plant growth promoting properties of halobacillus sp. and ...

Research Paper

Plant growth promoting properties of Halobacillus sp. andHalomonas sp. in presence of salinity and heavy metals

Prithviraj Desale, Bhargav Patel, Sukrit Singh, Aakshi Malhotra and Neelu Nawani

Dr. D. Y. Patil Biotechnology and Bioinformatics Institute, Dr. D. Y. Patil Vidyapeeth, Pune, 411033, India

Salinity and heavy metal stress are challenging problems in agriculture. Here we report theplant growth promoting ability of three moderate halophiles, Halobacillus sp. ADN1, Halomonassp. MAN5, and Halobacillus sp. MAN6, in presence of both salinity and heavy metal stress.Halobacillus sp. ADN1, Halomonas sp. MAN5, and Halobacillus sp. MAN6 can tolerate 25, 21, and29% NaCl, respectively and grow in presence of 1 mM cobalt, cadmium, and nickel and 0.04 mMmercury and 0.03 mM silver. Halobacillus sp. ADN1, Halomonas sp. MAN5, and Halobacillus sp.MAN6 produced 152.5, 95.3, and 167.3 mg/ml indole acetic acid (IAA) and could solubilize 61, 53,and 75 parts per million (ppm) phosphate, respectively in the presence of 15% NaCl. Theproduction of IAA and solubilization of phosphate was well retained in the presence of salinityand heavy metals like 1 mM cadmium, 0.7 mM nickel, 0.04 mM mercury, and 0.03 mM silver.Besides, the strains showed amylase and protease activities and could produce hydrogen cyanideand ammonia in presence of salinity and heavy metals. A mixture of three strains enhanced theroot growth of Sesuvium portulacastrum under saline and heavy metal stress, where the rootlength increased nearly 4.5 � 0.6 times and root dry weight increased 5.4 � 0.5 times ascompared to control. These strains can thus be useful in microbial assisted phytoremediation ofpolluted saline soils.

Abbreviations: PGPR – plant growth promoting rhizobacteria; PGPH – plant growth promotinghalobacteria; ppm – parts per million; dNTPs – deoxynucleotide triphosphates; IAA – Indole acetic acid;IBA – Indole butyric acid; ANOVA – analysis of variance; LSD – least significant differences

Keywords: Halophiles / Halobacillus / Halomonas / Plant growth promotion / Sesuvium portulacastrum / Heavy metals/ Salinity

Received: December 12, 2012; accepted: February 17, 2013

DOI 10.1002/jobm.201200778

Introduction

Two major problems affecting agricultural productivityand economy worldwide are salinity and accumulationof toxic heavy metals in soil. Industrial activities andimproper agricultural practices have rendered manysoils unusable. The organic matters in the sediments ofsalt marshes tend to accumulate metals which couldeventually result in loss of estuarine vegetation [1–3].Heavy metals arrest plant root and shoot growth anddisrupt the soil communities affecting signal and

nutrient exchange leading to reduced crop yield orplant cover. Salinity inhibits seed germination, retardsplant growth, hampers native soil flora, and interfereswith transformation of nutrients and their avail-ability [4, 5]. Conventional remedial measures forsalinity and heavy metal removal include leaching,irrigation management, landfill soil amendments,electroreclamation, and others [6]. Past few years havewitnessed the successful use of plants with phytoreme-diation potential in the removal of heavy metals byabsorption, concentration, or precipitation [7]. Most ofthe hyperaccumulator plants however suffer from poorgrowth rate in high concentration of heavy metals. Thisproblem can be alleviated by microbial assistedphytoremediation [8, 9] or by using rhizobacteria for

Correspondence: Neelu Nawani, Dr. D. Y. Patil Biotechnology andBioinformatics Institute, Dr. D. Y. Patil Vidyapeeth, Pune 411033, IndiaE-mail: [email protected]: þ912065101871

Environment � Health � Techniques

Plant growth promotion in presence of salinity and heavy metals 1

� 2013 WILEY-VCHVerlag GmbH & Co.KGaA,Weinheim www.jbm-journal.com J. Basic Microbiol. 2013, 00, 1–11

promoting crop productivity in metal-polluted soils [10].Plant growth promoting rhizobacteria (PGPR) stimulateplant growth directly by production of plant growthregulators or indirectly by making the root environmenthostile in terms of nutrient availability and throughenhanced nutrient uptake [11].

