Sustainable synthesis of amino acids by catalytic fixation of molecular dinitrogen and carbon dioxide

Article abstract of DOI:10.1039/c7gc02911j

The industrial process of nitrogen fixation is complex and results in a huge economic and environmental impact. It requires a catalyst and high temperature and pressure to induce the rupture of the strong N-N bond and subsequent hydrogenation. On the other hand, carbon dioxide removal from the atmosphere has become a priority objective due to the high amount of global carbon dioxide emissions (i.e. 36 200 million tons in 2015). In this work, we fix nitrogen from N₂ and carbon from CO₂ and CH₄ to obtain both glycine and alanine (d/l racemic mixture), the two simplest amino acids. The synthesis, catalyzed by polarized hydroxyapatite under UV light irradiation and conducted in an inert reaction chamber, starts from a simple gas mixture containing N₂, CO₂, CH₄ and H₂O and uses mild reaction conditions. At atmospheric pressure and 95 °C, the glycine and alanine molar yields with respect to CH₄ or CO₂ are about 1.9% and 1.6%, respectively, but they grow to 3.4% and 2.4%, when the pressure increases to 6 bar and the temperature is maintained at 95 °C. Besides, the minimum temperature required for the successful production of detectable amounts of amino acids is 75 °C. Accordingly, an artificial photosynthetic process has been developed by using an electrophotocatalyst based on hydroxyapatite thermally and electrically stimulated and coated with zirconyl chloride and a phosphonate. The synthesis of amino acids by direct fixation of nitrogen and carbon from gas mixtures opens new avenues regarding the nitrogen fixation for industrial purposes and the recycling of carbon dioxide.

Full text of DOI:10.1039/c7gc02911j

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DOI: 10.1039/C7GC02911J
Sustainable synthesis of amino acids by catalytic
fixation of molecular dinitrogen and carbon
dioxide
Manuel Rivas1, Luís J. del Valle1,2, Pau Turon1,3*, Carlos
Alemán1,2*, and Jordi Puiggalí1,2*
1 Departament d’Enginyeria Química, EEBE, Universitat Politècnica de Catalunya, C/
Eduard Maristany, 10-14, 08019, Barcelona, Spain.
2 Barcelona Research Center for Multiscale Science and Engineering, Universitat
Politꢀcnica de Catalunya, C/ Eduard Maristany, 10-14, Ed. C, 08019, Barcelona, Spain
3 B. Braun Surgical, S.A. Carretera de Terrasa 121, 08191 Rubí (Barcelona), Spain
*Correspondence to: pau.turon@bbraun.com, carlos.aleman@upc.edu and
jordi.puiggali@upc.edu
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Abstract
Industrial process of nitrogen fixation is complex and results in a huge economic and
environmental impact. It requires a catalyst, high temperature and pressure to induce the
rupture of the strong N–N bond and subsequent hydrogenation. On the other hand, carbon
dioxide removal from the atmosphere has become a priority objective due to the high
amount of global carbon dioxide emissions (i.e. 36,200 millions of tons in 2015). In this
work, we fix nitrogen from N2 and carbon from CO2 and CH4 to obtain both glycine and
alanine (D/L racemic mixture), the two simplest amino acids. The synthesis, catalyzed by
polarized hydroxyapatite under UV light irradiation and conducted in an inert reaction
chamber, starts from a simple gas mixture containing N2, CO2, CH4 and H2O and uses
mild reaction conditions. At atmospheric pressure and 95 ºC, the glycine and alanine
molar yields with respect to CH4 or CO2 are about 1.9% and 1.6%, respectively but they
grow up to 3.4% and 2.4%, when the pressure increases to 6 bars and temperature is
maintained at 95 ºC. Besides, the minimum temperature required for the successful
production of detectable amounts of amino acids is 75 ºC. Accordingly, an artificial
photosynthetic process has been developed by using an electrophotocatalyst based on
hydroxyapatite thermally and electrically stimulated and coated with zirconyl chloride
and phosphonate. The synthesis of amino acids by direct fixation of nitrogen and carbon
from gas mixtures opens new venues regarding the nitrogen fixation for industrial
purposes and the recycling of carbon dioxide.
2

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Introduction
Nitrogen fixation is an extremely relevant process from an industrial perspective.1 It is
difficult to overestimate the impact of the Haber-Bosch process that yields ammonia and
further on results in more than 450 million tons of fertilizers per year, using more than
1% of global energy supply.2 Moreover, the synthesis of ammonia from nitrogen is
constantly under research and only in the last decade new catalysts based on ruthenium
and zirconium have been developed.3 However, the environmental impact of the Haber-
Bosch process is not negligible as natural gas or coal are used as an energy source (i.e.
