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Differential graphene functions on two photosynthetic microbes

To cite this article: Anirban Bose et al 2020 Adv. Nat. Sci: Nanosci. Nanotechnol. 11 015004

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 | Vietnam Academy of Science and Technology Advances in Natural Sciences: Nanoscience and Nanotechnology

Adv. Nat. Sci.: Nanosci. Nanotechnol. 11 (2020) 015004 (10pp) https://doi.org/10.1088/2043-6254/ab6164

Differential graphene functions on two

photosynthetic microbes
Anirban Bose1, Sanhita Ray1, Vivek Kumar Singh2, Abesh Banerjee1,
Chumki Nayak3, Achintya Singha3, Amartya Bhattacharyya4,
Dipankar Chattopadhyay4, Amlan Chakrabarti5, Santanu Das2 and
Anjan K Dasgupta1,6
1
Department of Biochemistry, University of Calcutta, Kolkata—700 019, India
2
Department of Ceramic Engineering, Indian Institute of Technology (Banaras Hindu University), Varanasi
—221 005, Uttar Pradesh, India
3
Department of Physics, Bose Institute, Kolkata—700 009, West Bengal India
4
Department of Polymer Science and Technology, University of Calcutta, Kolkata—700 009, West
Bengal, India
5
A K Chaudhury School of Information Technology, University of Calcutta, Kolkata—700106, West
Bengal, India

E-mail: adbioc@caluniv.ac.in

Received 26 July 2019

Accepted for publication 13 October 2019
Published 8 January 2020

Abstract
Graphene, a Dirac material, permits the free flow of electrons on its surface. The interface of
graphene with different bio-materials is the emerging interest. In this paper, we describe
interfaces of graphene variants and two photosynthetic species (belonging to the class of alpha-
proteobacteria),Rhodobacter spand Rhodopseudomonas sp, both using photon capture in their
respective electron transport process. When grown on graphene oxide (or its exfoliated forms
obtained after microwave treatment) the bacterial species show differential pigment excretion
patterns, which is a measure of their photon driven electron-transport-chain activity. The
responses are measured by hydrodynamic diameters of pigment clusters and steady-state
quantum yield and time-dependent fluorescent emission patterns. The responses carry
fingerprints of graphene-specific effects on the respective microbes. Interestingly, there is a
reciprocal relationship between the size of the pigment cluster formed in the presence of
graphene (which varies for the two microbes) and the rate of fluorescence emission change. The
report opens up the possibility of developing photo-sensing and light-harvesting devices
exploiting the richness and diversity of this interface of the free-flowing electrons of this 2D-
material (graphene)and these cells,undergoing graphene-specific dynamics of pigmentation.
Keywords: graphene, photosynthetic bacteria, fluorescence enhancement, defect density
Classification numbers: 1.00, 5.15, 6.09

1. Introduction
The literature on graphene bacteria interactions is rife with
seemingly conflicting reports. Some groups have shown the
antimicrobial effects of graphene surfaces whereas others
have shown the functionality of graphene hybridised bacteria
in bioelectronics devices. The usefulness of graphene-bacteria
hybrids can be fully exploited in biosensors, solar cells and
6
Author to whom any correspondence should be addressed.
bio-diode type devices only under the conditions in which the
photosynthetic bacteria stay alive and remain functional [1].
While reports are available on cytotoxic effects of oxidised
forms of graphene on photosynthetic microorganisms [2], we
intend to study the different effects of oxidised forms of
graphene on the two species of photosynthetic purple non-
sulfur bacteria. Graphene, a Dirac material, is known to
contribute to electron transport chemistry including the
transfer of electrons from graphene to other species [3–5]. We
have recently reported how the graphene surface can

