Role of ACC and Exopolysaccharides Producing Plant Growth Promoting Rhizobacteria in

Phytoremediation of Industrially Contaminated Soil

ABSTRACT

Release of contaminants by various industrial activities is the major source of heavy metals. Heavy metals
are considered as persistent organic pollutants which are deleterious to animals as well as human beings
because of its ability to amass in the food chain. Present study was conducted to determine the effect of
heavy metal stress on plant biomass, plant chlorophyll and proline content, antioxidant enzyme production
and metal accusation by plants. Ten PM20, 33D, 33C, PM18, PM14, PM15, PM19, 23A, PM16 and
PM11 previously isolated from sugarcane were screened at different heavy metal concentration (50mg/l to
1000mg/l) to check their heavy metal tolerance capacity and also plant growth promotion characteristic
such as ACC, indole acetic acid production, Exopolysaccharide production (EPS) and nitrogen fixation
ability. Out of these ten strains the most competent PGP strains were (PM14,andPM11). These two strains
were further used to analyze their in-vitro potential via pot experiment. In this study flax (Linum
usitatissium L.) was used. Plant was grown in both non-contaminated and industrial contaminated soil. The
experiment comprised of six different treatments for Linum plant. Each treatment was comprised of 3
replicates and kept in growth chamber. The plants were harvested after 45 days of sowing . Results revealed,
that root, shoot length, proline content, chlorophyll content, carotenoid content, fresh weight, SOD and
POD enzymatic activity were greater in PGPR inoculated plants as compared to non-inoculated plants both
in non-contaminated and in industrial contaminated soil. The results indicated that inoculation of PGPR
such as Bacillus xiamensis, and Bacillus gibsonii increased plant growth and minimized stress in industrial
contaminated soil. Bacterial strains B. xiamensis and B. gibsonii enhanced phytoextraction potential of
Linum plant Thus, mentioned PGPR in current study can be used as potent tool to decrease the toxicity of
metals in contaminated.
INTRODUCTION
Soil contamination with heavy metals has become a serious issue all over the world for
the last few decades due to anthropogenic activities including use of fertilizers, pesticides,
polluted irrigation sources etc. (Chandrasekaran et al., 2015; Kumar et al., 2016). Particularly,
the issue of heavy metal contamination in agricultural soil is of extreme dangerous, because
heavy metal from soil get accumulated in plants used for vegetation purpose, thus easily entering
the food chain (Kaur et al., 2018). The most lethal heavy metal which are known to be the major
risk to the environment including Arsenic (As), Cadmium (Cd), Lead (Pb), Copper (Cu),
Chromium (Cr), Nickel (Ni), Zinc (Zn), Aluminum (Al) and Manganese (Mn) have known to be the
major threats to the environment (Dhanarani et al., 2016; Karthik et al., 2017). These metal
therefore pose serious threats to both human health and ecosystem (Ullah et al., 2015). Wide spread
use of agrochemicals such as NPK fertilizers, pesticides, weedicides etc. has caused pollution of
soil, ground water and vegetation with harmful metals (Kaur et al., 2018). Thus, to remediate the
heavy metals problem of soil is of much concern to protect the hostile effect on human health and
to keep the environment clean from pollutants for future generation (Glick, 2010). Both organic
and inorganic are either by physical, chemical and or biological treatments are available to clean
up the contaminated environment (Baldwin et al., 2015). However, the main disadvantage or
limitation of physical and chemical method is recognized as high cost and labor intensiveness.
Beside these, chemical methods of processes caused pollution and particularly costly since they
generate heaps and sludge (Tangahu et al., 2011). Phytoremediation serves as promising,
ecofriendly, economically cheap and as an emerging approach to degrade, extract, and immobilize
contaminants from soil surface and ground water, sediments etc. (Gupta et al., 2018).
Phytoremediation, the use of green plants for metal recovery includes various methods such as
phytoextraction, phytostabilization, rhizofiltration and phytovolatilization is cheap, improves
biodiversity and reduces erosion, reduced energy consumption leading to decreased CO2 emission
(Ali-Zade et al., 2010; Ghosh and Singh, 2005). Phytoextraction process is mainly based on the
usage of hyperaccumulator plants, which have potential to tolerate and store high concentration of
metals. The required characteristics for hyperaccumulator plants include deep root system, rapid
growing nature and large biomass (LeDuc and Terry, 2005). On the other hand phytostabilization
technique uses metal resistant plants for the purpose of mechanical stabilization of contaminated
land but required plants that take up small amount of metals so that to prevent their transfer into
the wild life food chain (Angle and Linacre, 2005). Additionally, phytostabilization may also be
the cost-effective methods to treat metal polluted soil (Dary et al., 2010). The efficacy of
phytoremediation may not only dependent on the plant but can be determined on the interaction of
plant with bacterial community and soil heavy metals concentration (Ma et al., 2009). Hence,
alternative phytoremediation technique that use the rhizospheric bacteria to deteriorate the toxicity
of metals on plants have been studied (Sun et al., 2010). Enhancing soil microbial diversity are
therefore particularly an important features in the success of phytoremediation (Al Mahmud et al.,
2018). Plant growth promoting rhizobacteria can affect the growth of plant either directly or
indirectly under heavy metal stress by showing resistance against abiotic stress, promoting root
growth, producing chelating agent for heavy metals and by enhancing soil microbial development
and nutrients availability (Bhattacharyya and Jha, 2012; Ma et al., 2016). The effectiveness of
phytoremediation required extensive root growth (Arshad et al., 2007). The plants inoculated with
ACC deaminase producing rhizobacteria of transgenic plants with ACC deaminase genes produce
larger root and greater root density (Safronova et al., 2006). This increase in root growth by ACC
deaminase thus enhance the efficiency of phytoremediation in polluted soil (Huang et al., 2004).
Exopolysaccharides producing (PGPR) in combination with plant is proposed recently as
ecofriendly techniques under heavy metal stress (Lal et al., 2018). In response to the exposure of
toxic heavy metals microbe producing EPS induce biofilm formation which helps in detoxification
of heavy metals by promoting metal tolerance capacity of microbial cell or by transforming toxic
metal ions into non- toxic metal ions (Gupta and Diwan, 2017).
Linum usitatissimum L. commonly flax/linseed is mainly cultivated for non-food and
industrial purposes is an ancient culture crop whose fiber is used in textile industry (Barkoula et
al., 2010). Flax/linseed served as heavy metal hyperaccumulators, because of their considerable
proportion used for non-food purposes (Angelova et al., 2004; Bjelková et al., 2011). Thus the
plant show a capacity of metal tolerance dependant on species, therefore ideal selection for
research (Bassuany et al., 2014). Therefore the main aims and objectives of the recent work were
to generate data on the efficiency of linum species in cleaning the heavy metal polluted soil via
phytoextraction and phytostabilization process.
 MATERIAL AN D METHODS
1) HEAVY METAL TOLERENCE
Heavy metals tolerant bacteria were secluded on nutrient agar added with different
concentrations of heavy metals such as cadmium, nickel, chromium and copper. Stress
concentrations for bacterial screening was started from 50mg/L and continue till the maximum
tolerance of bacteria against specific heavy metal(Mustapha and Halimoon, 2015)

