Applied Clay Science 134 (2016) 1925

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Applied Clay Science

journal homepage: www.elsevier.com/locate/clay

Research paper

Nano-biohybrids of engineered nanoclays and natural extract for

antibacterial agents
Hyoung-Jun Kim a, Tae-Hyun Kim a, Hyoung-Mi Kim a, In-Kee Hong b, Eun-Ji Kim b, Ae-Jin Choi c,
Hyun-Jin Choi c, Jae-Min Oh a,
a
Department of Chemistry and Medical Chemistry, College of Science and Technology, Yonsei University, Wonju, Gangwondo 26493, Republic of Korea
b
Radiant INC., 1143, G-tech village, Geodu-ri, Dongnae-myeon, Chuncheon, Gangwondo 24398, Republic of Korea
c
Postharvest Research Team, National Institute of Horticultural and Herbal Science (NIHHS) of RDA, Wanju, Jeollabukdo 55365, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: We have prepared nano-biohybrids between engineered nanoclays and antibacterial natural extract. Agrimonia
Received 25 November 2015 pilosa Ledeb (AP) was treated with either hot water or ethanol to obtain two kinds of extract, and we found
Received in revised form 5 April 2016 that water extract was more efcient in bacterial inhibition. Pore volume and specic surface area of three
Accepted 3 May 2016
clays, such as illite, scoria and hydrotalcite, were engineered with acid or heat, before hybridization. Nano-
Available online 17 May 2016
biohybridization was carried out with engineered nanoclays powder and extract solution. According to
Keywords:
thermogravimetry and dry weight method, it was determined that extract content in hybrid were between
Nano-biohybrid 11.6 and 39.5%. X-ray diffraction patterns showed that the crystal structure of engineered nanoclay was pre-
Engineered nanoclay served after hybridization. Scanning electron microscopy, extract content analyses and zeta potential measure-
Natural extract ment suggested that the surface of nanoclays were evenly and thoroughly covered with extract moiety with a
Antibacterial activity few molecular layers. Differential thermal analyses on hybrid indicated broad and lowered combustion temper-
ature of extract components in hybrids compared with extract only, suggesting possible molecular rearrange-
ment of extract moiety on nanoclay surface. We then evaluated bacterial colony forming inhibition of extract
and nano-biohybrids of which result indicated higher antibacterial efcacy of hybrids than extract itself.
2016 Published by Elsevier B.V.

1. Introduction biohybrids, it is important to develop various nanomaterials with

Nano-biohybrids, which are nano-scale combination of engineered
nanomaterials with bio-active substances, have been highlighted in a
bio-medical application elds such as drug delivery system, nano-
diagnosis, functional cosmetics, and nanofood (Yang et al., 2003; Choi
et al., 2012; Kim et al., 2013; Ruiz-Hitzky et al., 2013). Engineered
nanomaterials can stabilize bio-substances which are vulnerable to de-
naturation and deactivation under harsh conditions (Choy et al., 2010).
High specic surface area of nanomaterials often provide nanospace for
bio-substances to rearrange their molecular orientation resulting in
modied thermal behavior or optical properties (Costantino et al.,
1999; Li et al., 2004). Appropriately controlled internal structure of
nanomaterials can modulate release kinetics of incorporated bio-
substances (Park et al., 2008; Kang et al., 2015). Surface modications
with chemical species, such as homing ligand attachment, traceable
moiety incorporation and polyethylene glycol coating, provide various
functionalities to nano-bio hybrids like targeted delivery, efcient track-
ing and prolonged circulation (Oh et al., 2009; Lipka et al., 2010;
Musumeci et al., 2010). In order to maximize applicability of nano-
Corresponding author.
E-mail address: jaemin.oh@yonsei.ac.kr (J.-M. Oh).
tailor-made physicochemical properties as well as to control interaction
between nanomaterials and bio-substances in the hybrids.
Various kinds of components can be utilized in preparation of nano-
biohybrids. Among them, naturally obtained materials, for both nano-
and bio-part, are getting interests according to recent trends of eco-
friendliness and bio-compatibility. One of the candidates for nano-part
is nanoclay, which is the most abundant natural resources on earth
crust and has long been utilized in human history for pottery and ce-
ramic preparation. Clays generally stand for soil or mineral particles
having a few micrometer size (Guggenheim and Martin, 1995); in nano-
scale, they have hierarchical structure of swelling nanolayers or small
particles with high specic surface area. Due to structural characteris-
tics, inter-layer or inter-particle spaces of clays can form nanospace
which serves as reservoir for bio-substances. Recent progresses of nano-
technology made it possible to control structure, composition and pore
of engineered nanoclays with simple chemical processes, and thus the
utility of clays in nano-biohybrid eld is expanding.
Natural bio-substances, such as plant extract, have long been utilized
as herbal medicine. Currently, variety of chemistry have been developed
to analyze and characterize phytochemicals, and thus researches on
natural extract having bio-activities such as antimicrobial, antioxidant,
anti-aging, and anti-inammatory properties are extensively carried