However, the problem of heavy metal removal insaline soils persists due to lack of effective ecofriendlymeasures. Halophytes capable of growing in salineconditions and endowed with heavy metal accumula-tion ability can offer a promising solution to thisproblem. Accordingly, plant growth promotion ofhalophytes with phytoremediation potential can beextremely useful in reclamation of saline soils contam-inated with heavy metals. Reports substantiating plantgrowth promoting attributes of halophilic bacteria orplant growth promotion in high salt and heavy metalconcentrations are lacking. Few halophytes like Sesu-vium portulacastrum, are reported to remove arsenic [12],lead [13], and cadmium [14] from contaminated soilsand have been used in phytodesalination [15]. Theirassistance by plant growth promoting halobacteria(PGPH) with an ability to enhance plant growth inpresence of salinity and heavy metals can nurse the soiland reduce its phytotoxicity besides improving theirmetal removing ability. Such bacteria can also help inreestablishment of devastated coastal habitats. Thisstudy demonstrates Indole acetic acid (IAA) produc-tion and phosphate solubilization ability of threemoderate halophiles, Halobacillus sp. ADN1, Halomonassp. MAN5, and Halobacillus sp. MAN6 in presence ofhigh concentrations of NaCl and heavy metals. ThesePGPH could enhance growth of S. portulacastrum in aheavy metal polluted saline soil suggesting theirpossible utility in reclamation of metal polluted salinesoils.

Materials and methods

Bacterial strains and their characterizationThe three strains used in this study were isolated fromsoil samples collected from mangrove rhizosphere. Soilsamples were serially diluted in saline (0.85% w/v NaCl)and plated on nutrient agar with 5, 10, 20, and 25% NaCl(w/v). The plates were incubated at 37 °C for 3 days.Colonies which appeared on nutrient agar with 20%NaClor higher were checked for purity and preserved onnutrient agar slopes with 15% NaCl (w/v) at 4 °C and assuspension in 20% glycerol at �80 °C. For all theexperiments, culture suspension (106 cfu/ml) was addedto the medium to achieve 0.1% inoculum and incubation

was carried out at 37 °Cwith shaking at 150 rpm for 24 hand turbidity was determined at 600 nm to measuregrowth, except where mentioned. The range of NaCl forgrowth was determined in nutrient broth with NaClconcentration varying from 1 to 30% (w/v) at incrementsof 1%. The pH range for growth was determined innutrient broth with optimal NaCl concentration. Themediumwas buffered to desired pHusing (g/l) KH2PO4 5.0(for pH 4.0–6.0); K2HPO4 0.7, Na2HPO4 0.56 (for pH 7.0–8.0), and Na2B4O7 · H2O 0.95, NaOH 0.18 (for pH 9.0–10.0). Temperature range for growth was determined innutrient broth at optimum pH and NaCl concentration.Incubation was carried out at 20, 30, 40, 50, and 60 °C for24 h at 150 rpm.

Molecular identification of the strains was doneby PCR amplification of nearly full-length 16S rRNAgene. Primers specific for domain bacteria were usedand PCR amplification was carried out in an Eppendorfgradient mastercycler as reported by Marchesi et al. [16].The reaction mixtures with final volumes of 50 mlcontained deoxynucleotide triphosphates (dNTPs)50 mmol/L each, primers 2.5 pmol each, 1.5 U TaqDNA polymerase, and 2.0 ml genomic DNA of teststrain. The thermal cycling included initial denatur-ation at 94 °C for 5 min, followed by 30 cycles ofdenaturation at 94 °C for 1 min, primer annealing at55 °C for 1 min, and primer extension at 72 °C for1.5 min. This was followed by an extension step at 72 °C for 10 min. DNA sequencing and analysis was doneon Avant 3100 gene analyzer, USA.