2) ACC deaminase enzyme activity in the presence of heavy metal stress

Qualitative Screening
All ten sugar cane isolates were grown in 5 ml of TSB medium at 28 °C for 24h with
shaking (120 rpm). Centrifuge the suspension for 5 min at 3000rpm and washed twice with sterile
0.1 M Tris–HCl (pH 7.5). Then, washed with 1 ml of 0.1 M Tris–HCl (pH 7.5). Then, on petri
plates spot inoculated, containing modified DF(Dworkin and Foster, 1958)minimal salts medium
[glucose, 2.0 g; gluconic acid, 2.0 g; citric acid, 2.0 g; KH2PO4, 4.0 g; Na2HPO4, 6.0 g;
MgSO4.7H2O, 0.2 g; micro-nutrient solution (CaCl2, 200 mg; FeSO4.7H2O, 200 mg; H3BO3,
15 mg;ZnSO4.7H2O, 20 mg; Na2MoO4, 10 mg; KI, 10 mg; NaBr,10 mg; MnCl2, 10 mg; COCl2,
5 mg; CuCl2, 5 mg; AlCl3,2 mg; NiSO4, 2 mg distilled water, 1,000 ml), 10 ml; distilled water,
990 ml] supplemented with 3 mM ACC as the source of nitrogen. DF minimal salts medium
without ACC were used as the negative control and positive control was (NH4)2SO4 (0.2 % w/v).
Then plates were incubated for 72h at 28˚C. After 72h, plates supplemented with ACC was
matched with plates that was used as positive and negative control and selected on the bases of
growth by utilizing ACC as the nitrogen source.

Quantitative Screening of ACC deaminase activity.

To calculate ACC deaminase activity, at 28oC all ten sugar cane secludes were grown in
5ml of TSB medium unless they stretched the static phase. Under non-stress and heavy metal stress
situations, the cells were collected by centrifugation, washed twice with 0.1 M Tris–HCl (pH 7.5),
dissolve in 2ml of modified DF (Dworkin and foster salt medium) minimal medium either added
with 3mM final concentration of ACC without stress or supplemented with 50 mg L−1of heavy
metal (either Cu, Pb, As, Ni, Cr, Cd, or Mn) stress (Belimov et al., 2005) and at 28˚C incubated
for 36-72h. ACC deaminase activity was measured by measuring the production of α-ketobutyrate
and ammonia produced by the breakdown of ACC by ACC deaminase (Penrose and Glick, 2003).
Centrifuge the samples at 3000rpm for 5min.After, centrifugation washed harvested cells two
times with 0.1M Tris -HCl with (pH 7.5), resuspended in 200µl of 0.1M Tris -HCl with (PH
8.5) and 5% toluene. Then at the highest speed Vortexed the mixture for 30s. From cell suspension
50µl was taken and mixed with 5µl of 0.3 M ACC in an Eppendorf tube then incubate at 28˚C for
30 min. 50µl of cell suspension without ACC used as negative control. While blank included, 50µl
of cell suspension mixed with 5µl of 0.3M ACC and 50µl of 0.1M Tris -HCl (PH 8.5).The samples
were mixed with 500µl of 0.56N HCl and vortexed at the high speed. Then cells were collected by
centrifugation at 12000 rpm for 5min. Then, 500µl of supernatant was taken and transferred this
supernatant to glass test tube. 0.56N HCl 400µl and 150µl of DNF solution (0.1g 2, 4-
dinitrophenylhydrazine in 100ml of 2N HCl) was added to reaction mixture. Before measuring its
optic density at 540nm 1ml of 2N NaOH was added to the samples.

α-ketobutyrate stranded curve

The absorption of α-ketobutyrate was calculated by comparison with a standard curve in

each sample. In 500µl aliquot different α-ketobutyrate solutions (concentration 0, 0.01, 0.05, 0.1,
0.2, 0.5, 0.75, 1mM) 400µl 0f 0.56N HCl and 150µ DNF solution was added. In this mixture 1ml
of 2N NaoH was added. Its optic density was observed at 540nm. The values for consumption
versus α-keto-butyrate assemblage (mM) were used to construct a standard curve. The most
effective strains were further used to test their conflict with heavy metal stress.