1.87 tons of CO2 are released per ton of ammonia produced and globally 245 million tons
of CO2 were released by the ammonia industry in 2010, equivalent to about 50% of the
UK CO2 emissions).4 Thus, the outstanding benefits of nitrogen fixation are shadowed by
their impact on environmental contamination. Furthermore, catalytic reduction of
dinitrogen under mild conditions has been achieved by using molybdenum catalysts.
Shilov, in the 1980s, obtained a mixture of hydrazine and ammonia5 and Schrock and co-
workers6,7 converted dinitrogen into ammonia with excellent yield using protons and
electrons. Very recently, Kuriyama et al.8 reported the catalytic reduction of molecular
dinitrogen into ammonia and hydrazine by using the iron complexes bearing an anionic
ligand as catalysts at mild reaction conditions. However, to our knowledge, the nitrogen
fixation into amino acids (AAs) using hydroxyapatite (HAp) has not been reported yet.
On the other hand, CO2 recycling is an absolute necessity for our society knowing that
whose accumulation in the atmosphere is now approaching 1 Tera ton.9 Significant steps
have been done towards the utilization of CO2 in order to convert it in valuable chemicals
(150 Mt urea, 100 Mt methanol, 70 Mt salicylic acid, 9.7 Mt formaldehyde and formic
acid 0.7 Mt are the most produced).10-12 In last years, catalytic reactions via carbon
dioxide fixation are gaining a prominent role as representative green processes with
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enhanced sustainability.13-16 However, by learning from nature, photosynthesis as
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performed by living organisms is the carbon fixation process par excellence. Efforts to
mimic it synthetically have been elusive through time. The development of
photosynthetic processes requires significant advances in new materials for light
harvesting and the development of fast, stable, and efficient electrocatalysts.17,18
In this work, we introduce a new catalyst based on permanently polarized crystalline
hydroxyapatite (p-cHAp) with enhanced electrical and photochemical properties that
allows the coupling of nitrogen and carbon fixation process. Such catalyst family opens
an interesting field of research where simple gas mixtures that usually do not react among
themselves are combined to yield basic organic molecules, such as AAs, one of the main
building blocks of life. More specifically, in this work we prove that glycine (Gly) and
alanine (Ala) are produced at atmospheric pressure through an artificial photosynthetic
nitrogen and carbon fixation reaction, starting from a weakly reducing atmosphere (N2,
H2O, CO2 and CH4) and using UV radiation as a source of energy. This reaction
represents a very simple alternative to the costly chemical and enzymatic processes used
to produce commercially Gly and Ala.
Results and discussion
The catalyst design arises from the properties induced by the thermally stimulated
polarization process to cHAp (discussed below). Specifically, p-cHAp exhibits electrical
and electrochemical properties, both associated with charge transport phenomena, that
remain practically unaltered with time (i.e. p-cHAp is a permanently polarization
mineral). In order to take advantage of p-cHAp properties, other materials were necessary
to complete the whole catalytic system. Thus, the requirements of the catalyst were, on
one hand, a material able to transform an energy source, as for example UV radiation,
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into electron/hole pairs and, on the other hand, materials to put in contact the gases used
as reactants with the catalyst (i.e. adsorb the gases), which was essential to conduct the
reactions. Obviously, the success of the catalyst was also based on avoid unfavourable
synergies among the materials when they were combined. After evaluate different
options, two materials, which correspond to those presented in this work, were selected:
zirconia and a phosphonic acid with chelating properties. Details about the roles played
by each component of the catalytic system are given through the whole work.