Determination of plant growth promotingproperties of halobacteria in presence of salinityand heavy metalsProduction of indole compounds was monitored undervarying concentrations of NaCl (1–30% w/v), tryptophan(0.1–1.0% w/v) and in presence of 1% w/v of differentsugars. Basal medium used had (g/L) yeast extract 1.0,mannitol or other sugars 10.0, di-potassium hydrogenphosphate 0.5, magnesium sulfate 0.2, and pH 6.5–7.0.Incubation was carried out at 37 °C for 7 days. Mannitolwas replaced with sugars like xylose, maltose, lactose,glucose, fructose, and galactose to check the effectof sugar on production of indole compounds. Estima-tion of IAA was done in the cell free broth by themethod of Gordon and Weber [17]. To confirm theproduction of IAA, extraction of acidified cell free broth(pH adjusted to 2.5 with 1 N HCl) using two volumes ofethyl acetate was carried out. The ethyl acetate fractionwas dried at 40 °C and the residue was dissolved inmethanol. The extracted compound was spotted on TLCplates (Merck silica gel 60 F254) developed in ethyl

2 Prithviraj Desale et al.


acetate:chloroform:formic acid (5.5: 3.5: 1) and detectedusing van Urk-Salkowski reagent [18]. Purification wascarried out on a Sugar-D column (Nacalai Tesque, Japan)with acetonitrile:water (70:30) as the mobile phase at aflow rate of 10.0 ml/min. The fractions were analyzedby TLC with IAA and indole butyric acid (IBA) asreference standards. The fractions having Rf valuecorrelating with that of the standards were concentrat-ed and subjected to IR analysis. Functional groups inthe purified compound were determined by FT-IRspectrum recorded on Shimadzu FT-IR 8400S. IAAproduction in presence of both 10% (w/v) NaCl andheavy metals was monitored in above-mentionedmedium. Choice of the metals was done based ontheir presence in the contaminated soil used further forthe growth of S. portulacastrum.

Phosphate solubilization was quantitated in NBRIPmedium containing (g/L) glucose 20.0, tri-calciumphosphate 5.0, magnesium sulfate 0.25, KCl 0.2, magne-sium chloride 10.0, ammonium sulfate 0.1, yeast extract1.0, pH 7.0. The concentration of NaCl was varied from 3to 29% (w/v) in the medium which was then inoculatedwith the test strains. Incubation was carried out at 37 °C,150 rpm for 10 days and the phosphate solubilizationwasmonitored by themethod of Mehta and Nautiyal [19].Phosphate solubilization was tested in presence of both10% (w/v) NaCl and heavy metals in NBRIP medium.Ammonia production was tested at different concen-trations of NaCl (5, 9, 13, 19, and 23% w/v) in peptonewater. After 48–72 h incubation at 30 °C and 150 rpm,0.5 ml of Nessler’s reagent was added to the culture brothand formation of yellow to brown color indicated apositive test [20]. Production of hydrogen cyanide wastested on nutrient agar supplemented with differentconcentrations of NaCl (5, 9, 13, 19, and 23% w/v) and0.44% glycine. After streaking the bacterial cultures, afilter paper strip soaked in mixture of 0.5% picric acidand 2.0% Na2CO3 was placed on the agar. Plates wereincubated at 30 °C for 4 days and were monitored fordevelopment of orange to red color indicating HCNproduction [21]. Iron chelation was tested on chromeazurol S agar [22] with varying concentrations of NaCl (5,9, 13, 19, and 23% w/v). The plates were spot inoculatedwith the bacterial cultures and incubated at 30 °C for 24–48 h. Dark coloration of the colonies indicates ironchelation ability.