Protein concentration determination

By the method of (Bradford, 1976),the protein absorption of toluene-labialized bacterial
cell was evaluated. From the toluene-labialized bacterial cell sample that was used for the ACC
deaminase enzyme analysis 26.5µl was taken and mixed with 173.5µl of 0.1M Tris-HCl (PH 8.0).
Then these samples are boiled with 200µl of 0.1N NaOH for 10 min. After that the samples were
cooled at room temperature. A 200µl of Bradford’s reagent was mixed with solution and
immediately observed protein consumption at 595nm. A standard curve was constructed by bovine
serum albumin.
3) EXOPOLYSAACCHARIDE PRODUCING BACTERIA
To check the Qualititive production of exopolysaccharide bacterial strains were streaked on
ATCC medium no.14. For three days incubate them at 28˚C under hygienic atmosphere.
Formation of slimy layer around the bacterial colonies confirmed the production of
exopolysaccharide.

For the quantitative assay of exopolysaccharide, bacterial strains were grown in 50ml (ATCC no
14 liquid medium). For next three days’ bacterial strains were incubate at 28˚C and at 2000 rpm.
By centrifugation cells were harvested at 10,000rpm for 20 min. After that two volumes of cold
isopropanol were added into it and kept overnight at 40˚C. Then, centrifuge at 10000 rpm for 20
min. Pellets were taken and dried at 1000˚C. After drying weigh the pellet to show which bacteria
showed high production of exopolysaccharide. Then at different environmental situations, strains
which showed the greater production was used for the exopolysaccharide production.

4)Indole acetic acid production in the presence of heavy metal stress and non-
stress condition.

IAA acid production was determined Qualitatively by colorimetric assay by the method of(Loper
and McCartney, 1986). IAA production was specified by development of pink color (Gordon and
Weber, 1951).After 30 min, the absorbance of the final pink color solution was measured 535 nm
in UV/Visible spectrophotometer.

Quantitative analysis of IAA was estimated by the technique of(Brick and Ravid, 1991). which
then improved by(Goswami et al., 2013). Bacterial strains were inoculated in L-tryptophan broth
(in 1000ml, 5g of NaCl, 10g of trypton, 5g of yeast extract and 1g of l-tryptophan). The culture
broth was supplemented with 50 mg L−1of heavy metal (either Cu, Pb, As, Ni, Cr, Cd, or Mn)
stress and without heavy metal stress. The culture broth was incubated for 72h at 36˚C-38˚C. Then,
centrifuge the bacterial solution at 3000 rpm for 30min.Then, 2ml of supernatant was taken and
mixed with 4ml of salkowski reagent (50ml, 35% of per chloric acid, 1ml 0.5M FeCl3 solution).
Two drops of ortho-phospheric acid added to the mixture. Appearance of pink color determines
the production of Indole acetic acid and its density was observed at 530nm. A standard curve of
IAA (H media) in the range of 10-100µgml was used to estimate the concentration of IAA
produced.
5) NITROGEN FIXATION
All bacterial isolates were screened for nitrogen fixation ability by inoculating them in nfb
semi solid media with addition of NH4Cl as a nitrogen source (Day and Döbereiner, 1976). For ten
days then incubate at 28˚C.

6) Analysis of industrial contaminated soil

From two different sites soil sample was collected.First soil, sample was collected from industrial
area of IsIamabad,contaminated Pak steel rerolling mills. Second soil sample was collected from
non-contaminated fields of QAU, Islamabad. Soil was autoclaved to remove the
containments(Nelson and Sommers, 1982).Physiochemical properties of soil like soil organic
matter (Bhuvaneswari et al., 1980) soil texture (McLean, 1982) pH, EC (Yang et al., 1996) and
heavy metal contents (Cd, Cr, Cu and Ni) were analyzed by atomic absorption spectrophotom eter.

7)METAL ANALYSIS OF SOIL BY WET ACID DIGESTION METHOD

For metal analysis of soil one gram of soil sample was taken and mixed with the 12mL
(9mLHCl+3mL HNO3) of aqua regia. Beaker was covered with the watch glass and kept the
solution overnight. Then, for 2 hours heated the samples on the hot plates. After that suspension
was allowed to cool down and filtered through what man No.42 filter paper. Then, by adding distal
water volume of the suspension was made up to 50Ml. Heavy metals were estimated through
atomic absorption spectrophotometer AAS model spectra AA Varian(Achakzai et al., 2017).

8) Inoculant preparation
The bacterial strains PM14: Bacillus xiamensis: and PM11 Bacillus gibsonia were taken
from Plant-Microbe Interactions Laboratory, Department of Plant Sciences, Quaid-i-Azam
University, Islamabad. Theses bacterial strains were grown in 250mL flasks that contained LB
broth. These culture broths were incubated for 48h at 30˚C for 15min at 80rpm. By centrifugation,
at 8000rpm for 15min bacteria were separated from liquid medium. In resulted pallet 0.1M of
phosphate buffer (PH 7.4) was added. The bacterial suspension was adjusted to 0.5A at 600nm to
obtain an approximate concentration of 1 ×109UFC mL-1. The bacterial culture solution was further
utilized to inoculate the seeds of Linum usitatissimum L. while non-inoculated seeds were soaked
in sterilized distilled water taken as control.
9)GREEN HOUSE EXPERIMENT
9.1) Seed sterilization and inoculation
Seeds of flax (Linum usitatissimum L.) was collected from National Agriculture Research
Center (NARC), Islamabad, Pakistan. Seeds were sterile by serial washing with 75% ethyl alcohol
for 5min and with 0.1% HgCl2 for 1min respectively. Subsequently, washed serval time with
autoclaved distal water. Then, Seeds were soaked in distal water.

The seeds of Linum plant was planted in both industrial contaminated soil and in non-
contaminated soil in pots.. Then in all pots 5 seeds were planted. Expreiment consist of six
treatment. In each experimental unit 20mLof bacterial suspension was added. In control 25mL of
phosphate buffer (PH 7.4) was added. The experimental units were placed in growth chamber for
45 days. After that, every other day 20ml water was carried out.

