ISOLATION in the occurrence of heavy metals,

ISOLATION
OF ACC DEAMINASE PRODUCING Pseudomonas
sp. FROM TEXTILE EFFLUENT

 

1.INTRODUCTION

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    Microbial
populations in heavy metal polluted environments contains microorganisms which
have been adapted to toxic concentrations of heavy metals and become resistant
to metal. Such microorganisms have developed diverse mechanisms for survival in
the occurrence of heavy metals, and acquired genetic properties that counteract
the effects of high metal ion concentrations. The use of heavy metal resistant
microorganisms for the decontamination of heavy metals from contaminated water
and soil has attracted growing attention because of several problems associated
with pollutant removal using conventional methods and this pollution cause the
soil bacteria to tolerate or adapt the condition and help the plants in good
manner.

    Many industrial practices
release toxic heavy metal ions in the environment, such practices include direct
application of industrial effluents that may contain high concentrations of
heavy metals to pond, river, agricultural land, or irrigating agricultural land
with untreated wastewater. Accumulated heavy metals in the environment
constitute potential health hazards to humans, harm to living resources and
ecology. Heavy metals including cadmium, lead, zinc, mercury, copper, cobalt
and nickel, which act as soluble compounds or exchangeable elements represent a
risk of toxicity depending on the rate of transfer from polluted areas to soil
solution, plants, ground water, soil microflora and to the food chains. the
textile effluents released from the industries can directly introduced into
rivers and lands which leads to toxic of the soil as well as eutrophication
happens in the water bodies due to excess nutrients availability.

    It
was reported that a small number of soil Pseudomonas possess the enzyme
1- aminocyclopropane 1 carboxylase (ACC) deaminase (Klce el al.,1991)
this enzyme cleaves ACC, the immediate biochemical precursor of ethylene in
plants, to ammonia and ? – ketobutyrate (Honma and Shimomura 1978). The use of
the enzyme by Klec el al., (1991) to alter gene expression in transgenic
plants carrying a functional ACC deaminase gene by lowering ethylene levels
prompted us to question whether plant growth promoting rhizobacteria (PGPR)
such as Pseudomonas putida (Lifshitz et al., 1987) might also
posses ACC deaminase activity and if they did, whether this enzyme was somehow
involved in the promotion of plant growth by these PGPR. Some of these strains
were able to produce the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC)
deaminase and the plant growth regulatory hormone indole-3-acetic acid (IAA).
Some strains were also able to chelate ferric iron and solubilize potassium,
phosphorus and zinc, and produce ammonia.

    The important drawback in agricultural is
the ripening of the fruits as early age by the ethylene gas which is naturally
produce by plants when is needed. Ethylene is a plant aging hormone. It is
naturally occurring and produce with ripening .It is responsible for changing
texture, taste, colour, softening and other process in ripening. Recent studies
suggest two different strategies used by Rhizobia to reduce the amount of
ethylene synthesized by their legume symbionts. One strategy utilizes the
compound through rhizobitoxine which acts to inhibit the enzyme ACC synthase and
hence ethylene biosynthesis. In addition, the enzyme
1-aminocyclopropane-1-carboxylate (ACC) deaminase which catalyzes the cleavage
of ACC to ?-ketobutyrate and ammonia decreases ethylene levels in host roots
and thereby enhances nodulation (Ma et al. 2003a; Ma et al. 2004).

1.1 Plant growth promoting bacteria

    Plant
growth promoting bacteria have a positive influence n plant growth and
development. Many of the bacteria found in soil are bound to the surface of
soil particles and are found in soil aggregates and interact specifically with
the roots of plants. The interaction between bacteria and the roots of plants
may be beneficial, harmful or neutral for the plant and effect of a particular
bacterium may vary as a consequence of soil conditions. For example, a
particular organism that facilitates plant growth by fixing nitrogen, which is
usually present in the soil in limited amounts, is unlikely to provide benefit
to a plant in a setting where exogenous fixed nitrogen is added to soil.

    PGPR can promote plant growth and development by indirect or direct
means (Glick et al. 1999; Nelson 2004). “Direct mechanisms can be demonstrated
in the absence of plant pathogens or other rhizosphere microorganisms, while
indirect promotion of plant growth involves these bacteria reducing he
deleterious effects of plant pathogens” (Nelson 2004). There are several ways
in which plant growth-promoting bacteria can directly enhance plant growth and development
(Glick 1995). For example, they chelate irons by producing siderophore,
solubulizes minerals, fix atmospheric nitrogen, produce hormones, and enzymes
which can enhance or inhibit the plant growth and development.

