Keywords:

Silica nanoparticles; Worms; Agricultural waste; Bioprocessing

Introduction

Agro-industrial wastes have recently attracted a great deal of attention as potential
sources of novel green alternatives such as biotransformation for fuels and other
materials. Many of these wastes contain amorphous silica that can be transformed into
crystalline nanoparticles of industrial interest.

Silicon is the most common element of the Earth’s surface after oxygen; this element
is released into the soil by chemical and biological processes [1]. Industrially speaking, silicon is the basis of semiconductors, glasses, ceramics,
plastics, elastomers, resins, mesoporous molecular sieves and catalysts, optical fibers
and coatings, insulators, moisture shields, photoluminescent polymers, fillers, cosmetics
and biomedical devices [2,3], among many other applications. The manufacture of these materials typically requires
high temperatures, high pressure and/or the use of caustic chemicals [4]. In contrast, in nature, silica architectures with delicate morphologies are generated
under ambient conditions [5]. Unicellular organisms, such as diatoms, use structuring and templating biomolecules
to produce silica shells that not only contain hierarchically ordered porous structures,
with dimensions ranging from the nanometer to the micrometer domain, but also possess
remarkable mechanical and structural properties [6-8]. Other approach involves the use Fosuarium oxysporum, a plant pathogenic fungus for the biotransformation of naturally occurring amorphous
plant bio-silica into quasi-spherical crystalline silica nanoparticles and its extracellular
leaching in the aqueous environment at room temperature [9]. An analysis suggested that extreme thermophilic bacteria within the genera thermus and hydrogenobacter are predominant components among the indigenous microbial community in siliceous
deposits. These bacteria seem to actively contribute to the rapid formation of huge
siliceous deposits [10].

In general, the biosilification products are commonly composed of amorphous silica
(opal-A, opal-CT and opal-C), and other crystal arrays such as cristobalite, trydimite
and quartz. In particular, amorphous silica is a dominant component in marine surface
sediment most of which is considered to be generated by the activity of living organism
[3,11]. Many scientists not only investigate the process underlying their formation, but
also aim to mimic these processes in order to obtain better control over the structure
and morphology of chemically produced silica [12-14]. The natural silica production receives increasing attention, since it holds the
key to the formation of silica morphologies with a dedicated organization of hierarchically
structure elements and the ability to synthesize such silica under ambient conditions
[15]. There exist many studies in silica bio-mineralization of simple aquatic life forms,
including unicellular organisms like diatoms, radiolaria and sinurophytes as multicellular
sponges [16-19]. In the soil, silica plays a major role in higher plants [20]. Many plants sequester silica in biogenic phytoliths and soils can accumulate significant
quantities of biogenic opal-A [21]. The silica absorbed for terrestrial plants is around a fraction of 1% of the dry
matter to several percent, and in some plants to 10% or even higher [22]. It is observed that in some grammineae as rice (Oryza sativa), silica constitutes 20–22% of its total production in the rice husk form [23]. Sugarcane bagasse contains around 5.08 to 7.08% of silica in dry matter basis [24], and coffee husk contains around 1 to 3% of silica in dry matter basis [25].