2043-6262/20/015004+10$33.00 1 © 2020 Vietnam Academy of Science & Technology

Adv. Nat. Sci.: Nanosci. Nanotechnol. 11 (2020) 015004
modulate a photosynthetic biofilm that has an in-built light-
driven electron transport machinery [6]. We show how
modulation of graphene surface can alter the electrical prop-
erties of active biomaterial derived from such an organism(s).
As photosynthetic materials involve light energy harvesting,
charge separation and charge transportation to catalytic sites
[7, 8], designed modulation of such properties may be of
important utilitarian value. We have previously reported the
presence of organised structures of self-assembled porphyrin
derivatives (extracellular protein-pigment complex, ePPC)
derived from purple nonsulfur bacteria under specific condi-
tions [9]. Besides we have recently reported the classification
of water samples (depending on the variation of the physical
properties) [10] where we have used the ePPCs as a probe [9]
and measured the electrical properties in the dissolved state.
Therefore, graphene-based interface of such material could be
used as a probe for obtaining more sensitive and stable sen-
sing platform [11]. This is one of the major inclinations of the
present study. For light harvesting pigment molecules, most
of the studies have been made on photosynthetic purple
nonsulfur bacteria, primitive photoheterotrophic system, well
known for their metabolic versatility and light energy utili-
sation [12, 13]. Their membrane is comprised of chromo-
spheres that are mainly associated with protein-pigment
complexes which harvest light to provide the necessary
energy source for their light dependent metabolic pathways
[14]. While efforts have been made to mimic photosynthetic
apparatus [15], artificial systems like self-assembled light
harvesting pigment molecules containing electron donor-
acceptor systems (in the supra-molecular organisation) have
shown promising attention [16]. Still, there are fewer reports
on how a Dirac material interface would modulate photon
capture [17, 18]. For light harvesting molecules, porphyrin
and its derivatives appear to be the main building block of all
natural pigments and could easily undergo covalent interac-
tion with other porphyrins or electron donor-acceptor moi-
eties to perform light energy conversions [19]. On the other
hand, recent advances have been made to study the effect of
graphene with light capturing accessories from photosynthetic
bacteria [4]. Though significant studies have been made on
the synthesis and photo-electrical properties of graphene
conjugated photosynthetic systems but how such properties
depend on the graphene defects and other qualitative factors
has scarcely been reported.
The report is an advancement of fluorescence emission
response from the ePPCs accumulated by the two different
genera Rhodobacter sp and Rhodopseudomonas sp (under the
class alpha-proteobacteria) with graphene oxide (GO) and
various forms of microwaved graphene oxide (mwGO). The
supersensitive nature of this photosynthetic component
interfaced with graphene material that has been reported for
the first time. The present paper seeks to discuss how photo
responses of light harvesting complexes may be controlled by
using an array of tuned graphene materials. The level of
control would determine the use of photosynthetic bacteria in
solar cells, light trapping and photosensitive bioelectronics
devices.
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A Bose et al
2. Experimental section
2.1. GO and mwGO preparation
Graphene oxide (GO) was synthesised using the improved
method as reported elsewhere [20], all the chemicals used for
this synthesis were purchased from Merck Millipore (India)
and Sigma-Aldrich (India). In detail, graphite powder and
potassium permanganate mixture (1:6 molar ratio) was reac-
ted with a strong acid solution containing sulphuric acid and
ortho-phosphoric acid (9:1 molar ratio) and kept for 10 h
stirring at 45 °C–55 °C. After completion of the reaction, the
mixture was cooled down to room temperature and hydrogen
peroxide was added in the presence of ice until the colour
appears yellow. The precipitate was filtered out, collected,
and washed properly with methanol, isopropanol, and dis-
tilled water followed by vacuum drying at 60 °C for 24 h. The
as-obtained dark brown colored GO was then further heat
treated in an ambient condition in a micro-oven (750 W) for
5, 10 and 20 min to obtain different microwave treated gra-
phene oxides - GO5, GO10, and GO20 respectively [21]. It is
already known from the literature that microwave treatment
results in GO reduction as well as bond annealing.
2.2. Bacterial sample preparation
Two strains of photosynthetic bacteria Rhodobacter sp
(MTCC NO. 8549) and Rhodopseudomonas sp (MTCC NO.
8756) were collected from Microbial Type Culture Collection
and Gene Bank, India. The bacterial cells were grown photo-
heterotrophically in yeast extract supplemented RCV medium
(4 g of malic acid, 1 g of NH4Cl, 120 mg of MgCl2, 75 mg of
CaCl2, 20 mg of sodium EDTA salt, 1 mg of nicotinic acid,
10 mM KPO4 buffer, 1000 ml H2O, 0.3 g of Yeast extract
powder, pH 6.8±0.2). The microbial cells were maintained
in transparent sealed containers to avoid air exposure at 30 °C
under 2100 lux from an incandescent bulb [22]. The log phase
bacterial cells were harvested by precipitating cell-biomass
and further incubated for 96–120 h with different mwGO, GO
and graphene samples dispersed in the growth medium. The
concentration range of GO and mwGO samples was studied
from 0.02 to 0.1 mg ml−1, here 0.06 mg ml−1 of carbon
material was added to 2.5×108 bacterial cells to study the
photo-physical properties of the samples. After incubation,
the photosensitive biomaterial was harvested by following
standard methods reported previously [9]. The harvested
biomaterial was further subjected to spectroscopic data