    PGPR (Plant growth promoting rhizobacteria)
can affect plant growth in two different ways, indirectly or directly. The
indirect promotion of plantgrowth occurs when PGPR lessen or prevent the
deleterious effect of one or more phytopathogenic organisms. The direct promotion
of plant growth by PGPR for the most part entails either providing the plant
with compound that is synthesized by the bacterium or facilitating the uptake
of certain nutrients from the environment.

 

2.REVIEW
OF LITERATURE

   2.1 Isolation of Pseudomonas fluorescence

    Microorganisms
are generally found in nature as mixed populations. To study the specific role
played by a specific microorganism in the environment, it should be isolated as
pure culture. And the pure culture should be maintained.

    The
isolation and screening of PGPR by the production of phytohormones like IAA
(Auxin). In addition, most PGPR has ACC Deaminase enzyme that cleaves ACC which
is a intermediate precursor of ethylene synthesis, to ?-ketobutyrate and
ammonia thereby lowers the ethylene level. This enzymes only produce when the
bacteria under stress such as salinity, heavy metal.

     ACC
deaminase was  first discovered in bacteria.
Some plant growth-promoting bacteria are capable of processing the plant-borne ACC
by converting it into ammonia and ?-ketobutyrate using the enzyme ACC deaminase
(HonmaandShimomura,1978). ACC deaminase was retrieved. Pseudomonas sp. strain
ACP (Honma and Shimomura,1978), Pseudomonas chloroaphis  6G5 (Klee etal., 1991), Pseudomonas putida
 GR12-2 (Jacobsonetal.,1994) and Pseudomonas
putida UW4 (Hontzeasetal.,2004). ACC deaminase containing bacteria can
reduce stress susceptibility of plants during flooding (Barnawal et al., 2012;
Li et al., 2013), drought (Mayak et al., 2004a), salinity (Mayak et al., 2004b;
Nadeem et al., 2007, 2010), flower senescence (Nayani et al., 1998; Ali et al.,
2012), metal pollution (Glick, 2010), organic pollution (Gurska et al., 2009)
and pathogens (Glick, 2014 and references therein). In addition, it has been
reported that the presence of ACC deaminase can increase the symbiotic
performance of Rhizobial strains (Ma et al., 2003).

     Pseudomonas fluroscencs strains such as ps.
fuorescens Pf2, Ps. fluroscens TDK1 & RMD1 were isolated from soil
sample  and the efficacy of the bacteria
to promote plant growth under stress condition (Kumar D S 2006). Enterobacter cloacae,  Pseudomonas putida and Pseudomonas
fluorescens were isolated from the soil near the rhizosphere where have the
more ACC deaminase producing bacteria with ACC as a nitrogen source in the
media.(Donna m 2002).

     Pseudomonas isolation agar, is a selective
media of pseudomonas auroginosa. as
well as pseudomonas fluorescence was
isolated by pigmentation and biochemical characters. ACCD producing pseudomonas fluorescence was isolated
directly by adding ACC into the medium, where the ACCD producing bacteria grow
using ACC as nitrogen. IN most of the articles Kings B medium and Tryptic soy
broth were used as selective media for pseudomonas
fluorescence. instead of the selective media we can use the Pseudomonas
isolation agar  where pseudomonas fluorescence grow as a white
colourless pigment whille pseudomonas
auroginosa produce greenish yellow pigmentation on media.

 

2.2
Ethylene Biosynthesis

  
 Ethylene
was discovered as a plant regulator in e work done in dark green pea
seedlings  showed reduced growth of
hypocotyl (Neljubov 1901). Biosynthetic pathway of ethylene was came upon when
S-adenosyl-L- methionine (SAM) was an intermediate between methionine and
ethylene( Adams & Yang 1977). The major discovery that made the methionine
cycle in plants unique from all other organisms, was the characterization of
1-aminocyclopropane-1-carboxylic acid (ACC) as the intermediate between SAM and
ethylene (Adams and Yang, 1979). The identification of ACC as the precursor for
ethylene by feeding experiments on apple tissue, using radio-labeled methionine.

     As mentioned above, ACC is produced from
SAM, this reaction is catalyzed by the enzyme ACC-synthase (ACS; Boller et al.,
1979). The ethylene synthesisis carrued out by two major enzymes are ACC
synthase and ACC oxidase. ACS is a gene which is encoded by plant involved in
ethylene biosynthesis, encoes ACC synthase 
converts the SAM  into ACC ( Van
DER straeten et al., 1992). The second enzyme is ACC oxidase (ACCO)  acts 
in the presence of oxygen,  converts ACC into ethylene. and it was
isolated by addition of ascorbic acid (vitamin C) to the media ( Ververidis
& John 1991).