It is important to mention that the mechanical strength of plants resides greatly
on the cell wall, enabling them to achieve and maintain erect habit conductive to
light interception. There exist a relation in plants stress and the increased rigidity
of cell walls of plants grown with ample available silica [26,27]. When plants die, the silica is reincorporated into the soil where microorganisms
play an important role in the degradation of organic matter and in the release of
minerals nutrients [28,29], other important source that raises mineralization rate is earthworms producing biohumus.
Humus contains principally carbon, oxygen, hydrogen and minor proportion of other
minerals. These elements vary within the humic material in order to define chemical
characteristics of the original basis. There exists a symbiotic interaction between
earthworms and microorganisms that breakdown and fragment the organic matter progressively,
finally incorporating it into water-stable aggregates. The mineral nutrients in earthworm
casts and lining earthworm burrows are in a form readily available to plants. There
is evidence that interactions between earthworms and microorganisms not only provide
these available nutrients, but stimulate plant growth indirectly in others ways [30]. The digestive system of earthworms consists of a pharynx, esophagus and gizzard
(zone reception) followed by an anterior intestine that secrets enzymes and a posterior
intestine that absorb nutrients. During progress through this digestive system, there
is a dramatic increase in numbers of microorganism of up to 1,000 times. The digestive
systems of earthworms from different species, genera and families differ in detail,
but their gusts have a common basic structure. In different species earthworms Eisenia foetida is peculiar for its degradation rate [28]. Most studies of digestive enzymes in earthworms have been limited to the lumbricids.
Protease, lipase, amylase, lichenase, cellulose and chitinase activities also have
been described [31]. A wide range of microorganisms, including bacteria, algae, protozoa, actinomycetes,
fungi and even nematodes, are found commonly throughout the length of the earthworms
gut. The species of microbes in the gut are usually very similar to those in the surrounding
soil or organic matter upon which the earthworms feed [32-34]. Eisenia foetida is considered a machine to produce humus in conditions environmental control and
the microorganism can live in it in anoxic effect raising productivity in the material
expel. The biological mechanism to earthworms transforms organic matter and even so
carries out biosilification even is uncertain. Understanding the mechanism of silica
nanofabrication in other organisms is supported by a precursor namely biosilica monosilicic
acid Si(OH)4[35,36]. Proteins have been isolated from diatoms, sponges and grasses that are proposed
to be responsible for biosilification and have been sequenced and some of the key
amino acids identified. Other authors have studied the role of homopolymers of various
amino acids that are key constituents of the proteins lysine, histidine, arginine,
cysteine, proline and serine in the process biosilification [37]. This biopolymer acts as gelating agents of silica oligomers in silicic acid and
as flocculation agents in silica sols [38,39]. Other researches have been focused toward the development of efficient and innovative
fabrication methods to obtain inorganic materials using microorganisms from potential
cheap agro-industrial waste materials and could lead to an energy-conserving and economically
viable green approach toward the large-scale synthesis of oxide nanomaterials [9]. Thus, we develop a novel process for synthesis of diverse nanometric materials with
specific crystal arrays as precursors to agro-industrial wastes employing annelids,
an approach not used before, that permit to rise natural sources dedicated to production
particles’ mean biosilification.

Experimental

Three sources derived from agro-industrial activity were used: rice husk, coffee husk
and sugarcane bagasse. These by-products were added to vermicompost separately. The
annelid specimen used was Eisenia foetida. The environmental conditions ideal to the reproduction and control of these specimens
were set up: temperature at 20°C, moisture around 60–85%, aeration conditions and
darkness. The stabilization time was around 1 month and the humus obtained was dried
in a room at a temperature between 30 and 40°C. Then, the humus was sieved to size
0.5 mm approximately. Next, the sample was calcinated to eliminate the organic matter.
Three temperature levels were used: 500, 600 and 700°C for each agro-industrial waste
by 19 h. Calcinations were carried out in a muffle Lindberg/Eurotherm model 847 with
energy consumption of 0.17 kcal/h cm3, considering that the average density of the nanoparticles is around 0.1380 g/cm3, the consumed energy in the calcinations is approximately 1.2318 kcal/h by each gram
of recuperated SiO2 particles. This energy could be considered low in comparison with other conventional
process where fumed silica is manufactured with a consumed energy of until 15.48 kcal/h
by each gram of SiO2 particles [40]. Thus, the samples were tried with nitric and hydrochloric acids (volume ratio 3:1).
For each gram of calcinated sample, 4 ml of acid mix was added in order to eliminate
impurities (calcium, potassium, magnesium, manganese, iron, boron and phosphorous).
Acid treatment was achieved at 40°C by 4 h with constant stirring. Then, samples were
filtrated and washed with distilled water to neutralize them. Solids obtained were
dried at room temperature. All reagents employed were provided by Sigma–Aldrich.

Also, as a reference, SiO2 was obtained from the agro-industrial wastes without employing vermicompost bioprocess.
The extraction process to recuperate SiO2 is the same as described previously using calcination and acid treatment. In addition,
commercial synthetic SiO2 Aerosil® 130 provided by Degussa AG was employed to compare size and structure with
SiO2 nanoparticles produced in this research. Aerosil® 130 particles are amorphous SiO2 nanoparticles produced by high-temperature hydrolysis of silicon tetrachloride in
an oxygen gas flame [41]. Also, this research compares the particle features based on biotransformation process
with those synthesized using chemical process.