9.2) Harvesting
After 45 days of pot experiment, L. usitatissimum plant was carefully harvested from soil
root, shoot length ,fresh weight, Chlorophyll, Proline content, SOD, POD activity of enzyme and
the heavy metals contents were measured by using AA spectrophotometer.

10) Analysis of plant after harvesting

10.1) Plant length (Root and Shoot Length)

L. usitatissimum plant was carefully harvested from soil after 45 days and then to remove the soil
particles from root surface ,plant roots were washed with deionized. The roots were then placed
in 20 mM solution of sodium EDTA for 15 minutes metals from root surface should be removed.
Afterwards, plant root and shoot length was assessed.

10.2) Fresh weight of Linum plant

After harvesting plants fresh weight was measured by using digital balance.

10.3) Determiantion of photosynthetic pigments in plant

Chlorophyll and carotenoid contents of leaves were determined by the method of(Arnon, 1949).

Total chlorophyll (a,b) and carotenoid content was calculated by means of following
equation(Arnon, 1949)

Chl (a ) (mg/g) = [12.7 (OD663)-2.69(OD645)] ×V/1000×W

Chl (b) (mg/g) = [22.9 (OD645)-4.68(OD663)] ×V/1000×W

V = Volume of the extract (ml), W = Weight of fresh leaf tissue (g)

Carotenoids (mg/g): (OD at 480) x 4

10.4) Antioxidant activity of enzymes

POD activity

Peroxidase activity was estimated by the process of (Reddy et al., 1985).

The change in optical density per minute at 430nm was taken as one unit of peroxidase

. POD =Final reading –initial reading

SOD activity

Superoxide dismutase assay of plants was estimated by the process of (Beauchamp and
Fridovich, 1971)

Following formula was used for calculating SOD Activity.

R1- O.D of Reference, R2-O. D of Blank, R3- O.D of Sample

R4 = R3-R2

A= R1 (50/100)

Final= R4/A
10.5) Determination of proline content of Linum plant

The proline content of leaves was estimated by the method of (Bates et al., 1973). Fresh plant
material (0.1g) was taken and grinded in 4mL aqueous solution of sulfosalicylic acid (3%).
Then,Centrifuge the reaction mixture at 3000rpm for 5min. Then, 2mL of supernatant was taken
and mixed with 2mL of acid nin-hydrin solution.At 100℃ placed the test tubes in water bath for
1h .Then allowed the sample to cool down and by adding 4mlof toluene reaction mixture was
extracted . At 520nm absorbance was observed by using toluene as blank. Proline contents of
leaves were calculated by using following formula.

Proline contents = k × dilution factor × absorbance/ sample weight

Where, K =19.6

Dilution factor=4

Sample weight=0.1

11)METAL ANALYSIS OF PLANTS BY WET ACID DIGESTION

METHOD.

Metal analysis of plants was determined by the method of (Xin et al., 2012). Heavy metals were
analyzed through atomic absorption spectrophotometer AAS model spectra AA Varian (Achakzai
et al., 2017).

12) STATISTICAL ANALYSIS

The data obtained in study was examined statistically by using statistic software (STATISCA
VERSION 8.1). ANOVA was used to determine the important differences between the mean based
on Fisher ̓s least significant difference (LSD) method.
RESULTS
3.1 PHYSIOCHEMICAL AND NUTRIENT PROPERTIES OF SOIL
Physiochemical and nutrient properties of soil are given in (Table 1). Soil texture of non-
contaminated soil was Loamy whereas; soil texture of industrial contaminated soil was Loamy
plus sandy. pH of the contaminated soil was 6.35 which are slightly acidic in nature whereas PH
of industrial contaminated soil was 7.60 which was alkaline in nature. EC values were 0.005and
0.015(dsm-1) respectively. Organic matter present in non-contaminated soil was 0.74 whereas in
industrial contaminated soil was 0.92. Available nitrate was 7.24 ppm in non-contaminated and
5.32 ppm in industrial contaminated soil respectively. Available potassium was 109 ppm and 63
ppm in non-contaminated and contaminated soil. Available phosphorus was 7ppm and 12ppm in
non-contaminated and industrial contaminated soil respectively. Atomic absorption for the
analysis of heavy metals in both soils showed that there was a higher concentration of metals
such as Cr, Cd, Mn, Cu and Zn in industrial contaminated soil as compared to non-contaminated
soil.

3.2 HEAVY METAL TOLERANCE

Heavy metals tolerant bacteria were grown on nutrient agar supplemented with different
concentrations of heavy metals such as Cr, Cd, Ni, and Cu. Stress concentration for bacterial
screening was started from 50mg/L to maximum 1000mg/L(Table 2). Ten bacterial strains were
screened at different heavy metal stress concentrations. Among these ten bacterial strainsPM14,
and PM11 showed maximum growth at 1000mg/L concentration of Cr and Cd respectively. These
two bacterial strains CM21 (Bacillus xiamensis) 11A (Bacillus gibsonii) were further used for in-
vitro experiment.

Plant Growth Promoting Traits

A ) ACC Production
In Qualitative screening to check the ability of strains to produce ACC enzyme, strains
were grow on agar plates either supplemented with Ammonium Sulfates, DF medium and ACC.
Ammonium sulfate was taken as a positive control and plates with DF medium taken as a negative
control. Growth was observed on all plates by comparing plates of positive and negative controls.
Plates supplemented with ACC show variation in growth patterns of all strains. All strains show
positive results for ACC production which are shown in (Plate 1.)

To check total amount of enzymes production by bacterial isolates quantitative screening was
performed. The activity was observed in Chromium stress conditions (50mgL-1). All strains
showed production of ACC between ranges of 0.63-0.91 µM/mg protein /h (Fig 1). Among these
ten strains three strains showed significantly higher activity of ACCD such as PM20, PM14 and
PM15.

