)






R opacus DSM 43205 (NR_0261861)






V soli GH9-3 (NR_0438111)







S koreensis

(AB1668851)

S plymuthica IC1270

(AY55133

21)


)


9821)


851)




11)lowast



71)lowast


)

L boronitolerans 10

a (NR_04

12761)

L paki

stanensis

NCCP-54(N

R_1131661)

Lysinibacill

us sp R18

(KJ4997861

)

Lysin

ibacil

lussp

POUD (J

Q0077241)

Lysin

ibacil

lussp

BAB-

4156

(KJ7941141

)


(KJ7252361

)

L fusifo

rmis DSM 28

98(NR_04

20721)

L fusiformis NBRC 1

5717

(NR_1126281)


4 (NR_114213


17 (NR_1125691

)



)

S malt

ophil

iaLM

G958-

T (X959231)

S m

altop

hilia

K279a

(NC_0109431

)

P pi

ctoru

mLM

G981

(NR_0419571

)

P hi

bisc

icola

ATCC

19867

(NR_0247091

)

S m

alto

phili

a AT

CC 19861

(NR_0408041

)

Sten

otro

phom

onas

sp 4

_O_7

(EF5405161

)

S n

itriti

redu

cens

L2(N

R_0253051

)

5P

7P

8P 12P

10

P

4P

13P 6P

1P

S hu

mi R-32729

(NR_0425681

)

607

887

514

685

999

995

551

605

873

563

51

668

999992

801

644

764

998 944815

968

993

661

84

583

594

664

527

961

835

577


34631)



2


3




Control

Days0 2 5 7

pH

05

10152025303540455055

666870727476788082848688


Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(a)

pH

70

72

74

76

78

80

82


Days0 2 5 7


Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

0

5

10

15

20

25

30

35

40

45

50

(b)pH

70

72

74

76

78

80

82


Days0 2 5 7


Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

0

5

10

15

20

25

30

35

40

45

50

(c)pH

70

72

74

76

78

80

82

86

88

84


Days0 2 5 7


05

10152025303540455055

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(d)

pH

70

72

74

76

78

80

82

86

88

84


Days0 2 5 7


05

10152025303540455055

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(e)

pH

706866

727476788082

8688

84


Days0 2 5 7


05

10152025303540455055

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(f)

Figure 4 Continued


pH

706866

727476788082

8688

84


Days0 2 5 7


0

5

10

15

20

25

30

35

40

45

50

55

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(g)







4 Discussion




MnCO3


MK pH 75 +LBB

MK pH 80 +LBB

MK pH 85 +LBB

MK pH 90 +LBB


MK pH 80 + MK pH 85 + MK pH 90 +


3


2

manganese oxide


2






22




10G10G + protease

12 24 36 48 60 720

Time (hours)

0

5

10

15

20

25

30

35

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)


(a)

8P8P + protease

12 24 36 48 60 720

Time (hours)

0

5

10

15

20

25

30

35

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)


(b)

13P13P + protease

12 24 36 48 60 720

Time (hours)

0

5

10

15

20

25

30

35

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)


(c)



SEMStrains

Bacil

luss

p1

G

(a)

SEMStrains + Mn(II)

Bacil

luss

p1

G

(b)

StrainsTEM

Bacil

luss

p1

G

(c)

Strains + Mn(II)TEM

Bacil

luss

p1

G

(d)

SEMStrains

Bacil

luss

p10

G

(e)

SEMStrains + Mn(II)

Bacil

luss

p10

G

(f)

StrainsTEM

Bacil

luss

p10

G

(g)

Strains + Mn(II)TEM

Bacil

luss

p10

G

(h)

SEMStrains

Sten

otro

phom

onas

sp

7P

(i)

SEMStrains + Mn(II)

Sten

otro

phom

onas

sp

7P

(j)

StrainsTEM

Sten

otro

phom

onas

sp

7P

(k)

Strains + Mn(II)TEM

Sten

otro

phom

onas

sp

7P

(l)

SEMStrains

Sten

otro

phom

onas

sp

8P

(m)

SEMStrains + Mn(II)

Sten

otro

phom

onas

sp

8P

(n)

