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Microencapsulation by spray drying of nitrogen-fixing bacteria associated

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Article  in  World Journal of Microbiology and Biotechnology (Formerly MIRCEN Journal of Applied Microbiology and Biotechnology) · May 2014
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World J Microbiol Biotechnol
DOI 10.1007/s11274-014-1662-8
ORIGINAL PAPER
Microencapsulation by spray drying of nitrogen-fixing bacteria
associated with lupin nodules
Daniela C. Campos • Francisca Acevedo • Eduardo Morales
Javiera Aravena • Véronique Amiard • Milko A. Jorquera •
Nitza G. Inostroza • Mónica Rubilar
Received: 11 February 2014 / Accepted: 1 May 2014
Ó Springer Science+Business Media Dordrecht 2014
Abstract Plant growth promoting bacteria and nitrogen-
fixing bacteria (NFB) used for crop inoculation have
important biotechnological potential as a sustainable fertil-
ization tool. However, the main limitation of this technology
is the low inoculum survival rate under field conditions.
Microencapsulation of bacterial cells in polymer matrices
provides a controlled release and greater protection against
environmental conditions. In this context, the aim of this
study was to isolate and characterize putative NFB associ-
ated with lupin nodules and to evaluate their microencap-
sulation by spray drying. For this purpose, 21 putative NFB
were isolated from lupin nodules and characterized (16S
rRNA genes). Microencapsulation of bacterial cells by spray
drying was studied using a mixture of sodium algi-
nate:maltodextrin at different ratios (0:15, 1:14, 2:13) and
concentrations (15 and 30 % solids) as the wall material.
The microcapsules were observed under scanning electron
D. C. Campos  F. Acevedo (&)  E. Morales  M. Rubilar
Technology and Processes Unit, Agri-aquaculture Nutritional
Genomic Center, CGNA, 4791057 Temuco, Chile
e-mail: francisca.acevedo@cgna.cl
D. C. Campos  M. A. Jorquera  N. G. Inostroza  M. Rubilar
Department of Chemical Engineering, Scientific and
Technological Bioresource Nucleus, BIOREN, Universidad de
La Frontera, Casilla 54-D, Temuco, Chile
J. Aravena
The Robert H. Smith Institute of Plant Sciences and Genetics in
Agriculture, The Robert H. Smith Faculty of Agriculture, Food
and Environment, The Hebrew University of Jerusalem,
76100 Rehovot, Israel
V. Amiard
Genomics and Bioinformatic Unit, Agri-aquaculture Nutritional
Genomic Center, CGNA, 4791057 Temuco, Chile
•
microscopy to verify their suitable morphology. Results
showed the association between lupin nodules of diverse
known NFB and nodule-forming bacteria belonging to Al-
phaproteobacteria, Betaproteobacteria, Gammaproteobac-
teria and Bacteroidetes. In microencapsulation assays, the
1:14 ratio of sodium alginate:maltodextrin (15 % solids)
showed the highest cell survival rate (79 %), with a micro-
capsule yield of 27 % and spherical microcapsules of
5–50 lm in diameter. In conclusion, diverse putative NFB
genera and nodule-forming bacteria are associated with the
nodules of lupine plants grown in soils in southern Chile, and
their microencapsulation by spray drying using sodium
alginate:maltodextrin represents a scalable process to gen-
erate a biofertilizer as an alternative to traditional nitrogen
fertilization.
Keywords Polymer  Sodium alginate  Maltodextrin 
Enterobacter  Diazotrophic bacteria
Introduction
Legumes are considered a rich source of protein for human
and animal nutrition, and yellow lupin (Lupinus luteus)
represents an economically relevant crop for agriculture in
southern Chile. Lupin plants are characterized by their high
protein content (30–57 %) (Petterson et al. 1997; Glencross
2001) in seeds compared with other legumes, such as peas
(Pisum sativum, 22 %) and beans (Vicia faba, 25 %)
(Lampart-Sczapa 2001). Unfortunately, the excessive and
continued use of chemical fertilizers to improve lupin
yields involves various environmental concerns, such as
low soil fertility, low soil biodiversity, eutrophication of
water bodies, etc. Thus, novel fertilization strategies are
needed to reduce fertilizer inputs and their environmental
123

