Annals of Agricultural Science (2013) 58(2), 111–118

Faculty of Agriculture, Ain Shams University

Annals of Agricultural Science

www.elsevier.com/locate/aoas

ORIGINAL ARTICLE

Effect of carrier materials, sterilization method, and

storage temperature on survival and biological activities of

Azotobacter chroococcum inoculant

Dalia A. Abd El-Fattaha, Wedad E. Eweda

b, Mona S. Zayed

b,*,

Mosaad K. Hassanein a

a Central Laboratory for Agricultural Climate, ARC, Giza, Egyptb Unit of Biofertilizers, Agric. Microbiol. Dept., Fac. Agric., Ain Shams Univ., Shoubra El-Kheima, Cairo, Egypt

Received 16 June 2013; accepted 25 June 2013

Available online 6 September 2013

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KEYWORDS

Azotobacter chroococcum;

Nitrogenase;

Phosphatase activity;

Potassium mobilization;

PGPR;

Carrier materials;

Sterilization

Corresponding author. Tel.-mail address: monaszayed@

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niversity.

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Abstract Azotobacter chroococcum (A101) was examined for some biological activities such as

nitrogenase, phosphatase, potassium solubilization, and production of some plant hormones such

as indole acetic acid, gibberellic acid, and cytokinin. Six different formulations were prepared, using

different carrier materials namely; peat moss, mixture of peat moss plus vermiculite 1:2 (w/w),

wheat bran, rice husk, clay, and sodium alginate. Each carrier material was packed using polyeth-

ylene pages, and then divided into three groups. The first group was sterilized by autoclaving at

121 �C for 20 min, and the second one was sterilized by gamma irradiation at a dose rate of

4.0 kGy for 1 h. However, the third group was left without sterilization. Half of the inoculated poly-

ethylene bags, containing the tested formulations either sterilized by autoclaving or gamma irradi-

ation, were incubated at 8 �C and the other bags were incubated at 30 �C for 6 months. The non-

sterilized bags were incubated under the same condition but only for 3 months. For testing the sur-

vival ofAz. chroococcum (A101), the prepared formulation samples were taken every month during

the storage period. Nitrogenase activity was evaluated in the prepared formulations which exerted

survival cells equal to or more than 108 CFU/ml after 6 months of storage period. Results revealed

that non-sterile formulations exerted high numbers of total fungi and bacteria along the storage per-

iod; however, Az. chroococcum (A101) numbers were decreased over incubation time. No contam-

22461516.om (M.S. Zayed).

lty of Agriculture, Ain-Shams

g by Elsevier

ng by Elsevier B.V. on behalf of Faculty of Agriculture, Ain Shams University.

.001

112 D.A. Abd El-Fattah et al.

inants have been detected in all sterilized carriers.Az. chroococcum (A101) inoculated on wheat bran

exhibited the highest densities among the tested carriers. Encapsulated formulation of alginate

exerted the high stability inAz. chroococcum (A101) densities up to the end of the incubation period

(6 months) at both 30 �C and 8 �C, being 11.905 log 10 CFU/g.

ª 2013 Production and hosting by Elsevier B.V. on behalf of Faculty of Agriculture, Ain Shams

University.

Introduction

The biofertilizers have the ability to convert nutritionally impor-tant elements from unavailable to available form through bio-logical processes (Vessey, 2003). Some bacteria are capable to

fix the atmospheric nitrogen. The free-living non-symbioticnitrogen-fixing bacteria those belonging to genus Azotobactersp. which is a heterotrophic, aerobic microorganism being

broadly dispersed in different environments, such as soil, water,and sediments (Palleroni, 1984).

Different investigations stated that inoculation with Azoto-

bacter sp. has beneficial effects on plant yields (Idris, 2003),due to the increase in fixed nitrogen content in soil (Mrkovacet al., 1996) and to the microbial secretion of stimulating hor-mones, like gibberellins, auxins, and cytokinins (Salmeron

et al., 1990; Gonzales Lopez et al., 1991). Also, Kumar andSingh (2001) established the ability of Azotobacter sp. to solu-bilizing phosphates.

Variety of materials used as carriers has been shown toimprove the survival and biological effectiveness of inoculantsby protecting bacteria from biotic and abiotic stresses (van

Veen et al., 1997). Suitable carrier should be cheap, easily used,mixable, packageable, and available. Also, the carrier mustpermit gas exchange, particularly oxygen, and has high organic

matter content and high water holding capacity as well (Ba-shan,1998; Ben Rebah et al., 2002). According to Somasegaranand Hoben (1994), the good carrier material must be non-toxiceither to the bacterial inoculants or to the plant itself. Further-

more, Stephens and Rask (2000) and Ferreira and Castro(2005) stated that the carriers should have near neutral orreadily adjustable pH, be abundant locally at a reasonable cost

and able to sterilize. These properties only indicate the poten-tial for a good carrier, while final selection of carrier must bebased on microbial multiplication and survival during storage,

the general method of planting, equipment used for planting,and acceptable cost.