The bacteria were studied for biochemical propertiessuch as ability to degrade polymers like xylan, pectin,cellulose, starch, and casein in presence of varyingconcentrations of NaCl and for antibiosis towardsBacillus subtilis and Fusarium sp. Production of ammoniaand HCN, iron chelation and degradation of polymers

was also tested at tolerant levels of both salinity andheavy metals.

Effect of halobacteria on growth of S. portulacastrumS. portulacastrum grows in sandy clay, coastal limestone,and salt marshes. Soil samples and stem cuttings of S.portulacastrum were obtained from coastal regions.Herbaceous stem cuttings of S. portulacastrum with nodesfor root formation were surface sterilized and planted inpolluted sandy soil collected from the coastal habitat. Thesoil was sterilized by autoclaving at 121 °C for 30 min fortwo consecutive days. The soil properties were analysedto ensure the extent of pollution and to confirm whethereffect of bacterial treatment on the plant was aloneresponsible for plant growth. Heavy metal content in soilwas tested by using Atomic Adsorption Spectrophotome-ter (Chemito AA203, Mumbai, India). Cultivation ofplants was carried out in 18 cm diameter pots (three potsfor each treatment) to ensure adequate sprawling. Thepots received 10 ml suspension (108 cfu/ml harvestedfrom mid-log phase and grown in medium supportingIAA production) of each of Halobacillus sp. ADN1,Halomonas sp. MAN5, and Halobacillus sp. MAN6. Tenmilliliters of suspension with total count of 108 cfu/ml(1:1:1 mixture) was used to check if the three PGPH couldcomplement each other’s plant growth promoting traits.Control pots did not receive any bacterial strains. Theplants were grown in a controlled atmosphere (25–35 °C,40% humidity and 12 h photoperiod) for one month. Thewatering of plants was done with saline water containing2% salt initially which was gradually increased to thelevel of Electrical conductivity of the soil as determinedbefore inoculation of PGPH. No additional heavy metalswere added in the soil and heavy metal content wasdetermined once every week and also at the end of28 days of incubation. After a month, the plants werecarefully removed from the pots to determine the rootlength, number of lateral roots, and dry weight of theroots. The experiment was repeated thrice for statisticalsignificance.

Statistical analysisRandomization of the pots was done each time andthree replicate measurements were done for everyparameter evaluated. Evaluation of data was done byanalysis of variance (ANOVA) using the statisticalsoftware package SPSS 17.0. t-Test was carried out forcomparison between the test and control and the meanswere compared by using least significant differences(LSD). Differences were considered to be significant at aprobability of (p < 0.05). The data are represented withstandard error.



Results

Growth characteristics of strainsHalobacillus sp. ADN1, Halobacillus sp. MAN6, andHalomonas sp. MAN5 were isolated from mangroverhizosphere soil and were identified based on morpho-logical, biochemical and 16S rRNA gene sequences. Thephenotypic characteristics tested for the three strains aregiven in Table 1. The phylogenetic matches based on 16SrRNA gene sequences are represented in the form ofphylogenetic tree (Fig. 1). Strain ADN1 and MAN6 areclosely related to the genus Halobacillus and strain MAN5to the genus Halomonas. The range and optimum NaClconcentration for growth indicates these strains aremoderate halophiles. They showed resistance to heavymetals, as indicated by the concentrations of heavymetals in presence of which these strains could grow.

Plant growth promoting characteristicsThe production of indole compounds from three strainsstarted after 24 h of incubation and progressivelyincreased till 7 days. Maximum production occurredafter 5 days incubation for all the strains. Indolecompounds were produced in presence of 3–17% (w/v)NaCl for Halobacillus sp. ADN1 and Halomonas sp. MAN5,and 3–23% (w/v) for Halobacillus sp. MAN6. Beyond thesevalues, production of IAA reduced. Indole productionwas 162.6 mg/ml for Halobacillus sp. ADN1 in presence of11% NaCl; 144.5 mg/ml for Halomonas sp. MAN5 in