consequences in lupin crops from agro-ecosystems. In this
context, nitrogen-fixing bacteria (NFB) have been sug-
gested as an attractive alternative to reduce the application
of N-based fertilizers on soils by farmers, with the con-
comitant environmental and economic benefits. NFB can
be found associated with roots (diazotrophic bacteria),
within of plant tissues (endophytic bacteria, EB) and
forming structures (nodules) in roots which play an
essential role in the acquisition of N from atmosphere in
legume plants (Martı́nez-Viveros et al. 2010). Currently,
biofertilizers based on NFB are widely investigated and
commercialized as alternative to traditional chemical fer-
tilization. However, the survival and activity of bacteria
used for plant inoculation is reduced rapidly due to con-
currence with autochthonous soil bacteria and/or inade-
quacy of soil characteristics (acidity, osmotic potential,
nutrient availability, etc.) (Martı́nez-Viveros et al. 2010).
Microencapsulation of EB and NFB in a polymer matrix
offer them protection and gradual release into the soil,
ensuring a higher prevalence of beneficial bacteria under
environmental conditions. Beneficial bacterial cells have
been encapsulated by several chemical and physical tech-
niques using principally alginate, starch, skim milk and agar
as polymer matrices (Schoebitz et al. 2013). Spray drying
represents a fast and economic process widely used in the
large-scale production of biofertilizers (Schoebitz et al.
2013). However, despite the great potential of this technol-
ogy in agriculture, the application of microencapsulated EB
and NFB to improve crops yields is scarce, particularly in
lupin plants where so far has not been explored. Ash-derived
volcanic soils of south Chile (Andisols) present low P
available, acidity (pH \ 5.5) and occurrence of phytotoxic
cations (Al3? and Mn2?) in soil solution, which may impair
growth of unadapted bacteria. For this raison we decided to
work with native or naturalized bacteria and expect these to
be well adapted to the soil conditions of south Chile. In this
context, the purpose of this study was to isolate and char-
acterize putative NFB from lupin plants grown in southern
Chile and to evaluate spray drying for bacterial cell micro-
encapsulation. Isolates were genetically characterized based
on partial 16S rRNA gene sequences. One of the bacteria
strain was used to evaluate the bacterial microencapsulation
by spray-drying using different mixture of alginate/malto-
dextrin as polymer matrices.
Materials and methods
Sampling and isolation of putative nitrogen-fixing
bacteria
Roots containing nodules were collected from L. luteus
grown at the Carillanca Experimental Station (38°410 4000 S;
123
World J Microbiol Biotechnol
72°240 5700 W) from the Instituto de Investigación Agraria
(INIA), Region of La Araucanı́a, Chile. Lupin nodules were
surface sterilized (70 % ethanol for 30 s followed by 4 %
sodium hypochlorite for 60 s) and aseptically processed
according to Langer et al. (2008). Appropriated dilutions
were then sampled onto yeast-extract mannitol agar (YMA;
0.2 g L-1 magnesium sulfate, 1.0 g L-1 mannitol,
0.5 g L-1 potassium phosphate dibasic, 0.1 g L-1 sodium
chloride, 0.4 g L-1 yeast extract; 1.5 % agar), supple-
mented with 0.25 % Congo red dye. YMA is a universal
culture medium used for isolation of NFB associated with
