Dr. Ying Ma Rhizobacterium Bacillus sp. SC2b and its role in phytoextraction 4 th International...
-
Upload
alicia-pope -
Category
Documents
-
view
213 -
download
0
Transcript of Dr. Ying Ma Rhizobacterium Bacillus sp. SC2b and its role in phytoextraction 4 th International...
Dr. Ying Ma
Rhizobacterium Bacillus sp. SC2b and its role in phytoextraction
4th International Conference on Earth Science & Climate Change
Centre for Functional Ecology
Phytoextraction
Definition: use of higher plants to remove contaminants from polluted soil.
Subtypes: phytoextraction, rhizodegradation, phytostabilization and phytovolatilization
Merits & Demerits: cost effective, no destruction of the soil. However, it’s a long-term process depending on plant growth, metal tolerance and bioaccumulation capacity.
Plant growth
promoting
rhizobacteria
(PGPR)
Emerging trend:
PGPR-assisted phytoextractio
n(Kloepper et al.
1978)
Interactions between plants and PGPR (Ma et al. 2011 Biotechnology Advances)
ACC synthase
Bacteria Mycorrhizae
Root IronIron binding siderophore
IAA
ACC
NH3 -ketobutyrate
Bacterial N source
Production of organic acids and chelating agents
Insoluble Iron
Siderophore
Iron-siderophore complex
Bacteria
Iron
Insoluble P minerals + metals
P solubilization, minerals and
metals
Root uptake of soluble P, minerals
and metals
Ethylene
Cell elongation and root growth
IAA
Acidification, chelationand exchange reaction
x ACC deaminase
Amino acids SAM
ACCACC oxidase
Root exudate
Growth promoting factors
IAA
Siderophore production
Decreased ethylene
PGPR
Cell elongation or division
N2 fixation Minerals
mobilization
ACC deaminase
Root proliferation
Sti
mu
late
pla
nt
grow
th
Biological control
Enhance nutrients bioavailability
Plant growth promoting mechanisms of PGPR
The role of PGPR in metal phytoremediation process
Production of siderophores and organic acids
Production of Enzymes
Transformation
Ion competition, reduction, acidification
or chelation
Volatilization Phytovolatilization
Phytostabilization
Phytoextraction
Production of inorganic ligands: HCO3-, H+
(e.g. sulfide, carbonate or phosphate ions)
Immobilization
Precipitation/alkalinization
Insoluble minerals and metals in soil
Soluble minerals and metals
Facilitating minerals andmetal uptake
Mobile metal cations in soil
Mitigating metal phytotoxicity
+
+
PGPR
Mobilization
Ma Y. 2012. Monograph ISBN: 978-3-8484-3421-3; Ma Y. 2015. Monograph ISBN: 978-3-659-69505-6
Mechanisms of plant growth promotion
N2 fixation
P solubilizationIncreasing mineral solubility
Decomposition of aluminosilicates
Secreting plant growth regulators
Modifying ethylene level
Producing antibiotics
Mechanisms of heavy metal resistance
Mechanisms of biocontrol
Mechanisms of detoxification
Inorganic P Organic P
Siderophore
Bioadsorption
Redox reaction
Bioleaching
Mechanisms affecting metal bioavailability
Mechanisms affecting metal translocation
Competition and colonization
Production of antibiotic
Induced systematic resistance Secretion of organic acid and
siderophores
Secretion of biosurfactants
Secretion of extracellular polymeric substances
NitrogenAmmonium nitrogen
Ferric ionFe-siderophore
AluminosilicatePotassium ion
Role of PGPR in
phytoremediation of metal
contaminated soil
Bacterial mobilization
ligand binding
Plant growth promoting traits
Secrete phytohormonesIAA ACC deaminase
Make nutrients bioavailableSiderophore N2 fixationP solubilization
Suppress phytopathogenBiocontrolISR
Altering metal bioavailability
Synthesize siderophoreComplexation
Precipitation
Produce antioxidant enzymesRedox reactionTransformation
Biosorb/Bioaccumulate
MethylateDegradate (organo-Hg)
Reduction (Hg2+)
RhizobacteriaEndophytic bacteriaPathogens
Phytovolatilization: release of metals into
atmosphere in volatile forms
MetalsNutrients
Stimulate plant growth
PGPR-assisted phytoremediation
Metal contaminated soil
Available for plant uptake
Alleviate metal phytotoxicity
Phytostabilization: metal uptake and
sequestration in roots
Phytoextraction: xylem-loading-mediated metal
translocation to shoots
Experimental plant
A new Zn-Cd hyperaccumulator Sedum plumbizincicola (Wu et al. 2008)
To isolate and characterize metal mobilizing PGPR that enhance multi-metal phytoextractionMa Y et al. 2013. Chemosphere; Ma et al. 2015. Journal of Environmental
Management
The Pb-Zn mine area in Chunan city, Zhejiang province, Southeast China
Study area Objective
Among 56 metal resistant strains, 8 PGPR were selected based on their ability to utilize ACC as the sole N source.