Among carriers that can sustain high levels ofmicrobial load,

the peat is considered the most widely used carrier (Burton,1967; Peterson and Loynachan, 1981 ) but is not universallyavailable (Tilak and Subba Rao, 1978). Alternatively, differentmaterials such as industry by-products, organic wastes, mineral

soils, plant by-products, coal, perlite, and agro-industrial wasteshave been tested as culture media for the microbial growth(Brockwell and Bottomley, 1995; Stephens and Rask, 2000).

Alginate is dry, synthetic, simple to use, uniform, biodegrad-able by soilmicroorganisms, andnon-toxic in nature. It containsa large uniform bacterial population and provides slow release

of the bacteria for long periods (Bashan, 1998). It causes no eco-logical pollution and can be produced on large scale by the prop-er industry. The beads can be stored for long periods in arelatively small volume without any apparent effect on the size

of the bacterial population (Bashan and Gonzalez, 1999).

It is of importance to support high number of bacterial inoc-

ulants on carrier during the storage period before use and to pre-vent undesirable spreading of pathogenic bacteria toagricultural field (Somasegaran and Halliday 1982). Different

methodswere used for the sterilization of carriermaterials to ob-tain the most suitable one without any effect on their quality.Strijdom and Deschodt (1976) reported that steam sterilization

by autoclaving is themost commonly used and has the superior-ity among all employedmethods due to low cost and its ability toallow absolutely pure culture of inocula to be prepared. How-ever, Strijdom and van Rensburg (1981) reported that gamma-

irradiation is the most suitable way for carriers sterilization, be-cause this process makes almost no changes in physical andchemical properties of the material and the final materials are

more quality.The present work was designed to study the effect of differ-

ent products and wastes as carrier materials for preparation of

Azotobacter chroococcum formulation and evaluating the sur-vival and biological efficiency of tested strain in these formula-tions at the end of storage period using two different methodsof sterilization and stored at different temperatures.

Materials and methods

Source of strain

Az. chroococcum (A101) was kindly provided from Unit of

Biofertilizer, Fac. Agric., Ain Shams Univ., Cairo, Egypt.

Materials used for formulation of solid state inoculants

Peat moss and peat moss and vermiculite 1:2 (W/W) were ob-tained from ‘‘Shama Company for Agricultural Require-ments’’, wheat bran was obtained from Mills & Bakeries,

north of Cairo, rice husk was obtained from El-Sharkia Gov-ernorate, Egypt, clay was obtained from the farm of Faculty ofAgriculture, Ain shams University, Cairo, Egypt. Alginate was

obtained from El Nasr Pharmaceutical Chemicals Co., Egypt.

Assessment of some physiological activities of Az. chroococcum(A101)

Az. chroococcum (A101) was activated in Modified Ashby’sbroth medium (Abd El-Malek and Ishac, 1968). After 7 daysat 30 �C, cell densities were adjusted to be 35 · 107 cfu/ml

and examined for the following metabolic activities.

a. Nitrogenase activity according to the method described

by Hardy et al. (1973).b. Phosphatase activity and potassium solubilization

according to the method described by Jackson (1973).

Effect of carrier materials, sterilization method, and storage temperature on survival 113

c. Indole acetic acid (IAA) according to the method

described by Glickmann and Dessaux (1995).d. Cytokinin according to the technique reported by

Fletcher and Mccullagh (1971).

e. Gibberellic acid according to the method described byUdagwa and Kinoshita (1961).

Preparation of different formulations

Rice husk, wheat bran, clay, peat moss, and mixture of peatmoss plus vermiculite 1:2 (w/w) were used. Each material

was dried at 70 �C for 24 h and then milled to pass through200 lm mesh sieves; pH of the peat moss was neutralized byadding calcium carbonate.

Each carrier material was packed using high density polyeth-ylene pages in two dimensions (30 · 15 cm) for the monthly ta-ken samples and (50 · 25 cm) to be used after 6 months at theend of the experiment and then divided into three groups. The

first was sterilized by autoclaving at 121 �C for 20 min; the sec-ond one was sterilized by gamma irradiation at a dose rate of4.0 kGy for 1 h. (Gamma irradiation was carried out at the Na-

tional Center for Radiation Research and Technology(NCRRT), EgyptianAtomicEnergyAuthority (EAEA).), whilethe third groupwas left without sterilization. Furthermore, each

group was sub-divided into 2 parts to be stored at 8 �C and30 �C. The moisture of the carriers was adjusted to 50% of thewater holding capacity (WHC).