legume nodules. The YMA plates were incubated at 30 °C
for 6 days and colonies showing a slightly raised, colorless
and opaque phenotype were selected. Colorless phenotypes
was taken as an indicator that Congo red had not been
absorbed by bacterial cells, which is a characteristic of NFB.
Characterization of putative nitrogen-fixing bacteria
Phenotype
Twenty-one representative colonies of putative NFB were
selected, re-streaked onto YMA and pure cultures were
stored at -80 °C in Luria–Bertani (LB; 10 g L-1 tryptone,
5 g L-1 yeast extract, and 10 g L-1 NaCl) broth supple-
mented with glycerol (30 %). The isolated bacteria were
observed under light microscopy and a Gram test was
carried out according to Hucker’s staining method (Murray
et al. 1994). The non-staining KOH method (Buck 1982)
was performed as confirmative gram assays.
Ribotype
Genetic characterization to genus level of each isolated
strain was performed by partial sequencing of the 16S
rRNA gene. Briefly, total DNA was extracted by using
Gentra Puregene Yeast/Bact. Kit (Qiagen, Inc., USA)
according to the manufacturer’s instructions. Fragments of
16S rRNA gene were amplified by polymerase chain
reaction (PCR) using the bacterial universal primer set: 27f
(50 -AGA GTT TGA TCC TGG CTC AG-30 ) and 1492r (50 -
TAC GGY TAC CTT GTT ACG ACT T-30 ) (Pearce et al.
2004). The PCR mixture [0.125 lL GoTaqÒ Flexi DNA
polymerase (Promega, Inc., USA), 20 pmol of each primer,
3 lL MgCl2 25 mM, 2.5 lL of 2 mM deoxyribonucleotide
triphosphate and 5 lL Green GoTaqÒ Flexi Buffer 59]
was made up to 50 lL with DNA-free water. Amplification
was performed as follows: hot-start at 94 °C for 5 min
followed by 35 cycles of denaturation at 94 °C for 1 min,
annealing at 52 °C for 1 min, and extension at 72 °C for
2 min. The 16S rRNA fragments were purified and
sequenced using Macrogen (Seoul, Korea). The sequences
obtained were analyzed by BLAST algorithm and
World J Microbiol Biotechnol
compared with those deposited in the GenBank database.
The phylogenetic affiliation, based on the 16S rRNA genes,
of isolated NFB in relation to representative bacteria
deposited in GenBank was performed using MEGA5
(http://www.megasoftware.net/).
Bacterial growth rate
The bacterial growth rate for each isolated strain was
studied by plotting the cell growth (absorbance at 600 nm)
versus the incubation time. For this purpose, strains were
inoculated in 250 mL Erlenmeyer flasks containing 50 mL
of yeast mannitol broth (YM) and incubated for 72 h at
30 °C with shaking (150 rpm). After incubation, 1 mL of
each culture was transferred to tubes containing 9 mL of
YM medium (in triplicate). The tubes were incubated at
30 °C with shaking (150 rpm) and the optical density
(600 nm) was recorded every 1 h for 12 h.
The growth rate was estimated as: Ln(A/A0) = -k 9 t,
where A0 is the absorbance value of bacterial culture at
time zero, A is the absorbance value of bacterial culture at
time t and k is the growth rate constant.
Microencapsulation and survival
Bacterial cell microencapsulation
An isolated bacterium, characterized as Enterobacter sp.
14, was chosen as the model for microencapsulation
because it exhibited a high growth rate and presented
characteristic phenotypic colony facilitating its count and
monitoring by plate-counting method.
Microencapsulation of Enterobacter sp. 14 cells with
sodium alginate and maltodextrin as wall material was