http://dx.doi.org/10.1016/j.clay.2016.05.003
0169-1317/ 2016 Published by Elsevier B.V.
20 H.-J. Kim et al. / Applied Clay Science 134 (2016) 1925
Fig. 1. Paper disc diffusion assay results for AP-W (a and c) and AP-E (b and d) on Bacillus subtilis (a, b) and Escherichia coli (c, d).
out (Khknen et al., 1999; Rauha et al., 2000; Vouldoukis et al., 2004).
Among various bio-activities, antimicrobial property is one of the com-
mon properties of phytochemical and can be widely applied to various
elds like food preservation or antibacterial agent in consumer
products.
In this study, we prepared several nano-biohybrids utilizing
engineered nanoclays and natural extracts having antibacterial proper-
ty. Agrimonia pilosa Ledeb (AP), was treated with either hot water or
ethanol to obtain natural bio-extracts. Natural clays such as illite and
scoria were treated with acid to enhance pore volume and specic sur-
face areas. Anionic clay, hydrotalcite was thermally treated to prepare
layered oxide with enhanced specic surface area. Three kinds of
engineered nanoclays were hybridized with AP extract and thus obtain-
ed nano-biohybrids were analyzed in terms of interaction between
nanoclays and bio-substances. Then we evaluated antibacterial activity
of nano-biohybrids compared with natural extract itself on gram-
positive Bacillus subtilis (B. subtilis) and gram-negative Escherichia coli
(E. coli), which are known to cause endocarditis, eye infection, food poi-
soning, and etc. (de Boer Sietske and Diderichsen, 1991; Alwi and Ali,
2014).
2. Experimental section
2.1. Materials
Natural clays, illite and scoria, were provided by NANOSPACE Co.,
Ltd. (Gyonggido, Korea). Synthetic hydrotalcite clay was prepared by
coprecipitating Mg(II)/Al(III) solution with NaOH/NaHCO3 solution
followed by hydrothermal treatment at 150 C, as reported (Oh et al.,
2002). In order to obtain pore volume and specic surface area en-
hanced engineered nanoclays, illite and scoria was treated with 6 M
HCl for 6 h at 60 C, and resultants were named IL and SC. Hydrotalcite
was calcined at 400 C for 9 h to obtain surface area enhanced
engineered nanoclays, layered oxide (LO). Antibacterial natural extract
was obtained from Agrimonia pilosa Ledeb (AP) by either hot water or
ethanol extraction. Typically, AP was dried 50 g of dried part was
smashed and treated in hot water (100 C) or ethanol/water = 7/3 sol-
vent for 90 min and 3 h, respectively. Then the solvent was evaporated
to get dried AP extract. Extract obtained by hot water and ethanol was
named AP-W and AP-E, respectively.
2.2. Paper disc diffusion assay of AP-W and AP-E extract
Antibacterial activities of AP-W and AP-E extract were investigated
by paper disc diffusion assay on gram-positive bacterium B. subtilis
and gram-negative bacterium E. coli. First, B. subtilis and E.coli was inoc-
ulated in 8 g/L of nutrient broth solution (Merck KGaA, Germany) and
incubated at 180 rpm for 24 h at 37 C. Solid agar culture medium was
prepared by 20 g of nutrient agar (Merck KGaA, Germany) and 3 g
agar powder dissolved in 1 L deionized water then 20 mL agar solution
was solidied in petri dish at room temperature. The bacteria inoculated
culture medium was diluted with broth culture medium until the absor-
bance reached 0.5 at 595 nm wavelength, and was spread on agar plate
with sterilized cotton swab then 8 mm paper disc was placed onto bac-
teria spread agar plate. Each extract (5 L) of 0.25%, 0.5%. 1%, 2% concen-
tration was dropped on paper disc, and then zone of bacterial inhibition
was measured after incubation at 37 C for 24 h.
2.3. Preparation of engineered nanoclays/extract nano-biohybrids
For the preparation of engineered nanoclay-extract nano-
biohybrids, we utilized three nanoclays and AP-W. For the
 H.-J. Kim et al. / Applied Clay Science 134 (2016) 1925 21