analysis.
2.3. Spectroscopic techniques
2.3.1. Absorption and fluorescence spectroscopy. UV-Vis
spectroscopy was carried out by measuring the absorption
spectra of samples by scan method setting, keeping
wavelength range from 350 nm to 900 nm, at 1 nm of
Adv. Nat. Sci.: Nanosci. Nanotechnol. 11 (2020) 015004
bandwidth and 600 nm min−1 of scan speed using Thermo
Scientific TMEvolution 300 UV-Vis spectrophotometer.
Fluorescence spectroscopy was done with Photon
Technology International (PTI) fluorometric setup (Quanta-
master TM 40). A 72 W Xenon lamp was used as an excitation
light source and the detection was preceded by passing the
emitted beam through an optical chopper and emission
monochromator. The bandwidth was set at 2 nm for both
excitation and emission.
2.3.2. FTIR spectroscopy. The Fourier transform infrared
spectroscopy (FTIR) was carried out by mixing samples into a
KBr pellet and spectra were recorded using a Perkin-Elmer
FTIR spectrophotometer.
2.3.3. Raman spectroscopy. Raman spectrum of graphene
powder samples was measured by taking GO/mwGO
nanoplatelet powders on a glass slide. The Raman
measurements were performed using a micro Raman set-up
consisting of the spectrometer (Lab RAM HR Jovin Yvon)
and a peltier controlled CCD detector. An air-cooled argon-
ion laser (Ar+) with a wavelength of 488 nm was used as an
excitation light source, and a 50X objective with a numerical
aperture (NA) of 0.75 was used to focus the laser on the
sample. A neutral density filter D 0.6 was used for attenuating
the laser power on the sample. Below 1 mW power was used
to avoid heating effect with 20 s integration time. D: G peak
intensity ratios were used to evaluate the average distance
between defects i.e. LD.
2.4. Electron microscopy
2.4.1. Scanning electron microscopy (SEM). SEM images
were obtained for various GO samples on a properly cleaned
silicon wafer with a scanning electron microscope (Model
ZEISS EVO-MA 10). Images were obtained without gold
sputtering on top of dried samples. For sample preparation,
various GO samples were well dispersed in Milli Q water and
drop casted on properly cleaned silicon wafer then dried at 50
°C overnight.
2.4.2. Transmission electron microscopy (TEM). The
morphology and structure of as-prepared GO and various
microwave treated GO were characterised using a
transmission electron microscope (TECNAI G2 20 TWIN,
FEI, USA). For the sample preparation, all the samples (both
MZF nanoparticles and MZF-PU) were dispersed in methanol
followed by dropcasting on TEM grids. All the TEM/selected
area electron diffraction (SAED) results were collected under
electron beam of 200 kV acceleration voltage whereas all the
data were analysed using a Gatan Microscopy Suite software
(Gatan, Inc, USA) in-built with TEM system.
2.5. Dynamic light scattering (DLS)
The hydrodynamic size of the samples was measured using
the DLS instrument (Zetasizer Nano Series; Malvern
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A Bose et al
Instruments, Malvern, UK) at 25 °C. The measurement was
taken in low volume disposable cuvettes at 25 °C.
3. Results
3.1. Graphene characterisation
3.1.1. Electron microscopy. Microwave treated GO samples
were studied using scanning electron microscopy (figure 1).
GO (figure 1(A)) is present as a multi-layered material. Upon
microwaving, mwGO (figures 1(B)–(D)) undergoes
exfoliation into fewer layer structures. However, increasing
microwaving results in simultaneous shriveling up of the
flakes. As observed in figure 1(D), mwGO20 shows most
exfoliation and wrinkling, followed by mwGO10 and
mwGO5. GO appears to be multilayer and the wrinkles are
rare. Graphene is known to shrink upon heating and form
wrinkles during high temperature synthesis as contrary to
other materials [23], so prolonged microwave treatment
increases degree of exfoliation but leads to shriveling up of
the flakes.
Figures 1(a)–(b) shows the TEM image incorporation of
wrinkles in the graphene, which may be due to the rapid
exfoliation of the graphene layers during the microwave
treatment. Furthermore, it was observed that the layer
exfoliation occurred in graphene with proportional to the
time of microwave treatment, which is attributed to the
increase in surface area of the mwGO with longer micro-oven
treatment time. We believe that some sorts of atomic defects
are incorporated during microwave treatments of those
samples. From selected area electron diffraction (SAED)
pattern of both GO and MwGO (figures 1(c) and (d)), we
obtained that both samples exhibit characteristics [100] and
[110] graphitic planes, which indicates the existence of sp2
bonded graphitic structures in all the samples even after
microwave treatment. Graphene oxide (GO) and a series of
microwave reduced forms of graphene oxide (mwGO) were
used to study the interaction with the two bacterial species.
The Reduction of GO by microwaving has been described
either in the presence of a reducing agent or in an inert gas
atmosphere [24, 25]. However, in our case, we hypothesised
that simple microwaving is also capable of annealing C–C
bonds. Hence, we used GO that had been microwaved for 5
(mwGO5), 10 (mwGO5) and 20 (mwGO5) min respectively,
without any inert gas.
3.1.2. Raman spectroscopy. The Raman spectral analysis of
the graphene samples (figure 2) showed variation in graphene
defects upon microwave treatment. The values of the
ID/IGpeak ratio and hence the average distance between
defects (LD) varied, differentially reduced forms of GO had
the lowest LD value among all the samples since it is the most
oxidised form [26, 27]. LDinitially increased with increased
microwave time, for 5 and 10 min proving that reduction and
C–C bond annealing had taken place. However, on further
increase in microwave time to 20 min, the graphene sample
Adv. Nat. Sci.: Nanosci. Nanotechnol. 11 (2020) 015004 A Bose et al