Synthesis of amino acids at atmospheric pressure from an electrophotocatalyst
based on polarized hydroxyapatite
The new catalytic system, hereafter denoted p-cHAp/Phos-ZC-Phos, is based on p-
cHAp that is, subsequently, coated with 3-layers. One of them, the intermediate coating
layer, made of zirconyl chloride (ZC), which probably hydrolyzes into zirconia, and two
of them made of amino tris(methylene phosphonic acid) (Phos). p-cHAp is a mineral with
electrical and electrochemical activity obtained by applying a thermally and electrically
stimulated polarization process to crystalline hydroxyapatite (cHAp). More specifically,
p-cHAp is prepared using a three-step process: (1) cHAp is obtained by adding
(NH4)2HPO4 in de-ionized water to Ca(NO3)2 in ethanol, and applying hydrothermal
conditions to the resulting suspension during 24 h; (2) sintered cHAp is achieved by
heating the cHAp synthesized in step 1 at 1000 ºC for 2 h in air; and (3) p-cHAp was
produced by sandwiching discs of sintered cHAp (step 2) between stainless steel plates
and polarizing for 1 h with a constant DC field of 3 kV/cm at 1000 ºC. The bulk
resistance of p-HAp (105 ·cm2) is significantly lower than that of as prepared HAp
(108 ·cm2), indicating that that the ionic conductivity increases by three orders of
magnitude when the thermally stimulated polarization process is applied. Moreover, the
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ionic conductivity is preserved along time (i.e. the bulk resistance increases bDyOoI: n10l.y10349%/C7GC02911J
after 3 months). Similarly, the electrochemical activity of p-HAp is 150% higher than
that of as prepared HAp, which evidences that the polarization treatment facilitates the
diffusion of ions through the mineral matrix. On the other hand, the adsorption capacity
of phosphonates onto p-cHAp is around four times higher than onto cHAp, ensuring an
efficient contact between the p-cHAp and the Phos-ZC-Phos 3-layer (Figure 1a). Besides,
ZC and Phos layers have been used to adsorb the gaseous reactants, essentially N2 and
CO2, facilitating their reactivity. Details about the preparation of the catalyst and its
components are provided the Supplementary Information.
The synthesis of AAs was carried out in an inert reaction chamber with a weakly
reducing atmosphere constituted by N2 (0.33 bar), CO2 (0.33 bar), CH4 (0.33 bar) and
liquid H2O, using an UV lamp directly irradiating the catalyst and gas mixture (Figure
1b). The formation of primary amines adsorbed into the solid substrate was shown by
positive ninhydrin tests through the development of purple spots inside the catalyst
recovered after reaction (Figure 1c, left). Amine compounds were well dissolved in an
acetone solution after vigorous stirring (Figure 1c, middle), contrasting with the
uncolored solid and solution (Figure 1c, right) observed in many other assayed reaction
conditions and catalytic systems (see below).
After 72 h at 95 ºC, the clean synthesis of both Gly and Ala is demonstrated by the
1
NMR spectra displayed in Figure 2. The H NMR spectrum of samples obtained by
dissolving the catalyst and products of reaction (Figure 2a) shows the presence of the
catalyst signal corresponding to Phos methylene group (doublet at 3.79-3.76 ppm) and
the signals corresponding to newly produced AAs such as methylene protons (singlet at
3.65 ppm) of Gly and both methine (quadruplet at 3.91-3.85 ppm) and methyl (doublet at
1.54-1.52 ppm) groups of Ala. The same compounds are also observed in the solid state
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13
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C NMR spectrum (Figure 2b), where only peaks assigned to the Phos (54.34DaOnI:d10.51033.90/C07GC02911J
ppm), Gly (171.95 and 41.26 ppm) and Ala (175.25, 50.25 and 16.01 ppm) units are
detected. 31P NMR spectrum (Figure 2c) shows the presence of the p-HAp (7.21ppm) and
Phos (-0.03 ppm) peaks, additional signals related to products coming from
decomposition of the catalyst were not detected. With respect to CH4 or CO2, the Gly and
Ala molar yields at 95 ºC after 72 h are 1.9% and 1.6%, respectively (Table 1). Instead,
after 24 h at 95 ºC no trace of Gly and Ala are detected by NMR. Furthermore, the
formation of AAs is unsuccessful without the sustained exposure to the UV radiation,
which appears to be a fundamental issue to generate radicals (e.g. CH3 and OH) needed
for further reaction intermediates towards the final yielding of Ala and Gly.
Photoemission spectroscopy (XPS) analyses show that amines in AAs come from the
molecular nitrogen and not from a hypothetical decomposition of the Phos. The N 1s
spectra registered for different representative samples (Figure 3a) indicate that the peak at
399.5 eV, which is ascribed to the C–N of Phos, is observed with practically the same
intensity when both negative and positive reactions (i.e. without and with sustained
exposure to UV radiation) are monitored. However, only in the latter case bands at lower
binding energies appear due to the formation of deprotonated and protonated amino
groups (401.2 and 404.5 eV, respectively).19 Furthermore, the amount of atomic nitrogen
increased from 0% to 2.75-2.97% when the Phos-ZC-Phos 3-layer coating was added to
the p-cHAp substrate. The nitrogen percentage, after positive reaction, was increased up
to 6.2% , which corroborates the fact that formed AAs remain adsorbed into the catalysts.