Strain
ACC deamina
seP IAA
Siderophore Cd
resistance CAS Catechol Hydroxam
ate
mg/L mg/L cm mg/L mg/L mg/L R2C2f + 77.1±6.2 45.3±2.6 nd nd nd 50
R2C2i +113.0±1.
237.9±2.2 nd nd nd 50
R2C3a +90.3±13.
128.7±4.2 1.1±0.2
152.4±23.2
8.6±0.4 250
R2C2a + 44.6±6.9 24.5±1.2 nd nd nd 450
R3Cb +109.0±4.
222.8±0.5 1.0±0.1
170.8±18.9
13.8±0.7 250
SC1a + 76.3±6.8 23.3±0.8 1.0±0.361.6±66.
34.8±0.7 250
R1C3a + 62.7±1.5 75.6±5.4 nd nd nd 450
SC2b +113.2±8
.6116.5±3
.21.2±0.2
198.3±18.0
13.2±0.2 300
Screening of beneficial rhizobacteria
Roll towel assay
TreatmentShoot length
(cm)Root length
(cm)Vigor index
(%)
Control 3.7±0.2 9.9±0.8 688
R2C2f 4.3±0.5 10.9±1.9 836
R2C2i 4.7±0.2 12.7±0.8 870
R2C3a 4.8±0.1 11.2±1.5 958
R2C2a 4.3±0.2 11.0±0.7 840
R3Cb 5.1±0.2 12.7±0.8 981
SC1a 5.2±0.2 10.8±0.8 879
R1C3a 5.1±0.5 13.2±0.3 1010
SC2b 6.1±0.3 13.7±1.1 1087
Vigor index = germination (%) × seedling length (shoot length + root length)
Morphological characteristics of SC2b
Colony morphology: colony diameters is 1-6 mm, round,
smooth surface and circular, whitish and
cream.
Cell morphology: rod-shaped
Under transmission electron microscope at 10,000 times magnification
A B
Biochemical characteristics of Bacillus sp. SC2b
Characteristics Parameter UnitBacillus sp.
SC2bMetal resistance Cd mg/L 300
Zn mg/L 750 Pb mg/L 1400
Antibiotic resistance Ampicillin mm 4 (R) Tetracycline mm 17 (S) Streptomycin mm 18 (S)
Chloramphenic
ol mm15 (I)
Kanamycin mm 17 (S)Plant growth promoting
featureACC deaminase μm α-KB/mg/hr 25 ± 3.6
P solubilization mg/L 56.6 ± 4.3 IAA mg/L 64.8 ± 2.0
Hydrogen cyanide +
Siderophore CAS cm 1.2 ± 0.2 Catechol mg/L 198.3 ± 18.0 Hydroxamate mg/L 13.2 ± 0.2
R, resistant (<10 mm); I, intermediate (10–15 mm); S, susceptible (>15 mm)
Physiological and genetic characteristics of SC2b
Physiological characteristics
Gram-positive, aerobic, oxidase-positive; hydrolyze pectin and cellulose.
Genetic identification
Based on morphological, physiobiochemical analysis, 16S rDNA sequence and phylogeny, SC2b was identified as Bacillus sp. (similarity 100%).