            

figure
1- Biosynthetic pathway of ethylene via intermediate precursors SAM and ACC.

2.3 ACC Deaminase

 
   ACC deaminase is an
enzyme which breaks down ACC. 
Specifically ACCDcleaves the cyclopropane ring of ACC and removes an
amino group to produce ?-Ketobutyrate and ammonia (Penrose and Glick, 2001).The
Enzyme has been only found in microorganisms. Yeast and plant growth promoting
bacteria have been studied so far.

     ACC
deaminase production may be helpful in the nodulation process and thereby
increase the nitrogen supply for legume plants due to a more effective
nodulation. This may be especially important when plants are growing under
stressful conditions so that ethylene may attain levels that are highly
inhibitory to nodulation(Nascimenti et al., 2016).

     It can
reduce ethylene generation in plants; in this way they can reduce the extent of
growth inhibition caused by high-level ethylene, particularly under abiotic
stresses (Glick, Penrose and Li 1998). ACCD-mediated plant growth promotion has
stimulated tremendous interest in research into the isolation and application
of ACCD-producing bacteria (Glick et al. 2007, 2014).

 

2.3 ACC deaminase enzyme activity assay

     The
assessment of bacterial ACCD activity requires growth conditions with the
induction of ACC deaminase. Activity of ACCD was assayed by measuring
?-ketobutyrate and ammonia, the hydrolysis product of ACC (khan A L 2016).
In
a series of known ?-ketobutyrate concentrations, 2 mL of the
2,4-dinitrophenyl-hydrazine reagent (0.2% 2, 4-dinitrophenyl-hydrazine in 2 moL
L-1 HCl) was added, the contents were vortexed and incubated at 30°C for 30
min, during which the ?-ketobutyrate was derivitized as aphenylhydrazine. The
color of phenyl hydrazine was developed by the addition of 2 mL, 2 moL L-1 of
NaOH, the absorbance of the mixture was measured after mixing by using
spectrophotometer at 540 nm.( khan A L 2016).

     The level of ACC deaminase activity that
is observed when strain P. putida GR12-2 is grown on minimal medium plus
ammonium sulfate represents a basal level of activity of ?5% of the total activity that is
measured in extracts grown on minimal medium containing ACC (as a nitrogen
source) instead of ammonium sulfate. A few other amino acids including L-alanine,
DL-alanine and DL-valine can also induce ACC deaminase enzyme activity, albeit
to a limited extent, while  ?-aminoisobutyric
acid can induce activity to nearly the same level that is found with ACC.
Optimum pH and temperature for activity is 8.0 and 30?C respectively (Shimomura
and Honma, 1978). ACC deaminase requires pyridoxyl phosphate as a cofactor
which represents the aminotransferase family of enzymes. It is also reactive
with other D-amino acids but is competitively inhibited by L-serine (Penrose
and Glick).

2.4 Objectives

    However,
most researchers has focused on the regulation of biosynthesis of ethylene. The
existence of ACC deaminase in soil bacteria has been discovered, it was assumed
that may play an important role to reduce growth inhibition caused by
high-level ethylene, particularly under abiotic stresses. ACCD producing pseudomonas sp. were identified as efficient strain
in pseudomonads thus the objectives are

 1. To isolate ACC Deaminase  producing Pseudomonas
sp from the textile effluent.

 2. To characterize Psuedomonas fluorescens
isolates from their morphological and biochemical properties.

 3. To assess the activity of the enzyme by
calorimetric assay.

 4. To sequence the 16S rRNA of pseudomonas to
assign the species.

 

3.MATERIALS
AND METHODS

 MATERIALS AND METHODS

  3.1. Sample

    Textile
effluent was collected from the textile industry, kerala; stored at 4?.

  3.2 Isolation of pseudomonas fluorescens

     For
isolation, 1 mL of effluent was diluted in the range of 10-1 to 10-6.  10-2  and 10-3  spread on sterilized pseudomonas isolation
agar (Peptic digest of animal tissue 20.0g, Magnesium chloride 1.4g,
Potassium sulphate 10.0g, Irgasan 0.025g, Agar 13.6g per litre).
Incubated at room temperatures for 24-48 hrs. The plates were examined daily
upto 3 days for visible colonies. Pure culture were obtained by the streak
plate methed.

3.3 Characterization of
isolate.

3.3.1. Morphological
characterization

    Morphological characteristics
of the colony of  isolate were examined
on

Pseudomonas  isolation agar medium. Cultural
characterization of  isolates observed by
different characteristics of colonies such as shape,  size, surface, margin, colour, pigmentation
etc., were recorded.