StrainsTEM

Sten

otro

phom

onas

sp

8P

(o)

Strains + Mn(II)TEM

Sten

otro

phom

onas

sp

8P

(p)

SEMStrains

Lysin

ibac

illus

sp

6P

(q)

SEMStrains + Mn(II)

Lysin

ibac

illus

sp

6P

(r)

StrainsTEM

Lysin

ibac

illus

sp

6P

(s)

Strains + Mn(II)TEM

Lysin

ibac

illus

sp

6P

(t)

SEMStrains

Lysin

ibac

illus

sp

13

P

(u)

SEMStrains + Mn(II)

Lysin

ibac

illus

sp

13

P

(v)

StrainsTEM

Lysin

ibac

illus

sp

13

P

(w)

Strains + Mn(II)TEM

Lysin

ibac

illus

sp

13

P

(x)







2



5 Conclusions










Acknowledgments


References
































2


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


100

100100

100

100

100100

100

100100

100

100

100

100100

100100100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100

100 100 100

100

100

100

100

100

100

100 1

00

100

100

100

100

100

100

100

100

100

100

100100

100

100

100

100

100

100

100

100

100

100

100

100

100100

100

100

100

100

100

100

100

100

10010

0

100100

100

100

100

100

100 100

100 100

100 100

100

100

100

100

100

100

100

100

100

100

100

100


62831)


(NR_0253421)S

terrae

R-32768T

(AM4035892)

S ter

raeR-32

768

(NR_0

425691)

S acid

aminiphilaCCUG 5

4933

(GU9455351)

S acidaminiphila

AMX 19

(NR_02

51041

)



S maltophilia IA

M12423

(NR_0415771)


(NR_0247081)

S maltophiliaR

551-3

(NR_0748751)


(GU9455371)

B an

thra

cis(N

R_0744531

)

6G

9G

1G

12G

10G

8G

B cereus G9842 (CP0011861)





5G



7G


0036871)


R_1217611)


11821

(NR_0246971)


(NZ_CM

0007531)

B mycoides

273

(NR_0368801)

B th

urin

gien

sisBM

B171

(CP0019031

)B

thur

ingi

ensis

IAM12077

(NR_0434031

)

B ce

reus

ATCC

14579

(NC_0047221

)

B cy

toto

xicu

sNVH

391

-98

(NR_0749141

)

B fu

nicu

lusN

AF001

(NR_0286241

)

B ab

yssa

lisSC

SIO15042

(NR_1096711

)






R opacus DSM 43205 (NR_0261861)






V soli GH9-3 (NR_0438111)







S koreensis

(AB1668851)

S plymuthica IC1270

(AY55133

21)


)


9821)


851)




11)lowast



71)lowast


)

L boronitolerans 10

a (NR_04

12761)

L paki

stanensis

NCCP-54(N

R_1131661)

Lysinibacill

us sp R18

(KJ4997861

)

Lysin

ibacil

lussp

POUD (J

Q0077241)

Lysin

ibacil

lussp

BAB-

4156

(KJ7941141

)


(KJ7252361

)

L fusifo

rmis DSM 28

98(NR_04

20721)

L fusiformis NBRC 1

5717

(NR_1126281)


4 (NR_114213


17 (NR_1125691

)



)

S malt

ophil

iaLM

G958-

T (X959231)

S m

altop

hilia

K279a

(NC_0109431

)

P pi

ctoru

mLM

G981

(NR_0419571

)

P hi

bisc

icola

ATCC

19867

(NR_0247091

)

S m

alto

phili

a AT

CC 19861

(NR_0408041

)

Sten

otro

phom

onas

sp 4

_O_7

(EF5405161

)

S n

itriti

redu

cens

L2(N

R_0253051

)

5P

7P

8P 12P

10

P

4P

13P 6P

1P

S hu

mi R-32729

(NR_0425681

)

607

887

514

685

999

995

551

605

873

563

51

668

999992

801

644

764

998 944815

968

993

661

84

583

594

664

527

961

835

577


34631)