Fig. 2. Thermogravimetry analysis of (a) AP-W, (b) IL/AP-W, (c) SC/AP-W, and (d) LO/AP-W.

hybridization, 1 g of dried AP-W was dissolved in 100 mL of 50 C deion-
ized water then insoluble solid was ltrated by 70 mm lter paper
(Advantec, Japan). Engineered nanoclays, IL, SC and LO, was mixed
with AP-W solution with clay:AP-W weight ratio of 1:1 and the mixture
was vigorously stirred for 12 h under dark. The obtained precipitates
were then centrifuged and lyophilized.
2.4. Characterization of nano-biohybrids
In order to conrm the crystal structure of nanoclay or nano-
biohybrids, the powder X-ray diffraction patterns were obtained with
Bruker AXS D2 Phaser with Cu K radiation ( = 1.5418 ). The powder
sample on quartz holder was scanned from 5 to 80 with 0.5/min time
step, utilizing 1.0 mm equatorial slit and 3.0 mm air scattering silt. For
investigation of morphology change, eld emission-scanning electron
microscopic images were obtained with Quanta 250 FEG. The suspen-
sion of nanoclay or nano-biohybrids were put on the silicon wafer
then coated with Pt/Pd for 60 s. Zeta-potential values of AP-W,
nanoclays and nano-biohybrids suspension (0.01 g/mL) were measured
by light scattered electrophoresis apparatus, ELSZ-1000 (Otsuka). Con-
tents of AP-W extract in nano-biohybrids were investigated by both
thermorgravimetric analysis and dried weight method. Thermogravi-
metric (TG) analysis of AP-W extract and nano-biohybrids was carried