presence of 9% NaCl and 168.7 mg/ml for Halobacillus sp.MAN6 in presence of 17% NaCl after 5 days (Fig. 2). Thetype of indole compound produced was determined byorganic phase extraction of acidified cell free broth andits purification by HPLC. The purification yielded asingle spot on thin-layer chromatography with an Rfvalue of 0.6 which correlated with 0.59 which was Rfvalue for IAA and IBA (Fig. 3). The IR spectrum ofpurified compound which exhibits characteristic sig-nals at n 3415.7, 3001.03, 2910.18, and 1701.1 thatcorrelates with IAA and indicates the presence of aminegroup, aromatic ring, and carboxylic acid group (Fig. 4).From the spectral, chromatographic, and biochemicaldata, it was interpreted that Halobacillus sp. ADN1,Halobacillus sp. MAN6, and Halomonas sp. MAN5produced the plant growth hormone, IAA in presenceof high NaCl concentrations.

IAA production was monitored in presence ofdifferent sugars which could be possible degradationproducts of polymers of plant or animal origin. IAAproduction decreased 20–25%, for all the strains, inabsence of mannitol. When mannitol was replaced withxylose, maltose, and lactose, 8–10% increase in IAAproduction was seen for all the strains. IAA productionwas best at 0.25% tryptophan for all the strains. IAAproduction was also tested in presence of heavy metals.It was highest in absence of heavy metals although forHalobacillus sp. ADN1, 50% production (80 mg/ml) wasretained and for Halobacillus sp. MAN6, 41% (64 mg/ml)

Table 1. Phenotypic and biochemical characteristics of Halobacillus sp. ADN1, Halobacillus sp. MAN6, and Halomonas sp. MAN5

CharacteristicHalobacillus sp.ADN1

Halobacillus sp.MAN6

Halomonas sp.MAN5

Gram character and morphology Gram positive rods Gram positive rods Gram negative rodsMotility þ þ þColor of colony Orange Orange CreampH for growth

Range 4.0–10.0 4.0–10.0 5.0–10.0Optimum 7.0 7.0 7.0

Growth temperature (°C)Range 20–45 20–45 20–45Optimum 30–35 30–35 30–35

NaCl for growth (% w/v)Range 3–25 3–29 3–19Optimum 13 13 13

Hydrolysis ofa

Cellulose þ (5, 0.6 Cd, 0.5 Ni) þ (5, 0.6 Cd, 0.4 Ni) –Pectin þ (5, 0.3 Cd, 0.4 Ni) þ (9, 0.1 Cd, 0.3 Ni) þ (5, 0.3 Cd and Ni)Xylan þ (5, 0.1 Cd and Ni) þ (5, 0.1 Cd, 0.2 Ni) –Starch þ (13, 0.6 Cd, 0.7 Ni) þ (13, 0.6 Cd and Ni) þ (13, 0.8 Cd, 0.4 Ni)Gelatin þ (9, 0.5 Cd, 0.3 Ni) þ (13, 0.7 Cd, 0.5 Ni) þ (5, 0.5 Cd, 0.3 Ni)

aParentheses indicate the concentration of NaCl (% w/v) and heavy metal (mM) at which activity was best, no activity was seen inpresence of 0.01 mM mercury and silver.



was retained in presence of 1 mM cadmium. IAAproduction reduced to 16% (21 mg/ml) of control inpresence of 0.8 mM cadmium in case of Halomonas sp.MAN5 (Fig. 5). Nickel could consistently reduce IAAproduction in Halomonas sp. MAN5 whereas in case ofHalobacillus sp. ADN1 and Halobacillus sp. MAN6, itreduced initially but showed an increase in productionbeyond 0.6 mM. Addition of mercury and silver reducedIAA production by 50% after which a steady decreasewas seen for all the strains. No growth was observedbeyond 0.03 mM silver for any of the strains.

The three PGPH could solubilize phosphate inpresence of NaCl (Fig. 6). Phosphate solubilizationgradually increased with time and the amount ofphosphate solubilized after 5 days is reported here.Halobacillus sp. ADN1 could solubilize 61 ppm phosphateat 15% NaCl and; Halomonas sp. MAN5 and Halobacillus sp.