Alginate was prepared under non-aseptic condition accord-ing to the method described by Bashan (1986).

Preparation of different formula of solid state inoculants usingvarious carrier materials

Broth culture of Az. chroococcum (A101) having a cell density1 · 1012 CFU/ml was used to inoculate, the prepared bags

either sterilized, or non-sterilized, using sterile plastic syringewith hypodermic needle until reaching 50% of WHC. The bagswere thoroughly mixed to ensure similarity in distribution and

absorption of the liquid culture into the carrier. Half of theinoculated polyethylene bags, containing the tested formula-tions either sterilized by autoclaving or gamma irradiation,

were incubated at 8 �C and the other bags were incubated at30 �C for 6 months. The non-sterilized bags were incubatedunder the same condition but only for 3 months.

Survival of Az. chroococcum (A101) during the storage period

Periodical samples were taken from the prepared formulationsevery month during the storage period. Plate count technique

Table 1 Assessment of some metabolic activities for Az. chroococcu

Strain Metabolic activities

Nitrogenase

(lmol C2H4/ml/h)

Available phosphate a

(PPM)

Az. chroococcum (A101) 133.900 18.625

a Total phosphate was 90.03 PPM.b Total potassium was 99.35 PPM.

was applied using potato dextrose agar medium (PDA)(Ahmed, 2001) and nutrient agar medium (Jacobs and Ger-stein, 1960) to determine total fungi and bacteria in respective

order, while MPN method was applied using Modified Ash-by’s broth medium to determine the total count of Az. chroo-coccum (A101) (Abd El-Malek and Ishac, 1968). Samples of

alginate beads were rehydrated with NaCl solution (0.8% W/V, pH 7) with shaking at 100 rpm for 30 min according to Iva-nova et al., 2005 before testing the survival of the strains.

Evaluation the biological efficiency of Az. chroococcum (A101)

on carriers after 6 months of incubation

The prepared formulations which exerted survival cells equalto or more than 108 CFU/g were assessed for their efficiencyto fix nitrogen according to Hardy et al. (1973).

Results and discussion

Assessment of some metabolic activities for Az. chroococcum(A101)

It is obvious from the data recorded in Table 1 that Az. chroo-

coccum (A101) was able to fix atmospheric N2 with133.9 lmol C2H4/ml/h and was able to produce IAA, gibberel-lic acid, being 1.2 and 1.73 mg/L in respective order and cyto-

kinin 2.407 lg/ml. Az. chroococcum (A101) also showedcapability for phosphate solubilization and potassium mobili-zation which gave 18.625 and 33.78 ppm in respective order.

These results are in agreement with those obtained by Kumarand Singh (2001) and Kukreja et al. (2004).

Survival of Az. chroococcum (A101) and proliferation ofcontaminants in different non-sterilized carriers during thestorage period at different temperatures

The quality of the carriers-based inocula depends upon the via-

ble cell count of Az. chroococcum and the presence ofcontamination.

Survival of Az. chroococcum (A101)

Data recorded in Fig. 1 show that the initial densities of Az.chroococcum in all the tested carriers ranged approximatelyfrom 11.99 to 12.06 log 10 CFU/g and then decreased over

time. The number of viable Az. chroococcum cells after3 months was approximately conserved at the average of5.6–5.8 log 10 CFU/g in all the tested carrier materials at 8

and 30 �C. These data are in agreement with those obtainedby Kumar and Gupta (2010).

m (A101).

Available

potassium b (PPM)

IAA (mg/L) Cytokinin

(lg/ml)

Gibberellic

acid (mg/L)

33.78 1.2 2.407 1.73

Fig. 1 Survival of Az. chroococcum (A101) on different non-sterilized carriers during 3 months of storage at different temperature at (A)

30 �C and (B) 8 �C.

Fig. 2 Total bacterial density in different non-sterilized carriers inoculated with Az. chroococcum (A101) during 3 months of storage at

(A) 30 �C and (B) 8 �C.

114 D.A. Abd El-Fattah et al.

Proliferation of contaminants

Data recorded in Figs. 2 and 3 show that at zero time, totalbacterial counts were generally higher than total count of fungiwhich ranged approximately from 12.9 to 13.027 log 10 CFU/g

and from 8.9 to 9 log 10 CFU/g in respective order. The micro-

bial populations increased progressively during the storageperiod.