2


3




Control

Days0 2 5 7

pH

05

10152025303540455055

666870727476788082848688


Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(a)

pH

70

72

74

76

78

80

82


Days0 2 5 7


Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

0

5

10

15

20

25

30

35

40

45

50

(b)pH

70

72

74

76

78

80

82


Days0 2 5 7


Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

0

5

10

15

20

25

30

35

40

45

50

(c)pH

70

72

74

76

78

80

82

86

88

84


Days0 2 5 7


05

10152025303540455055

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(d)

pH

70

72

74

76

78

80

82

86

88

84


Days0 2 5 7


05

10152025303540455055

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(e)

pH

706866

727476788082

8688

84


Days0 2 5 7


05

10152025303540455055

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(f)

Figure 4 Continued


pH

706866

727476788082

8688

84


Days0 2 5 7


0

5

10

15

20

25

30

35

40

45

50

55

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(g)







4 Discussion




MnCO3


MK pH 75 +LBB

MK pH 80 +LBB

MK pH 85 +LBB

MK pH 90 +LBB


MK pH 80 + MK pH 85 + MK pH 90 +


3


2

manganese oxide


2






22




10G10G + protease

12 24 36 48 60 720

Time (hours)

0

5

10

15

20

25

30

35

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)


(a)

8P8P + protease

12 24 36 48 60 720

Time (hours)

0

5

10

15

20

25

30

35

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)


(b)

13P13P + protease

12 24 36 48 60 720

Time (hours)

0

5

10

15

20

25

30

35

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)


(c)



SEMStrains

Bacil

luss

p1

G

(a)

SEMStrains + Mn(II)

Bacil

luss

p1

G

(b)

StrainsTEM

Bacil

luss

p1

G

(c)

Strains + Mn(II)TEM

Bacil

luss

p1

G

(d)

SEMStrains

Bacil

luss

p10

G

(e)

SEMStrains + Mn(II)

Bacil

luss

p10

G

(f)

StrainsTEM

Bacil

luss

p10

G

(g)

Strains + Mn(II)TEM

Bacil

luss

p10

G

(h)

SEMStrains

Sten

otro

phom

onas

sp

7P

(i)

SEMStrains + Mn(II)

Sten

otro

phom

onas

sp

7P

(j)

StrainsTEM

Sten

otro

phom

onas

sp

7P

(k)

Strains + Mn(II)TEM

Sten

otro

phom

onas

sp

7P

(l)

SEMStrains

Sten

otro

phom

onas

sp

8P

(m)

SEMStrains + Mn(II)

Sten

otro

phom

onas

sp

8P

(n)

StrainsTEM

Sten

otro

phom

onas

sp

8P

(o)

Strains + Mn(II)TEM

Sten

otro

phom

onas

sp

8P

(p)

SEMStrains

Lysin

ibac

illus

sp

6P

(q)

SEMStrains + Mn(II)

Lysin

ibac

illus

sp

6P

(r)

StrainsTEM

Lysin

ibac

illus

sp

6P

(s)

Strains + Mn(II)TEM

Lysin

ibac

illus

sp

6P

(t)

SEMStrains

Lysin

ibac

illus

sp

13

P

(u)

SEMStrains + Mn(II)

Lysin

ibac

illus

sp

13

P

(v)

StrainsTEM

Lysin

ibac

illus

sp

13

P

(w)

Strains + Mn(II)TEM

Lysin

ibac

illus

sp

13

P

(x)







2



5 Conclusions










Acknowledgments


References
































2


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Control

Days0 2 5 7

pH

05

10152025303540455055

666870727476788082848688


Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(a)

pH

70

72

74

76

78

80

82


Days0 2 5 7


Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

0

5

10

15

20

25

30

35

40

45

50

(b)pH

70

72

74

76

78

80

82


Days0 2 5 7


Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

0

5

10

15

20

25

30

35

40

45

50

(c)pH

70

72

74

76

78

80

82

86

88

84


Days0 2 5 7


05

10152025303540455055

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(d)

pH

70

72

74

76

78

80

82

86

88

84


Days0 2 5 7


05

10152025303540455055

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(e)

pH

706866

727476788082

8688

84


Days0 2 5 7


05

10152025303540455055

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(f)

Figure 4 Continued


pH

706866

727476788082

8688

84


Days0 2 5 7


0

5

10

15

20

25

30

35

40

45

50

55

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(g)