Figure 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences indicating the positions of Halobacillus sp. ADN1,Halobacillus sp. MAN6, and Halomonas sp. MAN5 with other related species. Bootstrap values based on 1000 replications and >50% areindicated at branch nodes. Bar, 0.02 substitutions per nucleotide position.

Figure 2. IAA production by Halobacillus sp. ADN1 (&), Halobacillussp. MAN6 (~), and Halomonas sp. MAN5 (*) at varying concen-trations of NaCl.



MAN6 solubilized 54 and 80 ppm phosphate respective-ly, at 17% NaCl. The phosphate solubilization activitywas much closer to the NaCl concentration optimumfor growth. Phosphate solubilization was also moni-tored in presence of heavy metals (Fig. 7). Halobacillussp. MAN6 could solubilize phosphate more effectively inpresence of cadmium than nickel and retained nearly50% activity in presence of 0.9 mM cadmium and0.4 mM nickel. Halobacillus sp. ADN1 retained morethan 50% phosphate solubilization at 1.0 mM cadmiumand nickel however; higher concentrations were limit-ing the bacterial growth. Similarly, 50% phosphatesolubilization with respect to control was retained forHalomonas sp. MAN5 in presence of 0.6 mM nickel and0.9 mM cadmium. Nearly 40% solubilization wasretained in presence of mercury and silver as depictedin Fig. 7. The plant growth promoting properties,qualitative detection of hydrogen cyanide (HCN) and

ammonia in presence of heavy metals and salinity aregiven in Table 2. The three moderate halophiles couldproduce HCN and ammonia in wide range of NaCl,Halobacillus sp. MAN6 displayed better production ofHCN and ammonia upto 23% NaCl. However inpresence of heavy metals these attributes were sup-pressed and were not detected in metal concentrations>0.5 mM (0.01 mM for silver and mercury). Ironchelation was seen for all the strains only in absenceof heavy metals. Halobacillus sp. ADN1 and Halobacillussp. MAN6 showed antimicrobial activity against the teststrains, whereas Halomonas sp. MAN5 displayed noantibiosis.

Amixture of three strains enhanced the root growth ofthe ecologically important halophyte S. portulacastrum,where the root length increased nearly 4.5 � 0.6 timesand root dry weight increased 5.4 � 0.5 times ascompared to control which did not receive anyhalophiles (Fig. 8). Paired t-test showed significantcorrelation between IAA levels and root length withcorrelation coefficient 0.92 and p-value 0.016. Table 3shows the reduction in heavy metal content of soilwhich received PGPH and was planted with S. portula-castrum. A decrease inmetal content was observed in soilwhich received only the plant or plant along with PGPH,than control soil which did not receive any inoculants orplant. Cadmium and nickel uptake was preferential overmercury and silver. A 40% reduction was seen incadmium content and 24% reduction in nickel contentof soil which received the PGPH and S. portulacastrum.

Discussion

Halobacillus and Halomonas are native to the mangrovesoils but are not well documented for plant growthpromoting abilities. Saline soils frequently chelateheavy metals from surrounding environment whichincreases their phytotoxicity particularly when heavymetal pollution occurs in the vicinity of such regions.Estuaries are prone to this, and discharge of heavymetal pollutants in the sea can be harmful to nativehalophytes thereby, disturbing these habitats. Nativemicrobial flora like halophiles play a crucial role inconditioning saline habitats however, their role innutrient recycling or plant growth promotion in salinesoils, is poorly documented. The moderate halophilesstudied here could prominently grow in presence ofheavy metals like cadmium and nickel. The objective ofplant growth promotion of phytoremedial halophytesprompted us to screen these halophiles for plantgrowth promoting attributes like IAA production and

Figure 3. Thin layer chromatograph of the purified indole relatedcompound. Lane 1, standard IBA; lane 2, standard IAA; lane 3,compound purified from Halobacillus sp. MAN6; lane 4, compoundpurified from Halomonas sp. MAN5; and lane 5, compound purifiedfrom Halobacillus sp. ADN1.



phosphate solubilization in presence of NaCl and heavymetals like cadmium, nickel, silver, and mercury whichwere found in polluted soil subsequently used for thecultivation of S. portulacastrum.

