Dr. Ying Ma Rhizobacterium Bacillus sp. SC2b and its role in phytoextraction 4 th International...
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Dr. Ying Ma Rhizobacterium Bacillus sp. SC2b and its role in phytoextraction 4 th International Conference on Earth Science & Climate Change Centre for Functional Ecology
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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
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Good morning, my name is Ying Ma from University of Coimbra, Portugal. I am going to talk about Rhizobacterium Bacillus sp. SC2b and its role in phytoextraction.
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)
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Mining activities and pedogenesis of ultramafic rocks have caused metal contamination of soils in many countries. As an emerging technology, phytoremediation offers great potential to remediate polluted environments. Phytoremediation is defined as the use of higher plants to remove contaminants from polluted soil. It includes phytoextraction, rhizodegradation, phytostabilization and phytovolatilization. Today I am going to focus on phytoextraction. However, it’s a long-term process that depends on plant growth, tolerance to metal toxicity and bioaccumulation capacity. Recently, the benefits of combining PGPR with plant for enhanced remediation of pollutants has been demonstrated.
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
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When talking about microbe-assisted phytoextraction, we need to know what exactly happened between plants and PGPR in soil. In general, plant root exudates offer soil microbes the carbon source, while microbes provide plant the fixed nitrogen and some other plant growth promoting substances, such as indole-3-acetic acid (IAA), 1-aminocyclopropane-1-carboxylate (ACC) deaminase and siderophore and solubilized phosphate.
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
Anonymous
As effective bioinoculants for microbe-assisted phytoextraction, the microorganisms must be able to promote plant growth. The plant growth promoting mechanisms by which PGPR support plant growth include producing plant growth-promoting substances, enhancing mineral nutrients bioavailability, biological control namely protecting plants from pathogens.
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 Ying
In addition to plant growth promoting potential, the metal resistant PGPR can also improve phytoextraction efficiency by mobilizing heavy metals in soils and transporting them to plant shoots through release of chelating agents, acidification and redox changes.
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
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Therefore, the proposed mechanisms for PGPR-assisted phytoextraction mainly include plant growth promotion, biocontrol, detoxification, heavy metal resistance, mechanisms affecting metal bioavailability in soils and metal translocation in plants.
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
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The success of phytoextraction depends not only on biomass production but also metal uptake by plants. As I mentioned above, PGPR can stimulate plant growth by secreting phytohormones, solubilizing insoluble mineral nutrients and suppressing phytopathogens. In addition, PGPR can also alleviate metal phytotoxicity and alter metal bioavailability through the production of siderophore and antioxidant enzymes and bioaccumulation of metals by bacterial cells.
Experimental plant
A new Zn-Cd hyperaccumulator Sedum plumbizincicola (Wu et al. 2008)
Ying Ma
In our study, we chose Sedum plumbizincicola as our experimental plant. As an newly discovered Zn and Cd hyperaccumulator, it has been proved by Wu et al. (2008) to be capable of hyperaccumulating Zn and Cd in polluted soil.
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
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Our study area was located in Pb-Zn mine area in Chunan city of Zhejiang province, southeast of China. The soils in this area have been reported to be highly polluted by Zn, Cd and Pb through mining activities. The objective of this study was to isolate and characterize metal mobilizing PGPR that could enhance phytoextraction of multi-metal polluted soil.
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
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Among 56 Cd-resistant strains isolated from the rhizosphere of Sedum plumbizincicola growing in mine area, 8 PGPR strains were selected based on their ability to utilize ACC as the sole nitrogen source. However, strain SC2b exhibited relatively higher levels of IAA and siderophore production as well as P solubilization, compared with other strains.
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)
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In the roll towel assay, we inoculated 8 rhizobacterial strains with Brassica napus grown in Petri dish. After 20 days, we found that inoculation of SC2b showed the maximum increase in plant shoot and root length as well as the vigor index, indicating that SC2b had the best plant growth promoting potential among the 8 strains.
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
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By morphological analysis, we found that after 48 h incubation on LB agar, the colony diameter of SC2b was 1-6 mm, round and circular. Under transmission electron microscope at 10,000 times magnification, bacterial cell was rod-shaped.