4 Discussion




MnCO3


MK pH 75 +LBB

MK pH 80 +LBB

MK pH 85 +LBB

MK pH 90 +LBB


MK pH 80 + MK pH 85 + MK pH 90 +


3


2

manganese oxide


2






22




10G10G + protease

12 24 36 48 60 720

Time (hours)

0

5

10

15

20

25

30

35

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)


(a)

8P8P + protease

12 24 36 48 60 720

Time (hours)

0

5

10

15

20

25

30

35

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)


(b)

13P13P + protease

12 24 36 48 60 720

Time (hours)

0

5

10

15

20

25

30

35

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)


(c)



SEMStrains

Bacil

luss

p1

G

(a)

SEMStrains + Mn(II)

Bacil

luss

p1

G

(b)

StrainsTEM

Bacil

luss

p1

G

(c)

Strains + Mn(II)TEM

Bacil

luss

p1

G

(d)

SEMStrains

Bacil

luss

p10

G

(e)

SEMStrains + Mn(II)

Bacil

luss

p10

G

(f)

StrainsTEM

Bacil

luss

p10

G

(g)

Strains + Mn(II)TEM

Bacil

luss

p10

G

(h)

SEMStrains

Sten

otro

phom

onas

sp

7P

(i)

SEMStrains + Mn(II)

Sten

otro

phom

onas

sp

7P

(j)

StrainsTEM

Sten

otro

phom

onas

sp

7P

(k)

Strains + Mn(II)TEM

Sten

otro

phom

onas

sp

7P

(l)

SEMStrains

Sten

otro

phom

onas

sp

8P

(m)

SEMStrains + Mn(II)

Sten

otro

phom

onas

sp

8P

(n)

StrainsTEM

Sten

otro

phom

onas

sp

8P

(o)

Strains + Mn(II)TEM

Sten

otro

phom

onas

sp

8P

(p)

SEMStrains

Lysin

ibac

illus

sp

6P

(q)

SEMStrains + Mn(II)

Lysin

ibac

illus

sp

6P

(r)

StrainsTEM

Lysin

ibac

illus

sp

6P

(s)

Strains + Mn(II)TEM

Lysin

ibac

illus

sp

6P

(t)

SEMStrains

Lysin

ibac

illus

sp

13

P

(u)

SEMStrains + Mn(II)

Lysin

ibac

illus

sp

13

P

(v)

StrainsTEM

Lysin

ibac

illus

sp

13

P

(w)

Strains + Mn(II)TEM

Lysin

ibac

illus

sp

13

P

(x)







2



5 Conclusions










Acknowledgments


References
































2


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


pH

706866

727476788082

8688

84


Days0 2 5 7


0

5

10

15

20

25

30

35

40

45

50

55

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)

(g)







4 Discussion




MnCO3


MK pH 75 +LBB

MK pH 80 +LBB

MK pH 85 +LBB

MK pH 90 +LBB


MK pH 80 + MK pH 85 + MK pH 90 +


3


2

manganese oxide


2






22




10G10G + protease

12 24 36 48 60 720

Time (hours)

0

5

10

15

20

25

30

35

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)


(a)

8P8P + protease

12 24 36 48 60 720

Time (hours)

0

5

10

15

20

25

30

35

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)


(b)

13P13P + protease

12 24 36 48 60 720

Time (hours)

0

5

10

15

20

25

30

35

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)


(c)



SEMStrains

Bacil

luss

p1

G

(a)

SEMStrains + Mn(II)

Bacil

luss

p1

G

(b)

StrainsTEM

Bacil

luss

p1

G

(c)

Strains + Mn(II)TEM

Bacil

luss

p1

G

(d)

SEMStrains

Bacil

luss

p10

G

(e)

SEMStrains + Mn(II)

Bacil

luss

p10

G

(f)

StrainsTEM

Bacil

luss

p10

G

(g)

Strains + Mn(II)TEM

Bacil

luss

p10

G

(h)

SEMStrains

Sten

otro

phom

onas

sp

7P

(i)

SEMStrains + Mn(II)

Sten

otro

phom

onas

sp

7P

(j)

StrainsTEM

Sten

otro

phom

onas

sp

7P

(k)