The amount of IAA produced by these moderatehalophiles is higher than reported values of IAA fromAzotobacter sp. [11], Pseudomonas sp. [23], Bacillus amylo-liquefaciens [24], and Rhizobium sp. [25, 26] in presence ofsalinity. It is remarkable to note that a gradual increasein amount of IAA production was seen in the broth,which indicates its stability in 10% NaCl, making the

three strains suitable for use in saline soils for ecologicalconservation of saline habitats like mangrove ecosys-tems. IAA production was maximum in absence ofheavy metals and decreased with increase in metalconcentration. This negative correlation between metalconcentration and IAA production explains the effect ofmetal on growth and concomitantly on induction of thehormone. Nickel was less toxic than cadmium henceIAA production was better in presence of nickel thancadmium. This could also be due to possibility ofaccumulation of nickel from the medium by the

Figure 4. FT-IR spectrum of IAA purified from Halobacillus sp. ADN1 and standard IAA over wave number range 4000–500 cm�1 (spectrum ofIAA purified from Halomonas sp. MAN5 was similar).



bacterial cells. IAA production in range of 13–19 mg/mlwas earlier reported in Bacillus in presence of 200 ppmnickel, cadmium, and zinc which is lesser than thatreported for Halobacillus sp. ADN1, Halomonas sp. MAN5,and Halobacillus sp. MAN6 in this study [27]. Higher IAAproduction was earlier reported in presence of heavymetals like nickel [28, 29]. However, these reportsindicate IAA production in presence of heavy metalsonly, which indicates superiority of Halobacillus sp.ADN1, Halomonas sp. MAN5, and Halobacillus sp. MAN6due to their ability to produce IAA in presence of bothheavy metals and salinity. Increase in heavy metalconcentration reduced phosphate solubilization. Rhizo-bacteria are reported to enhance metal tolerance ofplants by improving absorption of phosphorus, hencephosphate solubilization in presence of heavy metalscan be a useful attribute [6].

Figure 5. IAA production by Halobacillus sp. ADN1 (&), Halomonas sp. MAN5 (*), andHalobacillus sp. MAN6 (~) in increasing concentrationof cadmium (A), mercury (B), nickel (C), and silver (D) found in the contaminated soil subsequently used for the growth of Sesuviumportulacastrum.

Figure 6. Amount of phosphate solubilized by Halobacillus sp. ADN1(&), Halobacillus sp. MAN6 (~), and Halomonas sp. MAN5 (*) atvarying concentrations of NaCl.



The root growth enhancing ability of PGPH demon-strated in this study can be useful parameter inmicrobial assisted phytoremediation of heavy metalpolluted saline soils. Higher concentrations of IAAenhance lateral root formation which increases thesurface area for heavy metal uptake by plants [30]. Theinoculation of polluted soil with PGPH reduced theheavy metal content which could be due to accumula-tion of metals by bacterial cells or due to enhancementof accumulation efficiency of S. portulacastrum. PGPHcan thus be important plant associates to enhance heavymetal removal from soils by influencing plant growth orby improving metal availability for uptake by plants.PGPH can themselves condition the soil via theirmetabolic activities, production of metal chelatorsand pH alteration affecting solubility of the metals.Collectively, all these attributes can increase thephytoremediation potential of plants, also reducingphytotoxicity of the contaminated soil [31, 32].

Figure 7. Phosphate solubilization by Halobacillus sp. ADN1 (&), Halomonas sp. MAN5 (*), and Halobacillus sp. MAN6 (~) in increasingconcentration of mercury (A), silver (B), cadmium (C), and nickel (D) found in the contaminated soil subsequently used for the growth ofSesuviumportulacastrum.