B) Exopolysaccharides Production
Exopolysaccharide production is an important feature of stress tolerant bacteria. Bacteria
tolerate stress by producing biofilm around their colonies. ATCC No. 14 medium was used to
check qualitative production of exopolysaccharide by bacterial isolates. All strains are positive for
exopolysaccharide production (Plate 2).

All strains were Quantitievly screened for Exopolysaccharide production. All strains
expect two (33A andPM16) shows higher production of Exopolysaccharide ranging between 0.5-
0.6 mg/ml (Fig 2). While 33A and PM16 shows dry matter EPS production up to 0.4 mg/ml.
Statistical analysis shows there is no significant difference between production of all bacterial strains expect
two (33A andPM14).

C) IAA production

Ten bacterial strains were, Qualitativeily Screened for indole acetic production in both
stress and non-stress conditions .In non-stress condition out of ten strains four strainsPM12,
PM18,PM16,and 23A shows ring formation in tubes after adding salkowaski reagent. While in
Chromium stress conditions four strainsPM12, PM16, PM14 and PM20shows ring formation after
treating with salkowaski reagent .

Whereas in Quantitative Screening Production of IAA was measured both in stress and non-stress
conditions. Strain 33C produce IAA equally in both stress and non-stress conditions. Statistically
production of IAA in both treatments is same(Fig 3). However other treatments produce different
concentration of IAA in both conditions e.g. 33D, PM19, PM18, PM11, PM16 Produce greater
quantity of IAA in non-stress conditions whilePM20, 23A, PM15and PM14 Secrete large quantity
of IAA in stressed conditions.

D) Nitrogen Fixation

Nitrogen fixation is an important feature of plant growth promoting bacteria. Ten bacterial
strains were checked for their ability to fix nitrogen on DF media. All strains showed positive
results for nitrogen fixation(Plate3).

Plant Growth and Biomass

After 45days plant was carefully harvested (Plate 4) to check the plant growth and biomass. Result
revealed that in stress and in non-stress condition inoculated Linum plant show one-fold greater
growth as compared to its non-inoculated stress and non-stress plant.There is a slightly difference
in growth pattern of Linum PGPR inoculated plant as compared to its non -PGPR-inoculated plant.
As PGPR inoculated plant increase the growth of linum up to a certain percent. In non-stress
condition inoculation of B. xiamensis increased the growth of shoot up to 56% and growth of root
up to 73%,inoculation of B. gibsonii enchanced the shoot growth(Fig 4) up to 84% and root growth
(Fig 5) 103% as compared to their non-PGPR inoculated non-stress control .Similarly heavy metal
stress decreased shoot and root length in linum plant while treatment with heavy metal tolerant
PGPR enchanced shoot and root in both treatments.B. xiamensis increased 43% shoot length and
53% root length of linum in stress condition. Whereas inoculation of B. gibsonii enchanced 52%
shoot and 69% root length in heavy metal stress condition.

Fresh weight of Linum

Results showed that there is an increase in Fresh weight of PGPR inoculated plant as
compared to their non-inoculated plant. In non-stress condition B. xiamensis increased fresh
weight of Linum up to 36% and inoculation of B. gibsonii increased fresh weight of plant up to
40% as compared to their non-PGPR non-stress plant (Fig 6 ). Moreover fresh weight of plant
decrased in heavy metal stressed plant but when Linum plant is treated with PGPR under stress
condition fresh weight of Linum enchanced as compared to its respective control. It was noticed
that inoculation of B. xiamensis increased frseg weight up to 87% and B gibsonii enchanced fresh
weight up to 111% respectively.
 Photosynthetic pigment contents

Result showed that in PGPR inoculated non-stress linum plant chlorophyll a ( chl a )
,chlorophyll b ( chl b ) and carotenoids content of Linum plant increased as compared to their non
PGPR inoculated non-stress plant.Inoculation of B. xiamensis increased chlorophyll a ( Fig 7) up
to 155% , chlorophyll b ( Fig 8) up to 60% and carotenoids content of plant up to 114%
(Fig9)Whereas inoculation of bacterial strain B. gibsonii in non- stress condition increased
chlorophyll a content 164% , chlorophyll b up to 98% and carotenoids content of plant up to 109%
as compared to their respective control in non-stress condition.

In heavy metal stress condition photosynthetic pigment of plant decreased but when Linum
plant is treated with PGPR in heavy metal stress condition chlorophyll a , chlorophyll b and
carotenoid contents of Linum plant increased.B. xiamensis increased chlorophyll a , chlorophyll b
and carotenoid of Linum plant respectively 62%, 91% and 34% as compared to their non-
inoculated stressed plant.Inoculation of B .gibsonii increased chlorophyll a 70% ,chlorophyll b
116% and carotenoid 94% respectively.

Activities of antioxidant enzymes ( SOD and POD)

PGPR inoculation enhanced production of enzymes as compared to un-inoculated control in

non- stress condition. Statistical analysis showed that B .xiamensis increased SOD and POD
activity up to 179% and 165% in non-stress condition whereas B. gibsonii showed 186 % increased
in SOD activity as compared to their control and 97% POD as compared to their respective control.

In heavy metal stress conditions plants ability to produce SOD ( Fig 10) and POD (Fig 11)
enzymes was reduced while plants inoculated with heavy metal tolerant strains shows greater
production of SOD and POD as compared to non-inoculated plants. Bacterial strain such as B.
xiamensis increased SOD activity up to 76% and B. gibsonii 411% respectively. Whereas POD
activity increased by inoculation of B. xiamensis was 98% and by inoculation of B. gibsonii was
25% as compared to it control. Hence, PGPR increased the production of enzymes under heavy
metal stress condition.