Strains + Mn(II)TEM

Sten

otro

phom

onas

sp

7P

(l)

SEMStrains

Sten

otro

phom

onas

sp

8P

(m)

SEMStrains + Mn(II)

Sten

otro

phom

onas

sp

8P

(n)

StrainsTEM

Sten

otro

phom

onas

sp

8P

(o)

Strains + Mn(II)TEM

Sten

otro

phom

onas

sp

8P

(p)

SEMStrains

Lysin

ibac

illus

sp

6P

(q)

SEMStrains + Mn(II)

Lysin

ibac

illus

sp

6P

(r)

StrainsTEM

Lysin

ibac

illus

sp

6P

(s)

Strains + Mn(II)TEM

Lysin

ibac

illus

sp

6P

(t)

SEMStrains

Lysin

ibac

illus

sp

13

P

(u)

SEMStrains + Mn(II)

Lysin

ibac

illus

sp

13

P

(v)

StrainsTEM

Lysin

ibac

illus

sp

13

P

(w)

Strains + Mn(II)TEM

Lysin

ibac

illus

sp

13

P

(x)







2



5 Conclusions










Acknowledgments


References
































2


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


MnCO3


MK pH 75 +LBB

MK pH 80 +LBB

MK pH 85 +LBB

MK pH 90 +LBB


MK pH 80 + MK pH 85 + MK pH 90 +


3


2

manganese oxide


2






22




10G10G + protease

12 24 36 48 60 720

Time (hours)

0

5

10

15

20

25

30

35

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)


(a)

8P8P + protease

12 24 36 48 60 720

Time (hours)

0

5

10

15

20

25

30

35

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)


(b)

13P13P + protease

12 24 36 48 60 720

Time (hours)

0

5

10

15

20

25

30

35

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)


(c)



SEMStrains

Bacil

luss

p1

G

(a)

SEMStrains + Mn(II)

Bacil

luss

p1

G

(b)

StrainsTEM

Bacil

luss

p1

G

(c)

Strains + Mn(II)TEM

Bacil

luss

p1

G

(d)

SEMStrains

Bacil

luss

p10

G

(e)

SEMStrains + Mn(II)

Bacil

luss

p10

G

(f)

StrainsTEM

Bacil

luss

p10

G

(g)

Strains + Mn(II)TEM

Bacil

luss

p10

G

(h)

SEMStrains

Sten

otro

phom

onas

sp

7P

(i)

SEMStrains + Mn(II)

Sten

otro

phom

onas

sp

7P

(j)

StrainsTEM

Sten

otro

phom

onas

sp

7P

(k)

Strains + Mn(II)TEM

Sten

otro

phom

onas

sp

7P

(l)

SEMStrains

Sten

otro

phom

onas

sp

8P

(m)

SEMStrains + Mn(II)

Sten

otro

phom

onas

sp

8P

(n)

StrainsTEM

Sten

otro

phom

onas

sp

8P

(o)

Strains + Mn(II)TEM

Sten

otro

phom

onas

sp

8P

(p)

SEMStrains

Lysin

ibac

illus

sp

6P

(q)

SEMStrains + Mn(II)

Lysin

ibac

illus

sp

6P

(r)

StrainsTEM

Lysin

ibac

illus

sp

6P

(s)

Strains + Mn(II)TEM

Lysin

ibac

illus

sp

6P

(t)

SEMStrains

Lysin

ibac

illus

sp

13

P

(u)

SEMStrains + Mn(II)

Lysin

ibac

illus

sp

13

P

(v)

StrainsTEM

Lysin

ibac

illus

sp

13

P

(w)

Strains + Mn(II)TEM

Lysin

ibac

illus

sp

13

P

(x)







2



5 Conclusions










Acknowledgments


References
































2


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


10G10G + protease

12 24 36 48 60 720

Time (hours)

0

5

10

15

20

25

30

35

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)


(a)

8P8P + protease

12 24 36 48 60 720

Time (hours)

0

5

10

15

20

25

30

35

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)


(b)

13P13P + protease

12 24 36 48 60 720

Time (hours)