The p-cHAp/Phos-ZC-Phos catalyst exhibits a rough and relatively irregular surface
morphology (Figure 3b), which changed after reaction due to the sporadic formation of
micrometric prismatic crystals with the hexagonal basal plane parallel to the disk surface
(Figure 3c). Although the nynhydrin test reflects the presence of AAs adsorbed inside the
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catalyst, micrographs also demonstrate the growth of AA crystals onto the surface of the
catalytic system. This behavior is consistent with the capacity of organophosphonate
films for inducing crystallization of oriented molecular sieves as proved by the growth of
stable, vertically oriented and one dimensional aluminum phosphate crystals.20,21 On the
other hand, the formation of AAs has also been proved by FTIR spectroscopy, which
shows intense absorption bands in the amine region of the corresponding spectra, and
characteristic X-ray diffraction patterns. Finally, chiral high performance liquid
chromatography (HPLC) analyses were carried out to quantify the ratio of D- and L-Ala
adsorbed into the catalysts. As it was expected, after dissolution of the catalysts after
reaction in a 0.1 M HCl solution with 50 mM NaCl, a racemic D-Ala:L-Ala mixture was
determined.
The effect of varying the pressure and the temperature
In order to increase the rate of reaction, the chamber pressure was increased up to 6
bar by introducing 2 bar of each feeding reaction gas, while temperature was kept fixed at
95 ºC. After 24 h, the molar yields, expressed with respect to CH4 or CO2, are 1.9% and
1.3% for Gly and Ala, respectively (Table 1), which are pretty similar to those achieved
at atmospheric pressure after 72 h. Moreover, at high pressure Gly and Ala molar yields
increase to 3.4% and 2.4%, respectively, after 96 h. Amazingly, the reported molar yields
of Gly and Ala, based on the same carbonaceous species, by the action of electric
discharges on a mixture of CH4, N2, water and traces of ammonia were only 0.26% and
0.71%, respectively, increasing to 1.55% when the sum of the 10 produced AAs was
considered.22
The effects of both the reaction time and the temperature in this catalytic process were
studied considering a chamber pressure of 6 bar. The variation of the Gly/Phos and
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Ala/Phos ratios, which were determined from the areas of signals correspondiDnOgI: 1t0o.10C39H/C27GC02911J
(Gly and Phos) and CH (Ala) protons in 1H NMR spectra, against the reaction time (from
2 to 96 h) reflects that Gly is produced first (Figure 3a), while Ala is subsequently
derived from this simple AA. Thus, the Gly/Ala ratio decreases from 5.4 to 2.2, while a
continuous increase of the Gly/Phos ratio with the reaction time is nevertheless observed
(i.e. from 0.8 to 4.5). On the other hand, the minimum temperature required for the
successful production of detectable amounts of Gly and Ala after 24 hours is 75 ºC
(Figure 3b). The Ala/Phos ratio grows progressively with the reaction temperature while
the Gly/Phos ratio decreases at 105 ºC due to the conversion of Gly into Ala, even though
the (Gly+Ala)/Phos ratio still increases.
Influence of changes on the catalytic system
Substitution of p-cHAp by p-aHAp, which is obtained replacing cHAp by amorphous
hydroxyapatite (aHAp) and applying the same thermally and electrically stimulated
polarization process, did not affect the production of AAs, even though the yield of Gly
and Ala decreases to 0.92% and 0.65%, respectively (Table 1). The latter has been
attributed to the partial thermal decomposition of aHAp during the sintering process,
which gives place to the formation of a -tricalcium phosphate23 causing a reduction of
the electrochemical activity with respect to samples obtained using cHAp. Conversely,
negative results were obtained when p-cHAp was replaced by sintered s-cHAp (i.e. non-
polarized), polarized silicates or aluminosilicates (mica), probing that the HAp and its
electrochemical activity plays an important role in the catalytic mechanism.
For the sake of completeness, the effectiveness of the substitution of the Phos-ZC-
Phos 3-layer coating by two possible combinations of 2-layer coatings (Phos-ZC and ZC-
Phos), by Phos monolayers or by Phos/ZC mixture were also assayed. In all cases,
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negative results were obtained demonstrating the importance of the 3-layered aDrcOhI:i1t0e.1c0t3u9/rCe7GC02911J
and thus discarding a process based on the photodecomposotion of Phos. In addition, both
the capability of incorporating water molecules24 and the effective role of
metal/phosphonate compounds as chelating ligands25 are noteworthy due to the presence
of free phosphonic acid groups. Results obtained after introducing the above mentioned
changes in the 3-layered p-HAp catalyst are summarized in Figure 4.