Fig. 4. X-ray diffraction patterns for (a) IL, (b) IL/AP-W, (c) SC, (d) SC/AP-W, (e) LO and
Fig. 3. Content of natural extract in nano-bio hybrids. (Black bar: content calculated by (f) LO/AP-W. (I, Q, Pl, Py, H and Pr next to (hkl) index stand for mineral phase of Illite,
thermogravimetry. White bar: content obtained by dry weight method.) Quartz, plagioclase, pyroxen, hematite and periclase, respectively.)
22 H.-J. Kim et al. / Applied Clay Science 134 (2016) 1925
Fig. 5. Scanning electron microscopic images for (a) AP-W itself, (b) IL, (c) SC, (d) LO, (e) IL/AP-W, (f) SC/AP-W and (g) LO/AP-W.
out with TA Instruments SDT2960 at Chungbuk University in the tem-
perature range of 251000 C and heating rate of 5 C/min. For the
dried weight method, the 1 mL of supernatant was gathered after reac-
tion and thoroughly dried at 80 C oven. Reacted amount of AP-W was
calculated by measuring unreacted AP-W in supernatant. In order to in-
vestigate thermal behavior of organic molecules in AP-W and nano-bio
hybrids, differential thermal analysis of AP-W and nano-biohybrids was
investigated at same condition and instrument with TG.
2.5. Colony forming inhibitory activity of engineered clay-antibacterial ex-
tract nanohybrids
B. subtilis and E.coli was inoculated in nutrient broth culture medium
and incubated 24 h. The AP-W or AP-W/nanoclay nano-biohybrids was
mixed with 1 ml of 1 104 CFU bacteria inoculated culture medium and
incubated in incubator at 37 C for 3 h. Then 10 L of cell suspension was
collected in each tube and spread on culture agar plate, which was fur-
ther incubated in incubator for 14 h at 37 C. The bacterial colony
forming inhibitory activity of samples was investigated by counting
number of bacterial colony on culture plate. Bacterial colony forming
rate of AP-W or hybrids treated agar plates was calculated by comparing
number of colonies of untreated agar plates (negative control).
3. Data and result
In order to evaluate antibacterial efcacy of AP extract, we carried
out paper disc diffusion assay with extract obtained by hot water (AP-
Fig. 6. Zeta-potential curves of AP-W, clays and nano-bio hybrids for (a) IL/AP-W, (b) SC/AP-W and (c) LO/AP-W. Open circle: engineered clay, dashed line: AP-W, solid line: nano-bio
hybrid.
W) or 70% ethanol (AP-E). Fig. 1 showed inhibition zone formed by
AP-W or AP-E treatment on B. subtilis or E. coli which were spread on
agar plate. It was revealed that AP-W was more effective in terms of an-
tibacterial activity than AP-E. AP-W showed 13.5 mm and 11.3 mm of
inhibition zone at 2% concentration for B. subtilis and E. coli, respectively,
while AP-E exhibited inhibition zone of 12.7 mm for B. subtilis only,
without signicant inhibition on E. coli. Biologically, E. coli and
B. subtilis are gram negative and positive bacteria, respectively. Gram-
positive and negative bacteria are different to each other in peptidogly-
can layer thickness on cell wall; layer of positive bacteria is thicker than
that of negative ones (Fayaz et al., 2010). Different extract solvent might
result in subtle difference in extract composition (Golet et al., 2002),
giving rise to different antibacterial activity on bacterial type. As our
purpose was to prepare nano-biohybrids having general antibacterial
activity, we chose AP-W for further hybridization.
In order to obtain pore and surface area modied clays for hybridi-
zation, we carried out acid treatment on illite (IL) and scoria (SC) as
well as calcination on hydrotalcite, which is referred to as layered
oxide (LO). According to nitrogen adsorption-desorption measurement
and BJH pore analyses (data not shown), IL, SC and LO showed en-
hanced pore volume and specic surface area upon acid and calcina-
tion, exhibiting SBET of 33.5 m2/g, 74.6 m2/g and 197.6 m2/g for IL, SC
and LO, respectively. The powdered engineered nanoclays were then
directly reacted with aqueous AP-W solution to get IL/AP-W, SC/AP-
W and LO/AP-W nano-biohybrids. For evaluation of AP-W content in
each hybrid, we carried out thermal analyses and dry weight calcula-
tion. As shown in Fig. 2(a), dried AP-W showed several steps of
 H.-J. Kim et al. / Applied Clay Science 134 (2016) 1925
Fig. 7. Differential thermal analysis curves of (a) AP-W, (b) IL/AP-W, (c) SC/AP-W, and
(d) LO/AP-W.
thermal decomposition with 94.7% of weight loss up to 850 C. Three
nano-biohybrids showed clearly distinguished two steps of thermal de-
composition: rst step from room temperature to ~200 C and second
step beyond ~200 C. According to previous reports on thermal analy-
ses of clays (Grim and Rowland, 1942), weight loss up to ~ 200 C
could be attributed to the removal of water molecules which are at-
tached at the pore surface of clays. The second steps of weight loss,
12.7%, 11.0% and 37.4% for IL/AP-W, SC/AP-W and LO/AP-W, respective-
ly, were assigned to the thermal decomposition of AP-W moiety in
each hybrid. Taking into account that ~5.33% of AP-W was remained
uncombusted, we multiplied (100% / (100 5.33)%) to each weight