0

5

10

15

20

25

30

35

Mn(

II) i

on co

ncen

trat

ion

(mgmiddot

Lminus1)


(c)



SEMStrains

Bacil

luss

p1

G

(a)

SEMStrains + Mn(II)

Bacil

luss

p1

G

(b)

StrainsTEM

Bacil

luss

p1

G

(c)

Strains + Mn(II)TEM

Bacil

luss

p1

G

(d)

SEMStrains

Bacil

luss

p10

G

(e)

SEMStrains + Mn(II)

Bacil

luss

p10

G

(f)

StrainsTEM

Bacil

luss

p10

G

(g)

Strains + Mn(II)TEM

Bacil

luss

p10

G

(h)

SEMStrains

Sten

otro

phom

onas

sp

7P

(i)

SEMStrains + Mn(II)

Sten

otro

phom

onas

sp

7P

(j)

StrainsTEM

Sten

otro

phom

onas

sp

7P

(k)

Strains + Mn(II)TEM

Sten

otro

phom

onas

sp

7P

(l)

SEMStrains

Sten

otro

phom

onas

sp

8P

(m)

SEMStrains + Mn(II)

Sten

otro

phom

onas

sp

8P

(n)

StrainsTEM

Sten

otro

phom

onas

sp

8P

(o)

Strains + Mn(II)TEM

Sten

otro

phom

onas

sp

8P

(p)

SEMStrains

Lysin

ibac

illus

sp

6P

(q)

SEMStrains + Mn(II)

Lysin

ibac

illus

sp

6P

(r)

StrainsTEM

Lysin

ibac

illus

sp

6P

(s)

Strains + Mn(II)TEM

Lysin

ibac

illus

sp

6P

(t)

SEMStrains

Lysin

ibac

illus

sp

13

P

(u)

SEMStrains + Mn(II)

Lysin

ibac

illus

sp

13

P

(v)

StrainsTEM

Lysin

ibac

illus

sp

13

P

(w)

Strains + Mn(II)TEM

Lysin

ibac

illus

sp

13

P

(x)







2



5 Conclusions










Acknowledgments


References
































2


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


SEMStrains

Bacil

luss

p1

G

(a)

SEMStrains + Mn(II)

Bacil

luss

p1

G

(b)

StrainsTEM

Bacil

luss

p1

G

(c)

Strains + Mn(II)TEM

Bacil

luss

p1

G

(d)

SEMStrains

Bacil

luss

p10

G

(e)

SEMStrains + Mn(II)

Bacil

luss

p10

G

(f)

StrainsTEM

Bacil

luss

p10

G

(g)

Strains + Mn(II)TEM

Bacil

luss

p10

G

(h)

SEMStrains

Sten

otro

phom

onas

sp

7P

(i)

SEMStrains + Mn(II)

Sten

otro

phom

onas

sp

7P

(j)

StrainsTEM

Sten

otro

phom

onas

sp

7P

(k)

Strains + Mn(II)TEM

Sten

otro

phom

onas

sp

7P

(l)

SEMStrains

Sten

otro

phom

onas

sp

8P

(m)

SEMStrains + Mn(II)

Sten

otro

phom

onas

sp

8P

(n)

StrainsTEM

Sten

otro

phom

onas

sp

8P

(o)

Strains + Mn(II)TEM

Sten

otro

phom

onas

sp

8P

(p)

SEMStrains

Lysin

ibac

illus

sp

6P

(q)

SEMStrains + Mn(II)

Lysin

ibac

illus

sp

6P

(r)

StrainsTEM

Lysin

ibac

illus

sp

6P

(s)

Strains + Mn(II)TEM

Lysin

ibac

illus

sp

6P

(t)

SEMStrains

Lysin

ibac

illus

sp

13

P

(u)

SEMStrains + Mn(II)

Lysin

ibac

illus

sp

13

P

(v)

StrainsTEM

Lysin

ibac

illus

sp

13

P

(w)

Strains + Mn(II)TEM

Lysin

ibac

illus

sp

13

P

(x)







2



5 Conclusions










Acknowledgments


References
































2


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




2



5 Conclusions










Acknowledgments


References
































2


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


























2


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article indirect manganese removal by ... · research article indirect manganese removal...

Documents