Effect of Heavy metal Tolerant Strains on Proline Content of Linum plant in non-stress
condition and under Heavy metal stress condition.

Non-inoculated non-stressed plant showed low amount of proline content as compared to

inoculated non-stressed plant. Inoculation of PGPR in non-stressed condition increased the
proline content of Linum. B. xiamensis increased 56% proline content and B .gibsonii increased
63% in non-stressed condition.

Linum plant couldn’t produce much proline in heavy stress condition but there is a significant
production of proline in PGPR inoculated stressed plant (Fig 12).When Linum plant was treated
with B .xiamensis it increased its proline content upto 120% as compared to its control.And when
Linum plant is treated with B. gibsonii it increased its proline content upto 100% as compared to
its non-PGPR stressed Linum plant.

ANALYSIS OF PLANT FOR UPTAKE OF HEAVY METALS

Heavy metal analysis of whole plants of Linum grown in industrial contaminated soils revealed
Linum plant showed comparatively better growth (Table 3). It was also noted that un-inoculated
Linum plants showed more phyto-extraction potential of heavy metals as compared to non-
inoculated. Bacterial strain B. xiamensis and B. gibsonii enhance the phyto-extraction of all heavy
metals in Linum plant. This revealed that along with enhanced growth of Linum plant bacterial
strains can also help to remediated contaminated soils via enhanced phyto-extraction of toxic
metals from soil.
Discussion
In soil one of the main cause of pollution is residues of heavy metals into the soil from industries.
Heavy metals have high acidification levels , high trace metal contents and very low organic matter
and nutrient availability therefore, heavy metals act as antagonistic for plant growth(Becerra-
Castro et al., 2012).Mining residues showed very high contents of Pb,Mn,As,Cd and Ni (Martínez
et al., 2016). And these heavy metals are very harmful for biochemical activities.Heavy metals
present in soil can be modify, mobilize and immobilize by plant growth promoting
bacteria(Ahemad, 2014). PGPR also prevent the entry of heavy metals into a plant by marks the
heavy metals to remain in the soil(Glick et al., 1998; Rajkumar et al., 2012).Through various
mechanisms of PGPR growth of the plant increase e.g production of IAA, nitrogen fixation,
phosphate solubilization, production of ACC deaminase and also by limiting the growth of several
microorganisms(Ahemad, 2015; Glick, 2010) The present study was conducted to identify heavy
metal tolerant strains which not only reduce toxic effects of heavy metals but also increase plant
growth in industrial contaminated soil. Ten strainsPM20, 33D, 33C, PM18, PM14, PM15, PM19,
23A, PM16 and PM11 previously isolated from sugarcane were used in this study.All ten strains I
have selected have the ability to produce ACC,IAA and Exoploysaccharide production in the
presence of heavy metal stress increase in plant growth may be due to production of
Exopolysaccharides, ACC or IAA. But out of these ten strains the most competent PGPR strains
were (PM14, Bacillus xiamensis and, PM11, Bacillus gibsonii). These strains were further used to
analyze their in-vitro potential (Ahemad, 2015) reported the production of IAA and ACC
deaminase in the presence of heavy metals which matches with the findings of ours. By appling
PGPR in stress condition plant root length, shoot length and fresh weight increaseswhich matches
with the findings of (Marques et al., 2013) where some bacteria are able to increase H. annulus
plant biomass in Pb and Zn mining residues.

Taking into account the information above, Bacillus xiamensis, Bacillus gibsonii may have
a potential use as bio- incubator in phytoremediation processes, since it may aid in the exogenous
IAA production that along with endogenous. IAA production may promote cell proliferation and
elongation as well as induction of ACC synthase to increase ACC production (Kende, 1993),
which is used by ACC deaminase to decrease ethylene production (Glick et al., 1998). As, the
concentration of heavy metals increased in soil growth and biomass of plants decreased. And, this
decreased is due to the change in the plant tissues which is also reported by (Ali et al., 2013; Gill
et al., 2015) that in heavy metal stress conditions plant growth has been decreased.

It has been found that in industrial contaminated areas, there is higher concentration of
heavy metals are present due which shoot length of plant growing in that area reduced (Faisal and
Hasnain, 2005).Application of plant growth promoting rhizobacreria such as B. xiamensis and B.
gibsonii increased the shoot length of plant.As PGPR reduced the uptake of hevy metal and
increase the uptake of nutrients from contaminated soil. Our results confirmed the previous results
(Paungfoo‐Lonhienne et al., 2014; Xin et al., 2009) that inoculation of PGPR increased the plant
growth and shoot length.

In present study, it was observed that fresh weight of plant was low in stressed plant as
compared to plants inoculated with PGPR. PGPR increased the fresh weight of plant in stress
condition. Under heavy metal stress condition decreased in fresh weight and biomass of the plant
is due to obstruction of different physiological procedures and decrease in the production of
enzymes. Excess amount of ROS abolish metabolic activities of the cell results in decrease growth
and reduction of plant biomass.In heavy metal contaminated soil, plants and microbes interaction
increase the phytoremediation ability of soil (Weyens et al., 2009).

Under a heavy metal stress condition Chlorophyll content was decreased in Linum plant.
Due to heavy metal stress decrease in Chlorophyll content was also reported by (Gill et al., 2015;
Singh, 2013).Heavy metal stress decreased Chlorophyll content, blocked gas exchange factors
(Ali, Khan et al. 2013) blocked photosynthetic pigments and alter ultra-structure(Gill et al., 2015)
in plants.It was also observed that in heavy metal stress condition ROS species decrease
chlorophyll contents of plants (Ehsan et al., 2014).