3.3.2 Gram’s staining

    A drop of sterile
distilled water was placed in the center of glass slide. A loopful

of inoculum from
young culture was taken, mixed with water, and placed in the center

of the slide.
The suspension was spread out on slide using the tip of inoculation needle

to make a thin
smear. The smear was dried in air and fixed through heating by

passing the
slide 3 to 4 times over the flame. The smear was then flooded with Crystal

violet solution
for 1 min and washed gently with flow of tap water. Then the slide was

flooded with
Iodine solution. After incubation at room temperature for 1 min, Iodine

solution was
drained out followed by washing with 95% decolorizer. After that, it was

washed with
water within 15 to 30 sec and blot carefully. The smear was incubated with

Safranin  solution for 1 min. The slide was washed
gently in flow of tap water and dried

in air. The
slide was examined under microscope at 100X power with oil immersion and

data was
recorded.

3.3.4
Biochemical characterization

    Motility,
IMViC, catalase, oxidase, nitrate reduction, gelatin hydrolysis were performed
to characterize the isolates and recorded.

3.3.5 ACC deaminase activity
assay

    ACC
deaminase activity was determined for 
isolated strain according to the protocol described by Penrose and Glick
(2003) with a standard curve of 
?-ketobutyrate between 0.1 and 1 µM. he number of µM of ?- ketobutyrate
produced were determined by comparing 
the absorbance at 540nm of the sample to the standard curve.10µM
?-ketobutyrate  used to generate standard
curve. Each in a series of known concentration of ?-keobutyrate  with 3 ml of 2,4- dinitroohenylhydrazine
reagent (0.2% of 2,4-dinitrophenylhydrazine in 2M HCl) is added, votexed and
incubated at 30?C for 30 mins, during this time ?-ketobutyrate is derived as
phenylhydrazone. The colour is developed by the addition of 2ml of 2M NaOH, after
gently mixed the absorbance at 540nm is measured.

3.3.6 Induction of ACCD
and Bacterial extract

    The enzyme activity is measured in
bacterial extracts in the following procedure. The bacteria are cultured in
sutable rich medium and transferred to the minimal medium with ACC as the sole
nitrogen source. Bacterial culture are grown to mid to late log phase in 15 ml
of rich medium 10 µl of culture strain. Incubated at suitable temperature. The
contents are centrifuged at 8000g for 10 min at 4?C. The pelleted cells are
washed with DF ( Dworkin & Foster 1958) salts minimal medium per litre ( 4g
KH2PO4, 6g Na2HPO4, 0.2g MgSO4.7HO,
2g glucose, 2g gluconic acid,2g citric acid, 1mg FeSO4, 10g H3BO3,
11.1g MnSO4. H2O,  124.6g      ZnSO4.H2O,
78.22g CuSO4. 5H2O, 10g MoO3, pH 7.2
& (NH4)2SO4 as a nitrogen source). Centrifuged
at 8000g for 10 min at 4?C. The cells are suspended in 7.5 ml of DF salts
minimal medium with 45 µl of 0.5 M ACC ( 
to obtain final concentration of 3 mM). the tube was incubated in
shaking incubator for 24hr. the cells are collected by centrifugation at 800g
for 10 min at 4?C. The cells are washed by 0.1 M Tris-HCl,pH 7.6 for enzyme
assay and suspended in 0.03 M MgSO4 for Gonotobiotic root elongation
assay.

 

3.3.7 Gonotobiotic root
elongation assay

    This method is used as a method of assessing
bacterial strain to produce ACC deaminase by inhibiting ethylene which in turn
producing long roots. ACC deaminase producing sol bacteria av been isolated was
assayed by root elongation assay and shown to promote can alo seedlings ( Glick
et al., 1995, Belimov et al., 2001). The bacterial cell pellet was suspended in
0.5 ml sterile 0.03 M MgSO4 and placed on ice. A 0.5 ml sample was
remoed and diluted 8-10 times in 0.03 M MgSO4; the absorbance is
measured at 600nm. This measured is used to adjust the absorbance to 0.15 with
0.03 M MgSO4.

     Canola seeds are disinfected before use. The
seeds are soaked in 70 % ethanol for 1 min followed by 1% sodium hypochlorite
for 10 mins and washed with sterile distilled water 5-6 times. Each petri dish
is filled with 12 ml sterile distilled water and 0.03 M  MgSO4. 6 seeds are
placed with bacterial strain, and without bacteria as control. The seed are
cultured at 25?C  with a cycle of 12hr
dark & 12hr light. The primary root length are measured after 5th
day f growth and recorded. Typically, the length of the roots of canola seeds
are 40 – 60%. Greater than the untreated seedlings.

3.4 Strain
identification

3.4.1 DNA isolation