Figure 8. Root length (&) and dry weight (&) of the roots ofSesuvium portulacastrum grown in contaminated soil inoculated withplant growth promoting halophiles. Standard error is displayed as errorbars (n ¼ 3). Different letters indicate statistically significant differ-ences from the control and/or between the samples at p < 0.05 asdetermined by LSD.



Thus, plant growth promoting halophiles capable ofenhancing plant growth particularly root develop-ment, offer a sturdy assisting bioinoculum forremoval of heavy metals from saline soils. Theycan also be used in reclamation of saline soils and in

plant growth promotion of halophytes, many ofwhich have medical and ecological significance.Restoration of contaminated or degraded wetlandscan be future large scale application of thesehalobacteria.

Table 2. Plant growth promoting traits of Halobacillus sp. ADN1, Halobacillus sp. MAN6, and Halomonas sp. MAN5

CharacteristicHalobacillus sp.ADN1

Halobacillus sp.MAN6

Halomonas sp.MAN5

Production of IAAa 11 (3–17) 17 (3–23) 9 (3–17)Solubilization of phosphatea 15 (3–23) 17 (3–25) 17 (3–19)Production of HCNa,b 13 (5–13) 5 (5–23) 9 (5–19)Production of ammoniaa,b 5 (5–19) 19 (5–23) 13 (5–19)Iron chelationa,c 13 (5–17) 13 (5–17) 13 (5–13)Production of IAA and solubilization of phosphate in presence of 10% (w/v) NaCl and heavy metalsd

Cadmium chloride þ(1 mM) þ(1 mM) þ(0.9 mM)Cobalt chloride þ(1 mM) þ(1 mM) þ(0.9 mM)Ferric chloride þ(4 mM) þ(3 mM) þ(2 mM)Nickel chloride þ(1 mM) þ(1 mM) þ(1 mM)Lead nitrate þ(1 mM) þ(3 mM) þ(1 mM)Manganese chloride þ(3 mM) þ(1 mM) þ(1 mM)Mercuric chloride þ(0.04 mM) þ(0.04 mM) þ(0.03 mM)Silver chloride þ(0.03 mM) þ(0.03 mM) þ(0.03 mM)Antimicrobial activity againstBacillus subtilis þ þ –Fusarium sp. þ þ –

aParentheses indicate the range of NaCl (% w/v) in which activity was seen; number of replicates was 3; figures outside parenthesesare NaCl (% w/v) concentrations at which best activity was displayed.bAttributes not seen beyond 0.5 mM concentration of any of the heavy metal (0.01 mM in case of silver and mercury) when tested inpresence of 10% (w/v) NaCl.cIron chelation was not observed in presence of heavy metals.dParentheses indicate highest concentration of the metal beyond which no growth was observed, amount of IAA produced andphosphate solubilized was less in presence of heavy metals than in their absence as discussed in the results section.

Table 3. Heavy metal content of soil used for cultivation of Sesuvium portulacastrum.

Heavy metal(mg/g of soil) Controla

Sesuviumportulacastrum

Sesuviumportulacastrumand mixture of ADN1,MAN5, and MAN6

Cadmium at day 0 0.49 � 0.012 0.51 � 0.008 0.51 � 0.01Cadmium after 28 days 0.45 � 0.005 0.39 � 0.002 0.31 � 0.007Nickel at day 0 114 � 1.1 111 � 1.7 109 � 2.1Nickel after 28 days 108 � 2.1 96 � 0.9 83 � 1.6Mercury at day 0 0.019 � 0.05 0.021 � 0.02 0.024 � 0.004Mercury after 28 days 0.02 � 0.02 0.019 � 0.01 0.023 � 0.06Silver at day 0 <0.01 <0.01 <0.01Silver after 28 days <0.01 <0.01 <0.01

aControl soil did not receive the plant and PGPH; readings are average of three replicates where plantation was repeated thrice,standard error is represented for each value.



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