evaluated. Sodium alginate and maltodextrin were mixed at
different ratios (0:15, 1:14, 2:13) and concentrations (15
and 30 % solids) (Table 1). In a 250 mL beaker, sodium
alginate was dissolved in 50 mL of distilled water at 50 °C
under continuous agitation. Maltodextrin was added and
mixed until a homogeneous solution was obtained. The
solution was cooled to 20 °C and mixed with 30 mL of the
bacterial suspension (106 UFC mL-1). Finally, distilled
water was added to get a final volume of 100 mL, and
homogenized at 18,000 rpm for 2 min with a benchtop
homogenizer (model 400DS; PRO Scientific Inc., USA).
The viscosity of polymer mixture was measured with a
double cylinder digital high speed viscometer (model VIS-
79 series; MRC, Israel) with different spindles depending
on the intermediate (spindle F) or low (spindle E) viscosity.
The spray-drying process was carried out with a LabPlant
SD-05 dryer (UK), containing a 1.5 mm nozzle diameter
and a spray chamber of 500 mm 9 215 mm. Polymer
mixtures were fed into the main chamber through a peri-
staltic pump. An air input temperature of 100 °C and an
outlet temperature of 65 °C were used, with a drying air
flow of 73 m3 h-1 and a feed rate of 5.3 g min-1.
The product yield (Y) of the spray-drying process was
estimated according to O’Riordan et al. (2001):
Y(%) = (Wm 9 100)/Wp, where Wm is the weight of the
recovered microcapsules (g) and Wp corresponds to the dry
weight of the polymer dispersion (g) in addition to the dry
weight of the bacterial suspension (g). In addition, the
moisture content of the microcapsules was measured
gravimetrically. Briefly, 0.5 g of microcapsule was
weighed, then dried at 105 ± 1 °C for 24 h, cooled in a
desiccator, and weighed a final time. The initial and final
weights were used to calculate the wet basis moisture
content. The experiments were carried out in triplicate.
Survival of microencapsulated bacterial cells
Enumeration of the free and microencapsulated Entero-
bacter sp. 14 cells was determined by the spread plate
method. One gram of microcapsule was dissolved in 9 mL
of sterile saline phosphate buffer and incubated at 30 °C
for 24 h with shaking (150 rpm). Serial dilutions (10-1–
10-7) of free cells or microencapsulated cells were made in
sterile saline solution (NaCl 0.85 %) and then 0.1 mL of
each dilution was spread onto YMA plates. Colony form-
ing units (CFU) were enumerated after incubation at 30 °C
for 48 h. The percentage of survival (S) with respect to
initial counts before drying was calculated according to
Rajam et al. (2012): S = (N/N0) 9 100, where N is the log
cell number of bacteria per g after drying and N0 is the log
cell number of bacteria per g before drying.
Morphology of microcapsules
The microcapsule morphology was visualized by scanning
electronic microscopy (SEM).
The dried powders of microcapsules were fixed on
aluminum studs and coated with carbon (double sided
carbon tape) and then metalized with pure gold using an
Edward S150 Sputter Coater, and observed in a scanning
electronic microscope (model JSM-6380LV; JEOL Ltd.,
Japan) at 10 kv voltage.
Statistical analysis
Results of this study were subjected to ANOVA followed
by Duncan’s test of multiple comparisons. Significance
was determined at 5 % confidence level. All the results
were presented as average value ± standard deviation of
three replicates.
123
 World J Microbiol Biotechnol