Results and Discussion

Figure 1 shows the FTIR spectroscopy analysis to the samples obtained from the agro-industrial
wastes: rice husk, coffee husk and sugarcane bagasse after vermicompost bioprocess,
calcinations and extraction process. The spectra present three important bands that
allow identifying the SiO2. At 1,080 cm−1, a band corresponds to stretching antisymmetric mode of Si–O–Si group. Around 800–810
cm−1, bending vibration mode is detected. This peak corresponds to Si–O group. Also, the
peak observed at 500 cm−1 corresponds to rocking mode of the Si–O group. At 3,500 and 1,640 cm−1 are observed stretching vibration mode of O–H and twisting vibration mode of H–O–H,
respectively. The peaks formed at different temperatures of calcination (500, 600
and 700°C) do not show important differences in the infrared analysis. The FTIR results
suggest that carbon from the organic matter is removed. Thus, silica is released,
and then the suboxides found after calcinations are separated correctly. In addition,
the SiO2 typical bands represent strong evidence of the efficiency in the synthesis process
based on biotransformation with annelids. Also, the bands corresponding to O–H vibration
mode and H–O–H twisting vibration mode indicate that particles synthesized remain
hydrated. As can be observed in Fig. 1, commercial sample Aerosil® 130 shows the same three bands corresponding to SiO2. However, the peaks at 3,500 and 1,640 cm−1 are weaker than the peaks belonging to the samples synthesized using vermicompost.
The latter allows to assume that nanoparticles obtained by vermicompost are further
hydrated than Aerosil®130 particles. This could be important for developing hybrid
materials, inasmuch as these types of materials use silanol groups to attach organic
moieties to inorganic Si [42-45].

Figure 1. IR bands to synthesis of nanoparticles SiO2 obtained for vermicompost. The Figure show the bands more representatives to SiO2 to in rice husk, sugarcane bagasse and coffee husk at temperature of calcination:
500, 600 and 700°C. Representative bands for Aerosil® 130 too are shown

Figures 2a–2f show TEM images for the SiO2 particles obtained using the synthesis by vermicompost. The morphology and structure
of these particles is not completely spherical. In addition, several particle clusters
are observed in these images. This agglomerates show different shapes of nanometric
size. Figure 2g–2h shows SiO2 particles obtained from the agro-industrial wastes without biotransformation. In
these pictures are observed elliptical particles with different diameter and a relatively
bigger size than particles analyzed in the Fig. 2a–2f. This reveals that bioprocesses employed contribute to reduce the size in SiO2 particles. We suggest that this size reduction is induced in earthworm gut with the
contribution from several microorganisms. Figure 3a–3b shows HRTEM images of SiO2 particles obtained through bioprocess. In these images, it is possible appreciate
the SiO2 particles in an atomic scale. Nanometric size and crystal structure are observed
in these images. Thus, it is shown that these particles present as well a crystalline
structure contrary to amorphous synthetic SiO2. In addition, the Fig. 3 shows the diffraction patterns and Miller index corresponding with these structures
in a selected area (A). Figures show the nanoparticles obtained from the precursors
that contain a higher content of SiO2, such as rice husk (Fig. 3a) and sugarcane bagasse (Fig. 3b). The indexation of nanoparticles obtained from rice husk shows SiO2 with crystal arrangement related to α hexagonal quartz with the next lattice parameters:
0.340 nm (1−1 0 0), 0.243 nm (1 1−2 0) and 0.198 nm (2 0−2 0). In the case of nanoparticles
obtained from sugarcane bagasse, SiO2 posses a tetragonal arrangement with the next lattice parameters: 0.200 nm (2 0 0),
0.199 nm (0 2 0) and 0.142 nm (2 2 0). In accord with these results, it is possible
to suppose an important contribution of the vermicompost bioprocess in order to give
crystalline phases in SiO2 nanoparticles.

Figure 2. TEM images to synthesis of SiO2 obtained with bioprocess (a–f) and without bioprocess (g–h). Figure 2 shows differences in size, form and dispersity to nanoparticles synthesized for both
methods employed, with bioprocess (a–f) and without bioprocess (g–h)

In order to confirm the TEM and HRTEM results, particle size was analyzed employing
dynamic light scattering (DLS). The results obtained by this technique are summarized
in Table 1. The mean diameter of particles synthesized employing bioprocess is around 81 nm
and do not show polydispersity. In contrast, the mean diameter of particles synthesized
without using bioprocess is between 152 and 254 nm with low polydispersity. This is
in agreement with TEM and HRTEM images and confirms the contribution of bioprocess
in particle size reduction to achieve a nanometric range. It is important to emphasize
the presence of agglomerates in the particles obtained without biotransformation,
which are not observed in the particles obtained by employing bioprocess. Thus, as
well as the bioprocess contribution to particle size reduction also provides specific
arrangement to the particles. It is suggested that this effect is produced by the
microorganisms which are part of metabolism in the earthworm. The size and morphology
of particle in the biosilification process are related with the concentration of inorganic
phosphate and polyamines, both compounds play an important role in order to catalyze
the polycondensation of silanol groups [46]. In vermicompost, the concentrations of these compounds vary with the microbial population.
Thus, it is supposed that the size and morphology of SiO2 nanoparticles could be changed in this kind of bioprocess.