Figure 1. (A)–(D) SEM images of GO, mwGO5, mwGO10 and mwGO20 are captured under same magnification and depicted to compare
the structural changes present. (a-b) TEM images show differences in surface morphology of GO (a) and mwGO (b). The surface of mwGO
(b) sample is wrinkled because of microwave treatment; (c) and (d) illustrates selected area electron diffraction pattern of GO and mwGO,
respectively; Both of the SAED patterns show the characteristics graphitic planes of (100) and (110), which suggests the existence of
graphitic planes in GO or mwGO even after microwave treatments.

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Adv. Nat. Sci.: Nanosci. Nanotechnol. 11 (2020) 015004
Figure 2. Raman spectral analysis of GO, mwGO5, mwGO10, mwGO20.
Figure 3. Normalised FTIR spectra of GO, mwGO5, mwGO10, mwGO20.
sustained thermal damage and hence LD, once again,
went down.
3.1.3. FTIR spectroscopy. The FTIR spectra of the graphene
oxide and microwaved GO samples are shown in figure 3. All
transmittance values are baseline corrected. Baseline
correction was done by normalising each spectrum by its
corresponding transmittance at 3900 cm−1. Major functional
groups are –OH at 3600–3300 cm−1, C=O and COOH at
1740–1720 cm−1, C=C at 1621 cm−1, epoxy groups at
1200 cm−1, alkoxy groups at 1000–1100 cm−1. From the
normalised figure, it may be observed that all hallmarks of
oxidation e.g. epoxy, hydroxyl, carbonyl and non-conjugated
C=C bonds are highest in case of GO and lower for all forms
of microwaved GO. These signature peaks also decrease upon
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A Bose et al
successive times of microwaving. The only exception is that
mwGO10 epoxide group intensity is pretty high indicating
damage by microwaving. Upon 5 and 10 min of microwaving
the C=O peak goes down implying reduction and bond
annealing. For greater microwaving time (mwGO20),
carbonyl peak reappears implying thermal damage and
oxidation of graphene. These data correspond well to the
Raman spectral analysis to prove that GO samples show
variation with the degree of reduction and exfoliation upon
microwave treatment.
3.2. Photo-physical response to graphene
Photosynthetic bacteria are capable of capturing photons from
almost every region of the light spectrum by their pigment
Adv. Nat. Sci.: Nanosci. Nanotechnol. 11 (2020) 015004 A Bose et al