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)
Ma Ying
By biochemical analysis, SC2b showed high resistance to heavy metals such as Cd, Zn and Pb and antibiotic ampicillin. In addition, it can also produce ACC deaminase, IAA, siderophore, hydrogen cyanide and solubilize phosphate.
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
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The results of physiological analysis showed SC2b was gram positive, aerobic, oxidase positive, hydrolyzes pectin and cellulose. Based on morphological, physiobiochemical analysis, partial 16S rDNA sequence and phylogeny, SC2b was identified as Bacillus sp. (similarity 100%).
Impact of L-tryptophan content (A) and pH (B) on growth of Bacillus sp. SC2b and its IAA
production
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We also studies the impact of L-tryptophan and pH on bacterial growth and its IAA production. As shown in Fig. a, SC2b did not produce IAA in the absence of L-tryptophan. It produced the highest amount of IAA when cultured in LB broth amended with 2 mg/mL L-tryptophan, whereas at higher L-tryptophan concentrations (3, 4 and 5 mg/mL) an adverse effect on IAA production was observed. In Fig. b, strain SC2b was also able to grow in the medium over a wide range of initial pH ranging from 4 to 10; however, the highest synthesis of IAA by SC2b was obtained when cultivated in acidic media at pH 6. Moreover, significant correlations were obtained between bacterial growth and IAA production at different L-tryptophan concentrations and pH.
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
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The P solubilization potential of SC2b was studied over 120 h by monitoring pH drop and available phosphorus in the culture medium. Maximum P solubilization (about 90 mg/L) was detected after 96 h incubation along with a significant pH decrease.
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
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The metal mobilizing isolate SC2b was tested for the ability to grow in DF salts minimal medium. We found that SC2b could grow in the presence of ACC, indicating that it can utilize ACC through hydrolyzing ACC to α-ketobutyrate and ammonia and use ammonia as nitrogen sources.
Biosorption of metals by Bacillus sp. SC2b cells
Order of absorption:
Zn2+> Cd2+> Pb2+
Incubation time (h)
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We also studied the biosorption of metals by Bacillus sp. SC2b cells in liquid media containing with three different concentrations of Cd, Pb and Zn. The resting cells of SC2b exhibited a high degree of metal biosorption potential. The order of absorption by SC2b is Zn>Cd>Pb. This is probably related to the radius of metal ion. Metal ions having a smaller radius are more easily adsorbed to bacterial cell surface.
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
*
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The effects of SC2b on mobilization of Cd, Zn and Pb in soil after 7 days incubation were evaluated. Compared with non-inoculated control, inoculation of SC2b significantly increased concentrations of water soluble Cd, Zn and Pb in soil by 16.8-, 4.6- and 5.7-fold, respectively.
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
Ying Ma
We conducted two types of experiments in order to study the effect of SC2b on plant growth. In phytagar assay, we used Brassica napus as a model plant. After growth in phytagar media for 45 days, the inoculation of SC2b greatly enhanced plant shoot and root length, vigor index and dry weight, compared to control. In pot experiment, after growth of S. plumbizincicola in multi-metal contaminated soil for 75 days, the inoculation of SC2b significantly increased plant biomass production and chlorophyll contents in plants.
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
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The metal uptake data showed that although SC2b decreased Pb accumulation in shoots and roots of S. plumbizincicola, however it significantly increased accumulation of Cd and Zn in plants by 15 and 13%, respectively. The decreased Pb uptake was probably due to direct dilution of Pb concentration by increased plant biomass, while the increased Cd and Zn uptake was corresponding to the metal mobilization ability of 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.
Ying Ma
To summarize, we found that rhizobacterium SC2b increased water extractable Cd, Zn and Pb concentrations in soil. It also improved the performance and metal uptake of S. plumbizincicola. Production of growth promoting/metal mobilizing metabolites/enzymes is a likely mechanism. Bacillus sp. SC2b is a good candidate 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
Ying Ma
This work was financed by FCT. Thanks for your attention!