Phylogenetic tree Bacillus horikoshii strain RB10 (GU232770.2)
Bacillus megaterium strain MB1-42 (KJ843149.1)
Bacillus flexus strain NY-1 (EU869200.1)
Bacillaceae bacterium MSB06 (FJ189761.1)
Bacillus aryabhattai strain NN31 (KJ542774.1)
Strain SC2b
Bacillus weihenstephanensis strain MC67 (DQ345791.1)
Bacillus cereus strain PR15 (JQ435675.1)
Bacillus thuringiensis strain BAB-Bt2 (AM293345.1)
Escherichia coli (J01859)
82100
100
0.02
Impact of L-tryptophan content (A) and pH (B) on growth of Bacillus sp. SC2b and its IAA
production
Phosphate solubilization by Bacillus sp. SC2b
0 6 12 24 48 72 96 120
0
20
40
60
80
100
120
P solubilization pH
Inoculation time (h)
Ph
osp
hat
e so
lub
iliz
atio
n (
mg/
L)
0
2
4
6
pH
Growth of SC2b on DF salts minimal medium
0.0
0.1
0.2
0.3
0.4
0 24 48 72 96 120 144 168
Opt
ical
den
sity
at 6
00 n
m
Inoculation time (h)
DFACC
Biosorption of metals by Bacillus sp. SC2b cells
Order of absorption:
Zn2+> Cd2+> Pb2+
Incubation time (h)
Effect of Bacillus sp. SC2b on soil metal mobilization
Bacillus sp. SC2b ↑ Cd: 16.8-fold; Zn: 4.6-fold; Pb: 5.7-fold
Cd µg/g Zn mg/g Pb mg/g0
1
2
3
4
5
20
25
*
*
Wat
er-e
xtra
ctab
le m
etal
ion
in s
oil
Heavy metal
Control SC2b
*
Effect of Bacillus sp. SC2b on plant growth
Values are means ± standard deviations of three samples
Phytagar assayNon-inoculated
controlBacillus sp.
SC2b % Germination 72 ± 4 b 89 ± 2 a
Shoot length (cm) 4.5 ± 0.3 b 5.2 ± 0.2 aRoot length (cm) 1.8 ± 0.3 b 2.5 ± 0.2 a
Shoot elongation rate (%) - 115.6 ± 35Root elongation rate (%) - 138.9 ± 24
Vigor index 454 ± 36 b 685 ± 42 aPlant dry weight (g) 2.3 ± 0.1 b 2.9 ± 0.1 a
Pot experimentNon-inoculated
controlBacillus sp.
SC2bShoot fresh weight (g) 47.9 ± 6.4 b 68.9 ± 7.2 aRoot fresh weight (g) 0.7 ± 0.0 b 2.2 ± 0.6 aShoot dry biomass (g) 4.4 ± 0.7 b 6.2 ± 0.5 aRoot dry biomass (mg) 154.8 ± 16.2 b 278.4 ± 18.9 a
Chlorophyll a (mg g-1 fw) 1.3 ± 0.2 b 1.9 ± 0.3 aChlorophyll b (mg g-1 fw) 0.5 ± 0.0 b 0.9 ± 0.1 a
Total chlorophyll (mg g-1 fw) 1.9 ± 0.3 b 3.0 ± 0.4 aBacterial colonization (105 CFU g-
1)nd 6.8 ± 0.1
Effect of Bacillus sp. SC2b on metal uptake by S. plumbizincicola
Bacillus sp. SC2b
Cd ↑ 15%Zn ↑ 13%
Root Shoot Plant0
30
60
90
120
150
*
*
*
Cd
conc
entr
atio
n (m
g/kg
dry
bio
mas
s)
Control RC2b
A
Root Shoot Plant0
30
60
90
120
150B
*
*
*
Pb
conc
entr
atio
n (m
g/kg
dry
bio
mas
s) Control SC2b
Root Shoot Plant0
500
1000
1500
2000C
**
*
Zn
conc
entr
atio
n (m
g/kg
dry
bio
mas
s)
Control SC2b
Conclusions
Rhizobacterium increased water extractable Cd, Zn and Pb concentrations in soil
Bacillus sp. SC2b improved the performance and metal uptake of S. plumbizincicola
Production of growth promoting/metal mobilizing metabolites/enzymes is a likely mechanism
Bacillus sp. SC2b could serve as an effective metal mobilizing and growth promoting bioinoculant for microbe-assisted phytoextraction.
Acknowledgements
The authors thankfully
acknowledge Fundação para a
Ciência e a Tecnologia (FCT)
research grant of Y. Ma
(SFRH/BPD/76028/2011). This
work was financed by national
funds through FCT within the
scope of Project:
UID/BIA/04004/2013.
Thanks for your attention!Governo da República Portuguesa