Molecular nitrogen and carbon dioxide fixation
Different experiments (Table S5) have also been performed in order to determine the
reactants that are essential to obtain AAs under the defined experimental conditions (UV
radiation, 6 bar, 95 ºC, and 24 h). Results indicate that N2, CH4, CO2 and H2O were all
necessary, because the removal of any of them led to negative ninhydrin tests as well as
an absence of the AA peaks in the corresponding NMR spectra (Figure 4). As it was
expected, the yielding of AAs systematically correlates with the pressure of N2 in the
reaction chamber (Table 1).
These observations support the idea that the nitrogen source for the AAs is N2 but not
Phos. Furthermore, the experiments performed without incorporating the coating to the
catalyst failed to produce AAs (Table S5), whilst XPS measures evidenced that N2 was
adsorbed by the Phos layers of the coating (Table S3). Taken together, these results
support that the catalyst coating facilitates both the adsorption and further reactivity of
N2. This point is particularly relevant since most of the reported AAs syntheses are based
on ammoniacal solutions as nitrogen source. It is well-known that N2 is fixed by means of
some specific bacteria by the action of enzymes that have metal-sulfide active sites.26
Numerous efforts have been made to develop metal-based catalysts to coordinate and
activate N2 through formation of metal–dinitrogen complexes with limited success.27 The
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splitting of the N–N bond in bridging dinitrogen complexes was first acDhOieI:v10e.1d039b/Cy7GC02911J
Cummins, who showed how three-coordinate molybdenum amido-complexes react with
dinitrogen at –35 ºC to yield an unstable dinitrogen bridging complex that, subsequently,
breaks down into two molecules of nitride-complex.28 Although some additional progress
has been made in this area, the fixation of atmospheric N2 remains a challenge because of
the limited reactivity and the harsh conditions required to convert dinitrogen into useful
nitrogen-containing organic compounds. The p-cHAp/Phos-ZC-Phos catalyst overcomes
such drawbacks rendering nitrogen containing compounds under relatively mild reaction
conditions.
Regarding carbon fixation, experiments performed without CH4 in the initial mixture
failed to produce AAs and, therefore, proves its role as a carbon source for the methyl
group of Ala and for the methylene group of Gly (i.e. it again discards a synthetic route
based on the decomposition of Phos). On the other hand, the carboxylic groups of the two
AAs would appear to come from CO2. The transformation of CO2 into organic moieties
under UV irradiation, mimicking photosynthesis, represents a very interesting approach
for the minimization of CO2 emissions and for developing sustainable industrial
processes. Thus, the catalytic process reported in this work fulfils the very ambitious
goals of artificial photosynthesis: to use earth-abundant and inexpensive materials
converting them into highly appreciated organic compounds (i.e. AAs) of industrial and
environmental significance. Although zirconium phosphonates are able to adsorb CO2,29
HAp has shown a noticeable capacity to incorporate carbonate and hydrogen carbonate
from atmospheric CO2 when dissolved in water23,30 facilitating the formation of
carboxylic groups. In this work, XPS experiments prove that both the p-cHAp substrate
and the ZC layer are responsible for the CO2 adsorption onto the catalyst, while Phos
layers do not contribute.
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Finally, water also displays a fundamental role in the synthesis of AAs (Figure 4). In
fact, water molecules do not appear as an element source for AAs but they have a
significant influence on the mechanism contributing to the formation of OH radicals and
dissolving CO2. Furthermore, hydration of Phos layers is also expected to play a major
role in ionic mobility through such Phos layers.31
Mechanism
Similar syntheses of AAs (mainly Gly and Ala) have been demonstrated when
ammoniacal solutions saturated with methane were exposed to near-UV irradiation ( <
405 nm) and even natural sunlight in the presence of a catalyst platinized TiO2
powder.32,33 The total yield of AAs after 72 h irradiation with a xenon lamp was 472
nmol, and after 312 h of exposure to sunlight was 2140 nmol, which represented a very
low conversion (molar yield: < 0.01%).32 The reaction mechanism proposed in that case
was based on the electron/hole (e-/h+) pair produced when TiO2 was irradiated with light
having higher energy of its band gap. However, results were negative when CO2 was
employed as a carbon source instead of CH4. The present work reveals an alternative way
by obtaining organic nitrogen from molecular nitrogen instead of ammonia.