Table 1 Phylogenetic assignment of selected putative nitrogen-fixing bacteria associated with lupin nodules
Isolates Taxonomic groupa Closest relatives or cloned sequences Similarityb (%) Accession no.
(accession no.)

5 Proteobacteria; Gammaproteobacteria; Enterobacter cloacae from ginger 99 KF750615

Enterobacteriales; Enterobacteriaceae rhizome (KC009683)
11 Proteobacteria; Alphaproteobacteria; Ochrobactrum antrophi from farm soil 99 KF750616
Rhizobiales; Brucellaceae (KC857470)
14 Proteobacteria; Gammaproteobacteria; Root endophytic Enterobacter ludwigii 99 KF750617
Enterobacteriales; Enterobacteriaceae from Panax notoginseng (JN700134)
15 Proteobacteria; Betaproteobacteria; Achromobacter xylosoxidans from 92 KF750623
Burkholderiales; Alcaligenaceae rhizosphere soil of peanut
(JN585718)
16 Proteobacteria; Alphaproteobacteria; Rhizobium sp. from nodules of 100 KF975673
Rhizobiales; Rhizobiaceae Phaseolus vulgaris (KF638351)
22A Proteobacteria; Alphaproteobacteria; Bradyrhizobium sp. nodulating Lupinus 100 KF975674
Rhizobiales; Bradyrhizobiaceae; consentinii from alkaline soils
(HQ233235)
22B Proteobacteria; Alphaproteobacteria; Bradyrhizobium sp. from root nodules 100 KF975680
Rhizobiales; Bradyrhizobiaceae of Cytisus villosus (EU561069)
23 Proteobacteria; Gammaproteobacteria; Nitrogen-fixing Enterobacter sp. from 99 KF750618
Enterobacteriales; Enterobacteriaceae rhizosphere of sugarcane
(KC833507)
25A Proteobacteria; Alphaproteobacteria; Rhizobium sp. from root nodule of 100 KF975675
Rhizobiales; Rhizobiaceae Cilliandra grandiflora. (JX855172)
26C Proteobacteria; Alphaproteobacteria; Sphingomonas sp. from cluster roots of 100 KF975681
Sphingomonadales; Lupin albus (JN590370)
Sphingomonadaceae;
27A Proteobacteria; Alphaproteobacteria; Mesorhizobium amorphae grassland 100 KF975682
Rhizobiales; Phyllobacteriaceae soil in Kawatabi experimental farm
(AB741445)
27B Proteobacteria; Alphaproteobacteria; Bradyrhizobium sp. from root nodules 100 KF975676
Rhizobiales; Bradyrhizobiaceae of Retama sphaerocarpa (KF357617)
27E Bacteroidetes; Sphingobacteriia; Sphingobacteriaceae bacterium 100 KF975677
Sphingobacteriales; kmd_062 from rhizosphere soil
Sphingobacteriaceae. (EU723098)
28E Proteobacteria; Alphaproteobacteria; Rhizobium sp. from potassium mine 100 KF975683
Rhizobiales; Rhizobiaceae soil (JF900025)
28F Proteobacteria; Alphaproteobacteria; Rhizobium sp. from root nodules of 99.7 KF975678
Rhizobiales; Rhizobiaceae Phaseolus vulgaris (JF318173)
28G Proteobacteria; Alphaproteobacteria; Rhizobium tropici from root nodule of 100 KF975679
Rhizobiales; Rhizobiaceae Ledespa sp. (GQ181030)
29 Proteobacteria; Gammaproteobacteria; Diazotrophic Stenotrophomonas sp. 99 KF750619
Xanthomonadales; Xanthomonadaceae from maize (JX174242)
33 Proteobacteria; Gammaproteobacteria; Endophytic Stenotrophomonas sp. from 99 KF750620
Xanthomonadales; Xanthomonadaceae peanut (JQ579644)
34 Proteobacteria; Gammaproteobacteria; Pseudomonas aeruginosa from 100 KF750621
Pseudomonadales; Pseudomonadaceae rhizosphere soil of pepper
(KF574910)
35 Proteobacteria; Gammaproteobacteria; Enterobacter ludwigii from rhizosphere 99 KF750622
Enterobacteriales; Enterobacteriaceae (JX122497)
36 Proteobacteria; Gammaproteobacteria; Stenotrophomonas maltophilia from 96 KF750624
Xanthomonadales; Xanthomonadaceae soil (EU927145)
a
The phylogenetic assignment is based on sequence analysis in GenBank database from NCBI (http://www.ncbi.nlm.nih.gov). It is given the
phylum as well as the lowest predictable phylogenetic rank
b
Based on partial sequencing of 16S rDNA gene and comparison with those present in GenBank by using Blastn (http://blast.ncbi.nlm.nih.gov/
Blast.cgi)