organisation. The present study concerns the extracellular

protein pigment complex (ePPC) accumulated by photo-
synthetic bacteria in the medium when grown photo-hetero-
trophically under certain conditions as mentioned in the
earlier study [9]. In response to UV excitation
(λmax∼395 nm), the ePPC shows fluorescence emission in
the red region (λmax∼613 nm), see supplementary figure S2
is available online at stacks.iop.org/ANSN/11/015004/
mmedia. Since the ePPC shows time dependent increase in
fluorescence emission, both the species in this present study
(lower panel of supplementary figure S2) showed similar UV
induced fluorescence amplification. As shown in this figure,
the time-based fluorescence scan is to some extent dependent
on the strain, as the photo-pigments that exhibit such fluor-
escence are found to undergo aggregation, such aggregation
dynamics lead to time dependent enhancement. In some
conditions, where the aggregation is impaired, fluorescence
emission intensity reduces with time and UV induced photo
bleaching occurs. This work distinctly shows how two pho-
tosynthetic bacterial strain (Rhodobacter sp and Rhodopseu-
domonas sp) interact with different oxidised forms of the GO.
Supplementary figure S1 shows how the ePPC accumulation
and bacterial growth are altered in the presence of GO and
mwGO samples. It is eminent from the figure that GO sti-
mulates growth and ePPC accumulation for both the strains Figure 4. Spectroscopic measurements: the fluorescence emission
though Rhodobacter sp shows a significant increase. Alter- rate (per unit time) difference between sample and control
(Δk=kS—kC, where kS represents the emission rate for sample and
natively, the ePPC accumulation and bacterial growth in the kC denotes the emission rate for control) is plotted for GO and
presence of different mwGO samples have discrepancy. The mwGO samples—GO, mwGO5, mwGO10 and mwGO20. The
ePPC accumulation for Rhodobacter sp is found to be higher upper panel shows the response from Rhodobacter sp and lower
in the presence of mwGO samples and almost two times panel shows the response from Rhodopseudomonas sp.
higher for mwGO5. Similar effect is found during bacterial
growth measurement. On contrary, Rhodopseudomonas sp
shows an only higher degree of growth and ePPC production
for mwGO20 only.
A time-based fluorescence measurement of ePPCs from accumulated by photosynthetic bacteria shows fluorescent
both the species shows fluorescence enhancement for Rho- enhancement during the dynamic equilibrium between
dopseudomonas sp and photo-bleaching for Rhodobacter sp monomeric and aggregated forms, the further insight of the
in the presence of GO. Whereas, in the presence of the hydrodynamic size measurement of the ePPCs is depicted in
mwGO, fluorescence enhancement is observed for Rhodo- figure 5. It is observed that, the aggregate size of the ePPCs is
bacter sp and photo-bleaching is observed for Rhodopseu- smaller for Rhodobacter Sp than Rhodopseudomonas sp in
domonas sp. The rate of fluorescence emission per unit time normal condition (panels (a) and (f) of figure 5). For Rho-
for both strains in the presence of GO and mwGO samples is dobacter sp, mwGO5 and mwGO10 shows two distinct
presented in figure 4. In this figure, Δk=kS—kC, where kS populations of aggregates in (figures 5(b) and (c)), one is
denotes the rate of fluorescence emission for sample and kC smaller and the other one is comparatively larger (ranging
denotes fluorescence emission rate for control. A comparative from 10–50 nm and 50–100 nm respectively). Whereas, in the
study is shown in figure 2 (inserted table) reveals that as presence of mwGO20, there is only one smaller population
Rhodobacter sp shows photo-bleaching in the presence of (figure 5(d)). But in the presence of GO, there are two
GO, the fΔk value is negative but in the presence of mwGO populations, and one of them appears larger (>100 nm) in
the Δk value reverts to positive and shows a systematic comparison to mwGO.
increase with increasing order of reduction time or degree of On the other hand, in the presence of mwGO samples,
exfoliation. On the other hand, Rhodopseudomonas sp shows Rhodopseudomonas sp shows only one comparatively bigger
only positive Δk value in the presence of GO. population of ePPC (figure 4). Whereas, in the presence GO
The dose of nanomaterial affects the photophysics of the species accumulates relatively small populations of ePPC
pigment complexes. For most types of mwGO, higher doses aggregates (figure 4). As the fluorescence enhancement
lead to a lower rate of enhancement, that is, slope is less steep. occurs during the dynamical process of aggregate formation,
For mwGO5, enhancement rates are higher than for control, it is quite possible to assume that the smaller the aggregates,
except the highest dose. For mwGO20, after 20 s, enhance- the greater the rate of aggregation size, the higher the fluor-
ment rates are lower than for control. Since, the ePPC escence enhancement rate (resulting in increased Δk value.)