Our first hypothesis for the reaction mechanism of the processes presented in this
work is the occurrence of water splitting photocatalysis on the surface of p-cHAp, in a
way that resembles the one on TiO2 electrodes early reported by Fujishima and
Honda’s.34 Thus, the reaction is not successful without water, which is consistent with the
fact that water plays an important role in the mechanism. The hydroxyl radical (OH)
photoproduction on those mineral surfaces is a well-known process that can be described
as follows.35 The photon energy is adsorbed and electrons are excited to the conduction
band, leaving holes in the valence band (h+). These electron-hole pairs travel to the
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surface of the substrate to participate in water splitting. Thus, the transfer of electron
from water molecules to valence band holes forms OH (Eq 1), whereas in the
corresponding reductive reaction the transfer of electrons to O2 molecules forms
superoxide radical anions (O2 ).
H2O  h OH H
(1)
Because of the electrical properties of p-cHAp, we have hypothesized a similar
process for the water photolysis on this mineral, regardless of possible variations in the
intermediate stages of the photooxidation and photoreduction reactions. It is worth noting
that p-cHAp presents vacancies that originate from the transformation of hydrogen
phosphate into phosphate during the thermally stimulated polarization process, as proved
by NMR experiments. Therefore, its catalytic role should be comparable to that of
activated TiO2 surfaces. Furthermore, Nishikawa36 demonstrated that, after heat
treatment, HAp becomes electro-conductive by UV irradiation, leading to the formation
of stable but very reactive O2 by altering its own PO34 groups. These features together
with electrochemical activity of p-cHAp, in combination with the Phos-ZC-Phos layers
could originate an electrochemical capacitor,37 explaining the electrophotocatalytic
activation of the p-cHAp/Phos-ZC-Phos system. Accordingly, the mobility of O2
,
which may originate OH in an aqueous environment, H+ and vacancies could be the
driving forces of this electrochemical mechanism. The high electrochemical activity of p-
cHAp in comparison to as prepared and sintered cHAp is proved in Figure 5, which
represents the voltammetric charge per surface unit (Q) against the number of
consecutive oxidation-reduction cycles. As it can be seen, Q is significantly higher for p-
HAp than for the two non-polarized samples, such difference increasing with the number
of redox processes.
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As the hydroxyl radicals are very oxidative in nature, we suggest several possible
reaction steps that could lead to production of carbonaceous intermediates required for
production of Gly and Ala:
(2)
(3)
CH4  OH CH3  H2O
CH3 OH :CH2  H2O
On the other hand, the one electron reduction of CO2 into its radical anion, CO2 , is
proposed as a net electron transfer process possible at the interface between p-cHAp and
Phos-ZC-Phos:
CO2  e  CO2
(4)
Although this process is expected to be disfavored, because of both the
thermodynamic stability of CO2 and the energy required for a stereochemical change in
geometry from a linear to a bent configuration, it has frequently been observed on HAp
surfaces.23,30 This steps is consistent with the analysis of volatile compounds, which
revealed the formation of small amounts of aldehydes. Thus, around 600 g/m3 of
acetaldehyde and 90 g/m3 of formaldehyde were detected after 16 h of reaction at 95 ºC.
Besides, a number of mechanisms have been reported to explain the electrochemical
transformation of N2 into NH3 through different steps that involve the N–N cleavage and
the N protonation onto the catalyst surface.38 Within this context, such a transformation
could follow associative or dissociative mechanisms. In the associative mechanism, N2
molecules adsorbed onto the catalysts are protonated sequentially without breaking the
N–N bond until the first NH3 molecule is produced, as it occurs in the reaction of N2-
fixing by enzymes,39 while in the dissociative mechanism, the N2 molecules dissociate
immediately and nitrogen radicals are protonated as they are in the Haber-Bosch
process.40 After this the resulting NH3 may be easily oxidized by OH to form NH2.41
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Then, Gly may be produced in successive free-radical reactions involving NH CO
DOI:21,0.1039/C7GC02911J
2
and CH3 and the intermediate species. Finally, the transformation of Gly into Ala is
possibly due to the -H–(CH) electrochemical removal and subsequent reaction with
CH3.
Conclusions
In summary, AAs are produced by using a new process based on an
electrophotosynthesis which uses a simple gas mixture. The process is able to fix nitrogen
and carbon dioxide into organic species under mild reaction conditions becoming an
efficient and environmentally friendly synthesis. The newly developed
electrophotocatalyst enhances the catalytic properties of cHAp to the extreme that
artificial photosynthesis is achieved thus opening a new era in the treatment of
contaminated atmospheres and nitrogen fixation research.