123
World J Microbiol Biotechnol
Results and discussion
Characterization and growth rate of putative nitrogen-
fixing bacteria
All isolate strains were rod-shaped cells, motile and gram
negative as revealed by light microscopy and Gram tests.
Respect to the ribotype, the genetic analyses of the 16S rRNA
gene sequences revealed a high similarity with known diazo-
trophic bacteria and NFB belonging to Alphaproteobacteria
(Ochrobactrum Rhizobium, Bradyrhizobium, Sphingomonas,
Mesorhizobium), Betaproteobacteria (Achromobacter),
Gammaproteobacteria (Enterobacter, Stenotrophomonas and
Pseudomonas) and Bacteroidetes (Sphingobacteria) among
others (Table 1; Fig. 1). The diversity of nodule-associated
bacteria in leguminous plants include (1) symbiotic NFB, such
as strains the genus Rhizobium, Bradyrhizobium, Azorhizobi-
um, Allorhizobium, Sinorhizobium and Mesorhizobium, (2)
and free-living NFB bacteria or bacteria associated with NFB,
such as Azospirillum, Enterobacter, Klebsiella and Pseudo-
monas (Hayat et al. 2010). In relation to the bacterial genera
found in our study, studies have also described genera such as
Enterobacter, Achromobacter Pseudomonas and Stenotroph-
omonas as diazotrophic bacteria associated with a wide variety
of plants (Ryan et al. 2009; Jha and Kumar 2009; Muthukumar
et al. 2010; Madhaiyan et al. 2013). Respect to lupin plants,
Chen (2013) reported the presence of Bradyrhizobium strains
in Lupinus angustifolius L. as revealed by sequencing of 16S
rRNA gene and the 16S-23S-internal transcribed spacer
region, whereas bacteria belonging to the genus Ochrobac-
trum have shown the ability to form nodules in Lupinus albus
(Trujillo et al. 2005). In relation to the occurrence of Rhizo-
biales, this bacterial order is known to harbor diverse symbi-
otic NFB (Rhizobium, Bradyrhizobium and Mesorhizobium),
which are widely studied due to their environmental signifi-
cance in biological N input in soils and soil fertility manage-
ment of cultivated lands (Bottomley and Myrold; 2007;
Olivares et al. 2013). In greenhouse conditions, dry matter
yield of diverse legume and non-legume plants (radish, maize,
wheat, barley, lettuce, etc.) is increased by inoculation with
members of Rhizobium, Bradyrhizobium and Sinorhizobium
(Antoun and Prévost 2005). Some of these authors also
mention the ability of these bacterial genera to (not exclu-
sively) (1) promote plant growth through the production of
phytohormone (i.e. indole acetic acid), (2) solubilize P from
insoluble P forms in soils, (3) protect plants against phyto-
pathogens (fungi, virus and insects) through the secretion of
secondary metabolism compounds, and (4) break down phy-
totoxic pollutants.
In relation to bacterial growth rates, the isolates Enter-
obacter sp. 5, Ochrobactrum sp. 11 and Enterobacter sp.
14 showed higher growth rates (ranging between 0.32 and
0.33 k h-1) compared to other assayed strains (ranging
between 0.13 and 0.26 k h-1). Although no significant
(p B 0.05) differences were observed in bacterial growth
rates among Enterobacter sp. 5, Ochrobactrum sp. 11 and
Enterobacter sp. 14, based on ANOVA followed by Dun-
can’s test of multiple comparisons, Enterobacter sp. 14 was
selected as the bacterial model for the subsequent micro-
encapsulation by spray drying due to its characteristic
phenotype colony. Also, Enterobacter strains are com-
monly found in rhizosphere soils and legume nodules, and
studies have shown that the inoculation of free and
encapsulated Enterobacter spp. can increase plant growth
(Vassileva et al. 1999; Morales-Garcı́a et al. 2011; Mad-
haiyan et al. 2013).
Microencapsulation and survival
Previous studies have demonstrated that it is possible to
obtain viable microencapsulated bacterial cells with spray
drying (Schoebitz et al. 2013). As shown in Table 2, the
polymer mixture containing sodium alginate and malto-
dextrin (treatments 1, 2, 3 and 4) presented a bacterial
survival greater than 76 %, which indicates that this mix-
ture provides a high protective action on bacteria during the
microencapsulation process. When alginate was absent
from the mixture (treatments 5 and 6), bacteria survival
was significantly (p B 0.05) lower with a value below
60 %. The survival rate of the bacteria depends of the
effects of operating conditions of the spray drying process.
In fact, Yu et al. (2010) reached a bacterial survival ranged
between 57.84 and 84.57 % for gram-negative bacteria
microencapsulated using as wall material gum arabic and
maltodextrin at different ratio (1:9), feed flow rate
(12.5 mL min-1) and inlet air temperature (160 °C).
Lower bacterial survival was obtained after the microen-
capsulation of microorganisms such as Rhizobium (John
et al. 2011) and Pseudomonas strains (Amiet-Charpentier
et al. 1998), probably due to the simultaneous dehydration
and high temperature inactivation. Formulations of poly-
mer mixtures used as vehicles are one of the most viable
options for the immobilization of beneficial microorgan-
isms in order to overcome the deficiencies of nitrogen in
legume crops. The mixture of sodium alginate and malto-
dextrin as a polymer matrix has been successfully used as
wall material for microbial microencapsulation with satis-
factory results (Bashan 1998; Both et al. 2012). In fact,
maltodextrin offers various advantageous properties as an
encapsulation matrix because it is osmotically inactive and
presents a low viscosity, providing protection, interspacing
for cells and strengthening the encapsulating matrix (Wil-
son and Shah 2007; Chavez and Ledeboer 2007). By
contrast, alginate possesses a high mechanical stability,
high porosity, viscosity and heat-resistance, and provides
123