loss value to estimate total AP-W content. Thus determined AP-W con-
tent was 13.4%, 11.6% and 39.5% for IL/AP-W, SC/AP-W and LO/AP-W,
respectively (Fig. 3). The AP-W content calculated from thermal analy-
sis was comparable with that obtained from dry weight method. Dry
weight method presented AP-W content of 15.8%, 11.5% and 32.6%, re-
spectively, for IL/AP-W, SC/AP-W and LO/AP-W (Fig. 3), showing that
the two quantication methods cross-conrmed quantication.
We then analyzed structure of nano-biohybrids utilizing X-ray dif-
fraction pattern in terms of structural change in engineered nanoclays.
Fig. 4(a) and (b) exhibited XRD pattern of IL and IL/AP-W hybrids, re-
spectively, showing characteristic peaks of illite at 8.84 (002), 19.9
(110), 34.9 (131) and those of quartz at 20.9 (100), 26.6 (101) and
42.4 (200). The peak position and intensity of IL were almost preserved
after hybridization, suggesting that hybridization did not seriously
Fig. 8. Bacterial colony forming (%) of (a) Bacillus subtilis and (b) Escherichia coli treated with AP-W and nano-biohybrids. Asterisks represent statistical difference with condence intervals
of 95% calculated by Student's t-test. Photographs above bar graphs are bacterial colony formed after treating with each sample.
23
induced structural change of nanoclays. Similarly, the structure of SC
was also maintained unchanged after hybridization, showing diffraction
peaks of plagioclase at 21.9 (202), 23.6 (112), 27.8 (021), that of
pyroxene at 29.7 (221) and those of hematite at 33.1 (1 0 4), 35.6
(1 1 0) (Fig. 4(c), (d)). Crystal structure of LO was also preserved show-
ing periclase peaks at 43.2 (2 0 0) and 62.5 (220) for both LO and LO/
AP-W (Fig. 4(e), (f)). Generally, it was known that LO with periclase
phase (usually Mg2AlO3.5) underwent phase recovery to hydrotalcite
structure, if sufcient water molecules and appropriate anion sources
were provided (Hibino and Tsunashima, 1998; Nyambo et al., 2009).
However, we could not observe signicant evolution of hydrotalcite
phase but decrease in periclase crystallinity. In our experimental condi-
tion, due to the lack of anionic species in AP-W, LO could not recover hy-
drotalcite structure. Furthermore, partial hydroxylation of layered oxide
during hybridization reaction might result in the decrease in periclase
crystallinity without serious structural change. From the XRD study,
we could conrm that the structures of engineered nanoclays were
well preserved during hybridization. Thus we expected that AP-W moi-
ety was adsorbed on the surface of nanoclays.
In order to investigate morphology change of nanoclays after hybrid-
ization, we carried out scanning electron microscopic analyses. As
shown in Fig. 5(a), dried AP-W showed large lumps of organic com-
pounds, exhibiting smooth surfaces as reported before (Hongal et al.,
2014). Compared with AP-W itself, engineered nanoclays showed
sharp morphology with distinguishable grain boundaries. The SEM re-
sult of IL exhibited characteristic morphology of layered clays, showing
large plate-like particles (Fig. 5(b)) (Liewig and Clauer, 2000). SC
showed agglomeration of small particles, which were considered hema-
tite, pyroxene and plagioclase nanoparticles (Fig. 5(c)). LO particles had
coin-like morphology with particle diameter ~200 nm, which was cor-
responding to our previous study (Fig. 5(d)) (Kim et al., 2014). After hy-
bridization, the surface morphology of nanoclays dramatically changed.
Sharp edge and plate-like shape of IL particles were camouaged by
smooth organic moiety of AP-W (Fig. 5(e)). Similarly, grain boundaries
of SC and LO became blur after hybridization (Fig. 5(f), (g)). These
changes in surface morphology was thought to be originated from sur-
face coating of engineered clays with organic moiety of AP-W, as the
surface of hybrids resembled that of AP-W itself.
The possible surface adsorption of AP-W moiety was further sup-
ported by the zeta potential measurement utilizing light scattered elec-
trophoresis. Fig. 6 revealed zeta potentials of nanoclays (open circles),
AP-W (dash lines) and nano-biohybrids (solid lines). Before hybridiza-
tion, each nanoclays showed average zeta potential value of 48.2,
46.1 and 8.32 mV for IL, SC and LO, respectively, while AP-W had
zeta potential of 21.5 mV. It was worthy to note here that not only av-
erage zeta potential but also the curve shape of nano-biohybrids were
24 H.-J. Kim et al. / Applied Clay Science 134 (2016) 1925
Fig. 9. Schematic diagram on molecular rearrangement of AP-W on surface of engineered nanoclay for antibacterial activity.
overlapped with those of AP-W, regardless of starting nanoclays. If the
AP-W moiety was located inside the nanoclays rather outer surface,
the zeta potential of nano-biohybrids would be between those of
nanoclays and AP-W. In this nano-biohybrids, the surface of individual
nanoclay particle seemed to be evenly and thoroughly covered by the
AP-W moiety, and thus the zeta potential value of hybrids became
same to AP-W.