Experimental methods
Preparation of polarized hydroxyapatite
The synthesis and characterization of aHAp and cHAp are described in detail in the
Supplementary Information. The sintering of aHAp, cHAp, and montmorillonite and
mica powders was carried out by heating them at 1000 ºC during 2 h at an air atmosphere.
After this, samples were uniaxially pressed at 620 MPa for 10 min. Discs of 100 mm of
diameter, 1.7 mm of thickness and sufficient mechanical consistence were finally
obtained for all sintered materials (s-aHAp, s-cHAp, s-Nanofil 757 and s-LM).
In order to get thermally and electrically stimulated minerals, preliminary assays were
performed with s-cHAp discs were performed using DC voltages that ranged from 250 to
2000 V. For this purpose, s-cHAp discs were sandwiched between stainless steel (AISI
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304) plates, heated in the furnace to 1000 ºC in air and, simultaneously, polarized for 1 h
under application of a constant DC voltage of 500 V. Subsequently, samples were cooled
to room temperature, maintaining the DC voltage. The best results, in terms of both
mechanical consistence and maximum adsorption capacity of phosphates and
phosphonates, were obtained at 500 V. After this, discs of s-cHAp, s-aHAp, s-Nanofil
757 or s-LM were polarized using the same procedure for 1 h under application of a
constant DC voltage of 500 V, the resultant polarized systems being denoted p-aHAp, p-
cHAp, p-N757 and p-LM, respectively.
Characterization of polarized hydroxyapatite
p-cHAp samples were characterized by cyclic voltammetry (CV), wide angle X-ray
diffraction (WAXD) and FTIR spectroscopy as detailed and discussed in the
Supplementary Information.
Deposition of phosphonate and zirconyl chloride layers
3-Layered systems consisting in the successive deposition of ATMP, ZC and ATMP
layers (Phos-ZC-Phos) onto a mineral substrate (i.e. s-cHAp, s-aHAp, s-Nanofil 757 or s-
LM before and after being submitted to the thermally stimulated polarization process)
were obtained by immersion in the corresponding aqueous solutions at room temperature
for 5 h. Detailed experimental conditions are described in the Supplementary
Information.
After different trials to evaluate the influence of the content of ZC (detailed in the
Supplementary Information) all the results displayed in this work, including the main
text, correspond to ZC layer deposited from a 5 mM solution (unless another
concentration is explicitly indicated).
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Synthesis of amino acids
A high pressure stainless steel reactor was designed ad hoc and employed to perform
the synthesis of amino acids (AAs). The designed reactor, which is discussed in the
Supplementary Information, was equipped with three independent inlet valves for N2,
CH4, CO2 and an outlet valve to recover the gaseous reaction products. A UV lamp
(GPH265T5L/4, 253.7 nm) was also placed in the middle of the reactor to irradiate the
catalyst directly, the lamp being protected by a UV transparent quartz tube. All surfaces
were coated with a thin film of a perfluorinated polymer in order to avoid any contact
between the reaction medium and the reactor surfaces, in this way discarding other
catalyst effects.
Regarding temperature range, the reactions were performed within 75-105 ºC for
reaction times between 2 and 96 h. Catalyst samples weighed approximately 150 mg and
0.5 mL of de-ionized liquid water were initially incorporated into the reaction chamber,
except for assessing the water effect. The chamber was extensively purged with the first
selected gas in order to eliminate the initial air content (i.e. N2 or CO2). After this, in
order to reach the target pressure with the right mixture, each selected gas was introduced
to increase the reaction chamber pressure (measured at room temperature) as described in
the Supplementary Information. The yields of AAs were determined by using the areas of
1H NMR signals corresponding to CH2 (Gly) and CH3 (Ala) protons, as it is described in
the Supplementary Information.
Measurements
Synthesis of AAs was routinely verified by the ninhydrin (2,2-dihydroxyindane-1,3-
dione) detection test for primary amines. On the other hand, quantitative analyses were
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performed by NMR spectroscopy and X-ray photoelectron spectroscopy (XPS).
Morphological studies were performed by scanning electron microscopy (SEM). Analysis
of volatile organic compounds has been carried out by gas chromatography. Details about
the equipment and experimental conditions for such analyses are provided in the
Supplementary Information.
Acknowlegements
Authors acknowledge MINECO-FEDER (MAT2015-69367-R and MAT2015-69547-
R) for financial support. Support for the research of C.A. was received through the prize
“ICREA Academia” for excellence in research funded by the Generalitat de Catalunya.