The total count of contaminants in different carriersstored at 30 �C was higher than those stored at 8 �C. Peatmoss exhibited the highest total fungal counts

Fig. 3 Total fungal density in different non-sterilized carriers inoculated with Az. chroococcum (A101) during 3 months of storage at (A)

30 �C and (B) 8 �C.

Fig. 4 Survival of Az. chroococcum (A101) on different carriers materials sterilized by autoclave and stored at (A) 30 �C and (B) 8 �Cduring storage period (6 months).

Effect of carrier materials, sterilization method, and storage temperature on survival 115

12.921 log 10 CFU/g (after 3 months of storage at 30 �C),while wheat bran recorded the highest total bacterial countsunder the same conditions 16.037 log 10 CFU/g. The lowest

total counts of fungi and bacteria were recorded at 8 �C after3 months of storage with rice husk and peat moss and ver-miculite 1:2 (W/W) which gave 11.859 and 15.306 log 10 C-

FU/g in respective order.

Survival of Az. chroococcum (A101) during the storage periodwas affected by sterilization methods, carrier materials, andstorage temperature

Total counts of Az. chroococcum (A101) affected by carrier

type, sterilization method, and storage temperature are re-corded in Figs. 4 and 5.

Fig. 5 Survival of Az. chroococcum (A101) on different carriers materials sterilized by gamma irradiation and stored at (A) 30 �C and (B)

8 �C during storage period (6 months).

Fig. 6 Nitrogenase activity lmol C2H4/ml/h of Az. chroococcum (A101) on the carrier materials after 6 months of incubation.

116 D.A. Abd El-Fattah et al.

Results presented in Figs. 4 and 5 show that there was notany contamination which has been detected in all sterilized car-

riers (either by autoclaving or by gamma irradiation) up to theend of the incubation period (6 months) at both storage tem-peratures 8 �C and 30 �C. These results are in agreement with

those obtained by Somasegaran and Halliday (1982).Az. chroococcum (A101) population either incubated at

8 �C or 30 �C were higher than those in the non-sterilized car-

riers. Gamma irradiation proved to be superior as compared tosteam sterilization by autoclaving in all the tested carriersresulting in higher survival density of Az. chroococcum

(A101) during the whole storage period for all the tested carri-ers. These findings are in line with those recorded by Strijdom

and van Rensburg (1981).Encapsulation using alginate exerted high stability in Az.

chroococcum (A101) densities up to the end of the storage per-

iod (6 months) at both 8 �C and 30 �C, being 11.905 log 10 C-FU/g. These results agreed with those of Bashan (1998) andBashan and Gonzalez (1999), who reported that PGPR can

survive in alginate beads for long periods.Following alginate, wheat bran sterilized either by gamma

irradiation or autoclaving achieved the highest density which

Effect of carrier materials, sterilization method, and storage temperature on survival 117

gave 10.81 log10 CFU/g. and 10.68 log 10 CFU/g at 30 �C,respectively. These results are coincided with those of Jacksonet al. (1991) and Paau et al. (1991), who reported that Peat,

wheat bran, rice husk, vermiculite, clay, and alginate can beused as good carriers. It is worth mentioning that sterilizedcarriers generally support higher populations and display

much longer shelf lives. They also proved that gamma-irradia-tion is the most suitable way of carrier sterilization, becausethis process makes almost no change in physical and chemical

properties of the material comparing to autoclaving at 121 �C(Kalra et al., 2010).

Biological efficiency of Az. chroococcum (A101) in differentinoculant carriers after 6 months storage

Nitrogenase activity was assessed in vitro for all the tested for-mulations after 6 months of storage period at 30 �C and 8 �C.Data recorded in Fig. 6 show that there was no change innitrogenase activity for Az. chroococcum (A101) in alginate mi-cro-beads at both 30 �C and 8 �C which gave 129.05 and

128.9 lmol C2H4/ml/h in respective order. As shown inFig. 6, these values are higher than those obtained by othercarrier materials. Where Az. chroococcum (A101) inoculated

either on Clay or rice husk sterilized with gamma irradiationand stored at 30 �C and 8 �C, respectively, recorded high nitro-genase activity, being 120 lmole C2H4/ml/h. The obtained re-sults are in agreement with those obtained by Bashan (1998)

and Bashan and Gonzalez (1999), who reported that alginatebeads can be stored for long periods in a relatively small vol-ume without any changes in the biological efficiency of the

bacterial population.From these results, it could be concluded that alginate is the

best carrier to be used for preservation and survival of Az.

chroococcum (A101), but it is expensive. The use of clay or ricehusk sterilized by gamma irradiation is preferable from thecost point of view.

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