In present day it was found that Chlorophyll (a,b) content of Linum plant increased by
appling PGPR such as B. xiamensis and B .gibsonii . This increased in photosynthetic pigment is
due to ability of plant growth promoting rhizobacteria to mobilize and solubilize the organic
minerals (Zayed and Terry, 2003) mostly nitrogen which is the important component of the
chlorophyll (Swan, 1971)which resulted in the increased chlorophyll contents(Bojović and
Marković, 2009). In non-inoculated stressed plant there was decreased in Chlorophyll content was
observed.
 In stress condition reduction in carotenoid was formerly stated in different plants (Ali et
al., 2013). Decrease in carotenoid content may be danger to the plant because during photo-
oxidative destruction carotenoid protects the plants (Middleton and Teramura, 1993). In present
study, Linum plant when treated with PGPR in stress conditions it enhance carotenoid content of
the plants as compared to non-inoculated stressed plants.

In present day study it was found that SOD and POD activity of plant decreased in heavy metal
stressed condition .By appling plant growth promoting rhizobacteria B. xiamensis and B. gibsonii
enchanced the activity of SOD and POD

The present study revealed that, in contaminated soil plant could be protected from the
effects of different heavy metals by the application of plant growth promoting bacteria. PGPR
reduce the uptake of metals and increase the uptake of nutrients and minerals which are essential
to plants growth and biomass. According to the result of (Ullah et al., 2015) who reported that
inoculation of Burkholderia sp increased growth of the plant, similarly our outcome result shows
that the growth of the Linum increased in PGPR inoculated stressed plants whereas in stress
condition growth of the Linum plant decreased. Hence, these PGPR can be used as potent tool to
decease the toxicity of metals in contaminated soil in order to increase the production of plant in
industrial contaminated areas.

CONCULSION
Current studies shows that growth of L. usitatisimum L. increased under normal soil conditions
due to PGPR. These bacteria also enhance growth of the plant even under heavy metal stress
conditions due to production of ACC deaminase, IAA, EPS and nitrogen fixation ability of PGPR.
It has been minutely observed and concluded in latest studies that by the application of plant
growth promoting bacteria B. xiamensis and B. gibsonii enhance phytoextraction potential of
Linum plant. It is concluded from results, PGPR inoculated plants are capable and strong enough
to endure under high concentration of heavy metals. Hence, linum inoculated with PGPR are very
useful to amend the toxicity of heavy metals. It is also beneficial to bring back soils and regions
for productivity.
 1.2

ACC um/mgprotein/h
1 a a
ab ab a
ab
0.8 b b ab
ab
0.6

0.4

0.2

0
FSA3 33* 33A FSD5 CM21 9B FAS11 23A B 11A
Strains

Fig 1= Quantitative production of ACC by Bacterial Isolates

0.7
a a a a a a a a
0.6
Dry Matter EPS mg/ml

0.5 b
b
0.4

0.3

0.2

0.1

0
FSA3 33* 33A FSD5 CM21 9B FSA11 23A B 11A
Srains

Fig 2=. Quantitative Production of Exoploysacchrides producing Bacterial isolates

 300 Non stressed stressed
I
Production of IAA (µMml-1)
250

200

GHI
150

GHI A
100
EFGHI DEF
CDE HI
GHIB HI C FGHIHI
50 EFG I A EFGH
HI CD

Bacterial strains

Fig 3=. Quantitative Production of IAA

Non-Contaminated soil Industrial Contaminated soil

18
AB
16
SHOOT LENGHT ( cm )

A
14 BC
C
12
10 BC D
8
6
4
2
0
C T1 T2
TREATMENTS

Figure 4. Effects of plant growth promoting bacteria B. xiamenensis PM14 and B.gibsonii PM11
Shoot length of L. usitatisimum in both non-contamiated soil and contaminated soil, ,Whereas C=
Control, (Non-contamiated soil and Industrial Contamiated soil), T1= B. xiamenensis PM14 innon-
contaminated soil and industrial Contaminated soil, T2= B.gibsonii PM11 in non-contaminated soil
and industrial Contaminated soil.
 Non-Contaminated soil Industrial Contaminated soil

10
A
9
ROOT LENGHT ( cm )

8 A A
A
7
6 A
A
5
4
3
2
1
0
C T1 T2
TREATMENTS

Figure5. Effects of plant growth promoting bacteria B. xiamenensis PM14 and B.gibsonii PM11
Roottlength of L. usitatisimum in both non-contamiated soil and contaminated soil, ,Whereas C=
Control, (Non-contamiated soil and Industrial Contamiated soil), T1= B. xiamenensis PM14 in
non-contaminated soil and industrial Contaminated soil, T2= B.gibsonii PM11 in non-contaminated
soil and industrial Contaminated soil.

Non-Contaminated soil Industrial Contaminated soil

1.4
A
1.2 B
1
FRESH WT/G

0.8
D
0.6 B B
0.4 C

0.2
0
C T1 T2
TREATMENTS

Figure 6. Effects of plant growth promoting bacteria B. xiamenensis PM14 and B.gibsonii PM11
Fresh weight of L. usitatisimum ,,Whereas C= Control, (Non-contamiated soil and Industrial
Contamiated soil), T1= B. xiamenensis PM14 in noncontaminated soil and industrial
Contaminated soil, T2= B.gibsonii PM11 in non-contaminated soil and industrial Contaminated
soil.
 Non-Contamiated soil Industrial Conatmianted soil
0.007 A
B
CHLOROPHYLL a (mg/g)

0.006

0.005 D C

0.004
E
0.003 F
0.002

0.001

0
C T1 T2
TREATMENTS

Figure 7. Effects of plant growth promoting bacteria B. xiamenensis PM14 and B.gibsonii PM11 on
Chlorophyll a content L. usitatisimum ,Whereas C= Control, (Non-contamiated soil and Industrial
Contamiated soil), T1= B. xiamenensis PM14 in non-contaminated soil and industrial
Contaminated soil, T2= B.gibsonii PM11 in non-contaminated soil and industrial Contaminated
soil.
 Non-Contaminated soil Industrial Contaminated soil
0.004
0.0035 A
CHLOROPHLL b (mg/g)

B
0.003
C
0.0025
D
0.002 E
0.0015 F
0.001
0.0005
0
C T1 T2
TREATMENTS

Figure 8.Effects of plant growth promoting bacteria B. xiamenensis PM14 and B.gibsonii PM11 on
Chlorophyll b content of L. usitatisimum,Whereas C= Control, (Non-contamiated soil and
Industrial Contamiated soil), T1= B. xiamenensis PM14 in non-contaminated soiland industrial
Contaminated soil, T2= B.gibsonii PM11 in non-contaminated soil and industrial Contaminated
soil.