This work is integrated within a wider research project supported by B. Braun Surgical
S.A., UPC, ICS and ICFO. Special thanks to Dr. J. F. Julián, Dr. J. Navinés, Mrs. Anna
M. Rodríguez, Mrs. Cristina Manjón, Dr. E. Rodríguez and Mr. Paul Wakely for their
valuable contributions and to Mr. A. Rodríguez for its contribution to the design of
Figures 1and 4 and to Mr. A. Lacasa for the design of the graphical abstract.
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Table 1. Influence of the experimental conditions (catalyst, pressure and time) in the
molar yield of Gly and Ala expressed with respect to CH4 or CO2. The weight of AA per
surface area of catalyst is also displayed.
Experimental conditions a
Yield
Mineral
P (atm)
1 b
Time
Gly
Ala
p-cHAp
p-cHAp
p-cHAp
p-cHAp
p-aHAp
p-cHAp
p-cHAp
24 h
72 h
24 h
72 h
72 h
24 h
24 h
-
-
1 b
6 c
6 c
6 c
5 d
7 e
1.9% (6.7 mg/cm2)
1.9% (6.6 mg/cm2)
3.4% (11.9 mg/cm2)
0.9% (3.2 mg/cm2)
1.5% (5.3 mg/cm2)
2.4% (8.3 mg/cm2)
1.6% (3.3mg/cm2)
1.3% (2.7 mg/cm2)
2.4% (5.0 mg/cm2)
0.7% (1.3 mg/cm2)
0.9% (1.9 mg/cm2)
1.6% (3.3 mg/cm2)
a Temperature was kept fixed at 95 ºC in all cases. b PN2= 0.33 bar; PCH4= 0.33 bar; PCO2
=
0.33 bar. c PN2= 2 bar; PCH4= 2 bar; PCO2= 2 bar. d PN2= 1 bar; PCH4= 2 bar; PCO2= 2 bar. e
PN2= 3 bar; PCH4= 2 bar; PCO2= 2 bar.
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Figure 1. Schemes describing (a) the preparation of the p-cHAp/Phos-ZC-Phos catalytic
system and (b) the reaction medium used to produce AAs. Details of the reactor are
provided in the Supporting Information. (c) Representative results of the ninhydrin test
for a positive reaction before stirring (left) and after stirring (middle) and a negative
reaction (right).
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Gly
CH2
A
C
Ala
CH3
p-cHAp
Phos
Phos
CH2
Ala
CH
Gly
CH2
B
Gly
CO
Ala
CH3
Ala
CO
Phos
CH2
Ala
CH
Figure 2. For samples obtained after reaction (95 ºC and 24 h) using a chamber pressure
of 1 bar (i.e. 0.33 bar of each feeding reaction gas): (a) 1H NMR spectrum of the solution
obtained after extraction of the AAs from the catalyst by dissolving the sample in
deuterated water containing 100 mM of HCl and 50 mM of NaCl; and solid state (b) 13C
and (c) 31P NMR spectra of the catalyst with the synthesized AAs.
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Figure 3. (A) N 1s high resolution XPS spectra for (1) p-cHAp, (2) p-cHAp/Phos-ZC-
Phos, and (3) p-cHAp/Phos-ZC-Phos after negative reaction (i.e. without exposure to UV
radiation) and (4) p-cHAp/Phos-ZC-Phos after positive reaction (24 h at 95 ºC). SEM
micrographs before (B) and after (C) successful reaction using the p-HAp/Phos-ZC-Phos
catalytic system (24 h at 95 ºC). Reactions were performed at 1 bar. Variation of
Gly/Phos (○), Ala/Phos (□) (Gly+Ala)/Phos (♦) and Gly/Ala (▲) ratios versus (D) time
for reactions performed at 95 ºC and (E) temperature for reactions performed during 24 h
using the p-cHAp/Phos-ZC-Phos catalytic system prepared as is displayed in Figure 1a.
Reactions were performed at 6 bar.
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Figure 4. Summary of the results obtained using different experimental conditions: green
and red boxes refer to successful and unsuccessful production of AAs, respectively.
Details about the 16 different experimental conditions are provided in the Supporting
Information.
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Figure 5. Variation of the electroactivity (voltammetric charge stored for reversible
exchange in a redox cycle) of as prepared cHAp, sintered cHAp and p-HAp. In all cases,
electroactivity measurements are expressed as voltammetric charge per surface unit (Q).
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Table of Contents: Graphic