Table 1. Diameter mean using DLS to particles SiO2 synthesized with bioprocess and without bioprocess

Table 2 shows the elemental analysis for SiO2 particles synthesized by bioprocess; a significant amount of silicon and oxygen weight
percent is observed in all samples obtained with the precursors employed: 41.62% Si
and 52.90% O for particles obtained from rice husk; 23.37% Si and 42.37% O in particles
synthesized from sugarcane bagasse and 41.68% Si and 55.39% O to particles extracted
from coffee husk. Also, in the composition are observed other elements with lower
percent than silica and oxygen, such as sodium, magnesium, aluminum, potassium and
calcium. These elements are typical in the biomineralization process and take part
in the crystalline phases growing that are produced with this biological mechanism.

Table 2. EDS to particles SiO2 synthesized with bioprocess and without bioprocess

Figure 4 shows the diffractrograms generated from the particles obtained using the three precursors:
rice husk (a–b), sugar cane bagasse (c–d) and coffee husk (e–f). Crystalline nanoparticles
are obtained using two different calcination temperatures: 500°C (a, c and e) and
700°C (b, d and f). Figure 4a shows the diffractrogram corresponding to particles obtained from rice husk at 500°C.
In this figure, it is observed an amorphous structure when the bioprocess is not used
during the synthesis. In contrast, crystalline phases are found when the bioprocess
is employed. In this diffractrogram, it is possible to identify typical peaks corresponding
to diffraction planes from quartz and other polymorphic structures of SiO2, such as trydimite. Also, erionite and albite diffraction planes corresponding to
aluminum silicate groups are found. These results support the proposal that biomineralization
mechanism is achieved through earthworms contributing in the atomic arrangement and
modifying the original structure toward specific crystalline structures. Figure 4b shows the diffractrogram corresponding to particles obtained from rice husk at 700°C.
In spite of the high temperature, even amorphous structure is observed when bioprocess
is not used and only one diffraction plane corresponding at low quartz is shown. This
crystal is formed probably by temperature effect. In the same Fig. (4b), it is shown the diffractrogram corresponding to the samples treated with bioprocess
at 700°C. In this figure, the crystalline structure is related to low quartz, trydimite
and albite.

Figure 4. X-ray diffraction to synthesis of nanoparticles SiO2 obtained with bioprocess and without bioprocess using as precursors: rice husk (a–b) sugarcane bagasse (c–d) and coffee husk (e–f) to two temperatures of calcinations: 500 and 700°C. It shows a change in the crystallinity
of the nanoparticles synthesized, since the different precursors used and varying
the temperature of calcination and employing or not employing the bioprocess

Figure 4c and 4d show the diffractrograms corresponding to particles obtained from sugarcane bagasse.
Crystalline phases are present in both procedures (with and without bioprocess). However,
some diffraction planes are not the same in both processes. This allows to assume
that the calcination temperature and earthworm metabolism play an important role to
generate different crystalline phases that involve not only silicon and oxygen atoms,
but other elements take part, as well. In Fig. 4c and 4d, by comparing the diffractrograms (samples obtained without bioprocess at 500 and
700°C), it is possible to observe that the same crystalline phases are found: quartz
(different crystallographic planes) and trydimite. However, the diffractrograms corresponding
to samples obtained via bioprocess show different crystalline phases, such as: zinc
phosphate, aluminum phosphate at 500°C and albite in 700°C.

It is important to mention that silica shows several polyphorms depending on temperature
and pressure. Thus, although bioprocess conditions employed to obtain SiO2 particles are favorable to inducing α hexagonal quartz (corroborated by X-ray diffraction),
it is possible to find others metastable polyphorms such as tetragonal arrangement
(268–1,470°C). This structure belongs to β crystobalite [3]. Therefore, some SiO2 nanoparticles obtained from sugarcane bagasse can be found with transitions of hexagonal
to tetragonal phase. Thus, tetragonal arrangement does not appear in X-ray diffraction;
however, in some nanoparticles characterized by HRTEM (Fig. 3b) it is identified.