Non-Contamianted soil Industrial Contaminated soil

6 A
B
C
CAROTENIODAS( mg /g)

4 E

3 D F

0
C T1 T2
TREATMENTS

Figure 9. Effects of plant growth promoting bacteria B. xiamenensis PM14 and B.gibsonii PM11 on
Caroteniod content of L. usitatisimum in both non-contamiated soil and contaminated soil,,Whereas
C= Control, (Non-contamiated soil and Industrial Contamiated soil), T1= B. xiamenensis PM14 in
non-contaminated soil and industrial Contaminated soil, T2= B.gibsonii PM11 in non-contaminated
soil and industrial Contaminated soil.
 Non-Contamianted soil Industrial Contamintaed soil

1.4 A
A A
1.2
SOD ACTIVITY

1
0.8
0.6
B B
0.4
C
0.2
0
C T1 T2
TREATMENTS

Figure 10. Effects of plant growth promoting bacteria B. xiamenensis PM14 and B.gibsonii PM11
on SOD activity of L. usitatisimum in both non-contamiated soil and contaminated soil,,Whereas C=
Control, (Non-contamiated soil and Industrial Contamiated soil), T1= B. xiamenensis PM14 in
non-contaminated soil and industrial Contaminated soil, T2= B.gibsonii PM11 in non-contaminated
soil and industrial Contaminated soil.

Non-Contamianted soil Industrial Contamianted soil

0.12

0.1 A
POD ACTIVITY

0.08 AB

0.06 BC

0.04 BC
C C
0.02

0
C T1 T2
TREATMENTS

Figure 11. Effects of plant growth promoting bacteria B. xiamenensis PM14 and B.gibsonii PM11
on POD activity of L. usitatisimum in both non-contamiated soil and contaminated soil, ,Whereas
C= Control, (Non-contamiated soil and Industrial Contamiated soil), T1= B. xiamenensis PM14 in
non-contaminated soil and industrial Contaminated soil, T2= B.gibsonii PM11 in non-contaminated
soil and industrial Contaminated soil.
 Non-Contaminated soil Industrial Contaminated soil
500
A E
450
PROLINE CONTENT ug/g

400 B
D
350
300 C
250
200 F
150
100
50
0
C T1 T2
TREATMENTS

Figure 12. Effects of plant growth promoting bacteria B. xiamenensis PM14 and B.gibsonii PM11
on Proline content of L. usitatisimum in both non-contamiated soil and contaminated soil, ,Whereas
C= Control, (Non-contamiated soil and Industrial Contamiated soil), T1= B. xiamenensis PM14 in
noncontaminated soil and industrial Contaminated soil, T2= B.gibsonii PM11 in non-contaminated
soil and industrial Contaminated soil.
Table 1; Physiochemical and nutrients properties of soil

Soil parameters Soil 1 (Non- Soil 2 (Contaminated)

contaminated)

Soil EC(dsm-1 ) 0.005 0.015

Soil pH 6.35 7.60

Soil texture Loamy Sandy loam

Organic matter 0.74 0.92

Nitrate-Nitrogen(ppm) 7.24 5.32

Available k(ppm) 109 63

Available p (ppm) 7 12

Cd(ppm) 0.005 0.681 11.739

Cr(ppm) 0.869 128.15

Mn(ppm) 0.569 125.25

Zn(ppm) 0.249 90.55

Cu(ppm) 0.075 5.78

Table 2;Screening of bacterial strains at different concentration of heavy metals
Bacterial Strains Cr mg/L Ni mg/L Cd mg/L Cu mg/L

CM21 1000 150 1000 100

9B 1000 150 1000 100

B12 700 150 1000 -

11A 1000 150 700 50

33C 700 150 500 50

33CD 700 150 500 100

23A 700 150 500 50

FSD5 50 - 50 -

FSA11 150 150 150 100

FSA3 50 - 150 -
Table 3; Role of PGPR in differential uptake of Heavy metals from contaminated industrial
soil

Treatments Zn Mn Cd Cr Cu Ni Pb

Linum

C 12.4± 0.01 11.7± 0.23 6± 0.15 16.7± 0.28 16.1± 0.13 20± 0.41 56± 0.02

T1 38.17±0.57 33.9± 0.47 10.5± 0.19 18.9± 0.37 23.1± 0.43 53± 0.19 86± 0.06

T2 22.58±0.51 17± 0.36 14.8±0.08 35.3± 0.81 31.4± 0.08 48.8± 0.30 85± 0.19

Bacterial strain B. xiamensis and B. gibsonii enhance the phytoextraction of all heavy metals in
Linum plant.

Plate 1 A (ACC deaminase activity of ten bacterial strains ) B ( ACC supplemented DF medium)
C (Nitrogen rich ammonimum sulphate medium)
Plate 2 Production of Exopolysaccharide by different bacterial strains
Plate 3 Production of by different bacterial strains on nfb media
Plate 4 Effect of PGPR on L. usitatissimum

Plate 1 A: (Effect of plant growth promoting bacteria on L. usitatissimum L. in industrial

contaminated soil)
B: (Effect of plant growth promoting bacteria on Linum usitatissimum L. in non-contaminated
soil)