Generally, natural extract are composed of carbohydrates, lipids and
several small molecules like tannic acid, avonoids, polyphenols and
etc. If we simplify those organic molecules as single plate or rod, they
have approximate area between 1000 and 2000 m2/g. Taking into ac-
count specic surface area of nanoclays and AP-W content in corre-
sponding nano-biohybrids, we could roughly calculate that AP-W
covered surface of engineered nanoclays with a few layers of which
thickness was only a few nanometers. Therefore, we could suggest the
molecular arrangement of AP-W in each state as follows. In solution
state, each organic components of AP-W are separated by hydration;
in dried powder state, organic molecules get together through strong
van der Waals attraction to make large lumps; on the other hand, in
nano-biohybrids, a few layers of organic molecules are re-arranged on
the nanospace which is provided by engineered nanoclays in the form
of particle surface. Similar molecular rearrangement of organic com-
pounds on clay was suggested by Costantino et al. (1999). According
to their study, molecular orientation of methyl orange adsorbed on or
intercalated in anionic clays were fairly different from those in solution
or solid, exhibiting different optical properties depending on molecular
arrangement.
In order to verify our hypothesis on AP-W moiety rearrangement,
we carried out differential thermal analysis on dried AP-W and clay/
AP-W hybrids. As shown in Fig. 7(a), AP-W exhibited strong and
sharp exothermic peak at 467 C, which was attributed to the thermal
decomposition of organic components. As the organic molecules were
strongly bound to each other through van der Waals interaction, they
might be abruptly combusted at a certain temperature. On the other
hand, nano-biohybrids showed broad exothermic peak at around
300 C, which was attributed to the gradual combustion of organic mol-
ecules adsorbed on the surface of nanoclays. The lower combustion
temperature of AP-W in hybrids revealed that the inter-molecular inter-
action in hybrids were less than that in dried AP-W, corroborating that
organic molecules of AP-W were rearranged to adsorb on nanoclays'
surface with only a few layers. These results corresponded with their
TG analysis results in Fig. 2.
According to molecular rearrangement in hybrids, antibacterial
components of AP-W could be more exposed in hybrids than in dried
powder, and the antibacterial components could be locally concentrat-
ed around nanoclay particles, possibly exhibiting enhanced antibacteri-
al efcacy. Fig. 8 displayed antibacterial efcacy of AP-W and clay/AP-W
hybrids on both B. subtilis and E. coli obtained by bacterial colony
forming inhibition assay. For B. subtilis, AP-W showed 7.89% of bacterial
colony forming, standing for 92.1% of inhibition. The inhibition effect of
IL/AP-W and LO/AP-W signicantly increased compared with AP-W
only, showing 98.2% and 100% inhibition. For E. coli, all the three
nano-biohybrids showed strong bacteria colony forming inhibition
compared with AP-W itself: inhibition efciency was 89.3%, 99.6%,
99.4% and 97.8% for AP-W, IL/AP-W, SC/AP-W and LO/AP-W, respective-
ly. The enhanced bacterial inhibition of nano-biohybrids might be due
to the molecular rearrangement of AP-W on surface of nanoclays,
which exposed antibacterial active components at surface with en-
hanced local concentration (Fig. 9.)
4. Conclusion
We prepared bio-nanohybrids consisting of natural extract (AP-W)
and engineered nanoclays (IL, SC and LO). Natural extract obtained
with hot water showed antibacterial efcacy on B. subtilis and E. coli,
and thus it was hybridized with engineered nanoclays which were
pre-treated with acid or heat for pore volume and surface area enhance-
ment. According to X-ray diffraction, scanning electron microscopy and
zeta potential measurement, we could expect that the AP-W moiety
mainly existed on the surface of nanoclays. Differential thermal analysis
suggested possible molecular rearrangement of AP-W components in a
few layers on nanoclays, while AP-W makes large lumps in solid state
due to strong inter-molecular interaction. Antibacterial assay utilizing
bacterial colony forming inhibition assay, we found that clay/AP-W
nano-biohybrids generally possess higher antibacterial activity than
AP-W itself. The enhanced antibacterial activity of nano-biohybrids
was considered from molecular rearrangement of AP-W on nanoclays,
which increased exposure and local concentration of antibacterial com-
ponents around nanoclays.
Acknowledgment
This work was nancially supported by Ministry of Science, ICT and
Future Planning (MSIP) in Korean government and a Korea Industrial
Technology Association (KOITA) as A study on the programs to support
a collaborative research among industry, academia and research insti-
tutes (KOITA-2014-3), by Rural Development Administration (RDA,
Korea) as Cooperative Research Program for Agriculture Science &
Technology Development (PJ01050201).
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