Diffractrograms corresponding to particles obtained from coffee husk are shown in
Fig. 4e and 4f. Significant changes in both processes (using bioprocess and without employing bioprocess),
which are produced by temperature and metabolism in earthworms, are observed. At 500°C,
by employing bioprocess, the diffraction peaks detected are related to quartz, trydimite,
sanidine and magnesium nickel hydride, meanwhile without bioprocesses diffraction
peaks appear, corresponding to trydimite, gypsum and calcium aluminim oxide hydrate.
At 700°C, by employing bioprocess, quartz, aluminim phosphate and caminite are found,
meanwhile without bioprocess appears: calcium aluminim oxide hydrate, trikalsilite
and quartz.

Table 3 shows the crystallization percent, considering precursor type and calcination temperature
employed. Crystallization degree is obtained for both: particles obtained via bioprocess
and particles synthesized without using bioprocess. These values are calculated by
considering mean low curve area in the peaks from XRD. It is observed that, for rice
husk, the effect in the temperature is not enough as to transform the amorphous phase.
However, the metabolic effect in earthworms increases the crystalline phase up to
28.47% (500°C) and 16.69%(700°C). In sugarcane bagasse, the effect is the opposite,
the crystalline phase is low for the samples obtained via bioprocess and crystallinity
percent increases for particles obtained without bioprocess. In addition, diffractrograms
obtained from sugarcane bagasse show that some crystalline phases do not correspond
to SiO2, such as zinc phosphate and aluminim phosphate; however, when the bioprocess is not
used, particles show a considerable number of SiO2 polymorphs. This suggests that most of the oxygen needed to form SiO2 may be consumed by the earthworms during their metabolic process. This assumption
is in agreement with EDS analysis, inasmuch as EDS results for particles obtained
from sugar baggasse show lower percent of oxygen than particles synthesized from rice
husk and coffee husk. With respect to the crystallization percent in the particles
obtained from coffee husk, there is not a clear tendency. For the samples obtained
using bioprocess at 500°C, the crystallization percent is lower than that of samples
synthesized without bioprocess. Probably, bioprocessing contributes to provide certain
array in the nanoparticles, depending on organic matter, oxygen and other inorganic
compounds present in the precursors. This would confirm that temperature is not the
most important factor for the crystallinity in these kinds of nanoparticles.

Table 3. Percent crystallization in particles SiO2 synthesized with bioprocess and without bioprocess

Conclusion

The microbial population in annelids is very important to achieve the biotransformation
of the amorphous silica naturally present in the analyzed agro-industrial wastes.
Characteristics of the organic matter exposed to a broad variety of microorganisms,
as well as the method employed for the fragmentation and minerals release, represent
key factors to understand the biocrystallization. Our results reveal a novel synthesis
method to obtain SiO2 crystalline nanoparticles using annelids’ biotransformation by employing agro-industrial
wastes. The approach represents an inexpensive and relatively eco-friendly technology
in comparison with standard chemical methods. By taking into account the biological
aspects of the production of SiO2 nanoparticles with specific crystal arrangement using annelids allows to extend the
number of living organism dedicate to biosilification, in addition to open an interesting
field toward the knowledge of new annelid bioprocesses with a primitive metabolism,
as potential alternative natural nanotechnology bioprocesses to synthesize nanoparticles
and nanostructures, for the particles obtained through biotransformation by annelids
show similar characteristics than synthetic SiO2 Aerosil®130 such as size, composition and polydispersity. While synthetic particles
possess amorphous structure the particles synthesized using vermicompost present different
disables characteristics such as crystalline (different polymorphism) and nanometric
dimension.

Acknowledgments

The authors are grateful to Ms. Maria de Lourdes Palma for her assistance in TEM,
to Dr. Genoveva Hernández-Padron for her assistance in IR analysis, to Dr. Eric Rivera
for his assistance in XRD, to CINVESTAV Querétaro, particularly to Dr. S. Jimenez
and Mr. F Rodriguez for their assistance in some measurements and to DGEST and Consejo
Nacional de Ciencia y Tecnologia (CONACyT), Mexico, for the economical support through
the projects P333-05 and JI-58232, respectively. Financial support from the National
Council for Science and Technology of Mexico (CONACYT) (PhD Scholarship to A.E.-G.)
is gratefully acknowledged.

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