Fig. 1 Phylogenetic tree
showing the affiliation of
isolated putative nitrogen-fixing
bacteria in relation to
representative bacteria taken
from GenBank database
(accession number is given in
parenthesis). The UPGMA tree
was constructed using MEGA5
and a bootstrap analysis was
performed with 1,000 trials
tolerance against adverse environmental conditions (Kai-
lasapathy 2002). Alginate is one of the most commonly
used polymers for encapsulation of plant growth promoting
bacteria in agriculture (Ivanova et al. 2005; Yabur et al.
2007). Pasin et al. (2012) indicated that the interaction
between alginate and other biopolymers such as malto-
dextrin forms highly versatile mixed systems suitable for
the formation of encapsulating microspheres. Vassileva
et al. (1999) also reported that the application of alginate-
encapsulated cells of Enterobacter sp. increased the growth
and phosphorus uptake by lettuce plants (Lactuca sativa).
In terms of product yield, despite treatments 3 and 4
presenting a similar survival rate than treatment 1 and 2,
these treatments showed much a lower microcapsule yield
(Table 2). This can be explained by the high viscosity of the
polymer mixture in these treatments, which is due to the high
(30 %) percentage of solids that does not allow the spray
dryer to perform well as described by Boza et al. (2004).
Based on our results of bacterial survival and product yield
after spray drying, the polymer mixture containing 1:14
sodium alginate:maltodextrin (15 % w/w solids) was
selected as suitable wall material for the microencapsulation
of isolated endophytic nodule-associated bacteria.
123
World J Microbiol Biotechnol
Morphology of microcapsules
The use of SEM provided information on the degree of
integrity and porosity of the microcapsules (Rosenberg
et al. 1985). As shown in Fig. 2, the diameter of the
spherical microcapsules obtained in this study ranged from
5 to 50 lm. The outer surfaces of the microcapsules were
characterized by the presence of indentation; however, no
cracks were observed. The absence of cracks in micro-
capsules in this study is essential to wall functionality in
limiting core deterioration and/or losses during storage
(Sheu and Rosenberg 1998).
The presence of indentation was previously reported for
microcapsules obtained by spray drying using polysac-
charides as wall material (Rosenberg et al. 1985). Choi
et al. (2010) attributed the presence of surface indentation
to the presence of polysaccharide in the polymer mixture.
Also, the presence of indentation could be due to the
summation of factors such as drying speed, the atomization
mechanism, uneven shrinkage, viscoelastic properties and
rapid solidification (before the expansion of the micro-
capsules) of the wall materials (Rosenberg et al. 1985;
Sheu and Rosenberg 1998).
World J Microbiol Biotechnol

Table 2 Microencapsulation of Enterobacter sp. 14 by spray-drying using different mixtures of polymer materials
Treatment A:M ratio (w/w) Solids (w/w) Viscosity (mPa*s) [spindle] Moisture content (%) Product yield (%) Survival (%)

1 1:14 15 20.5 [E] 1.28 ± 0.35 27.01 ± 0.96 78.86 ± 8.81a

2 2:13 15 55.2 [E] 0.71 ± 0.19 25.09 ± 0.12 76.62 ± 11.03a
3 1:14 30 72.4 [F] 0.38 ± 0.05 10.97 ± 0.11 79.90 ± 9.38a
4 2:13 30 64.1 [F] 0.95 ± 0.47 2.97 ± 0.01 91.01 ± 11.62ab
5 0:15 15 \1.0 [E] 0.16 ± 0.14 12.33 ± 0.03 58.94 ± 7.77bc
6 0:30 30 23.7 [E] 0.22 ± 0.05 23.27 ± 0.02 53.95 ± 7.78c
A: sodium alginate; M: maltodextrin: E: low viscosity; F: intermediate viscosity
Values represent mean ± standard error (n = 3). Different letters in the same column denote significant difference (p B 0.05, comparisons of
means were carried out for each pair with Duncan’s test)

feasibility of the spray-drying process for microbial inocu-

lants encapsulation on an industrial scale in agriculture.

Acknowledgments This research was supported by funding from

FIA through project FIA PYT-2012-0088.

Conflict of interest The authors report no declarations of interest.

The authors alone are responsible for the content and writing of this
article.

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Fig. 2 Scanning electronic microphotographs of microcapsules of
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