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Adv. Nat. Sci.: Nanosci. Nanotechnol. 11 (2020) 015004
Figure 5. Hydrodynamic size measurement of extracellular protein pigment complex (ePPC). Left panel: size measurement for Rhodobacter
sp. Right panel: size measurement for Rhodopseudomonas sp.
4. Discussions
Photosynthetic bacteria are capable of capturing photons from
almost every region of light spectrum due to their pigment
organisation. The present study concerns the modification of
the structural and photo-physical characteristics of the extra-
cellular protein pigment complexes (ePPC) when exposed to
graphene surfaces. There are two aspects of the problem being
explored: one is the nature of the graphene materials used; the
other is the differential photo\physics of pigment systems
from the two microbes under investigation. Owing to their
differential cell specific effects, graphene like materials and
few layer graphene have been put in use in various biome-
dical applications [28]. Alteration of photo-physical proper-
ties of the ePPCs has been highlighted here depending on the
interface of graphene surface (e.g. GO and its altered forms
achieved by microwaving). If we consider graphene as a 2D
atomic network with decorated functional groups and defects,
the change in free energy of graphene surface electrons due to
surface area change, can be grossly approximated to [29]:
dG = - SdT + DAg + å mj dNj , (1 )
where, μjis chemical potential of the jth component (in this
case we assume it is jth lattice site) and γ is a surface potential
7
A Bose et al
like constant characteristic of the graphene surface or the
functional groups attached therein.
The area change may be caused by either of the two
factors. If a microbial species is able to have a cross talk with
the graphene surface via its electron transport chain (graphene
being a bed of free flowing electrons), a local stress (e.g.
defect dependent) or a global stress (dependent on the chiral
vector change due to microbial growth) is possible. Now for a
wide range of electron and proton transfer reactions like
photon capture by the pigments, the rate can be expressed by:
KT ⎛ -DG a ⎞
kAB = k exp ⎜ ⎟. (2 )
 ⎝ KT ⎠
If we assume that the growth of the bacterial species induces
stress in the graphene material, ΔA or area changes in the
graphene would imply that the activation energy ΔG awould
be distinctly affected. Where, ΔG a represents the activation
energy for aggregation at temperature T and k is the trans-
mission barrier.
The DLS data as shown in figure 5 clearly implies that
for the two bacterial species Rhodobacter sp and Rho-
dopseudomonas sp, the sizes of the porphyrin based pigment
aggregates are different. This implies that a function F exists
Adv. Nat. Sci.: Nanosci. Nanotechnol. 11 (2020) 015004
Figure 6. PCA analysis of ePPC from Rhodobacter sp and Rhodopseudomonas sp with GO, mwGO5, mwGO10, and mwGO20.
such that:
DG a photo ‐ aggregation = F (DAgraphene , ggraphene).
The functional nature is evident from the two opposite
arrows of the PCA as shown in figure 6, Rp and Rb (Rho-
dobacter sp and Rhodopseudomonas sp respectively) vectors
show opposite vector directions, the principal component is
primarily guided by the kinetics of fluorescence changes, and
the kinetics is determined by the activation energy expressed
in equation (2). The graphene specific constant like LDwhich
represents the spacing of the defects (determining graphene
surface dependent constant like γ) seems to have an inde-
pendent direction (figure 6) indicating that the differential
microbial responses are due to global distortion in the struc-
ture rather than being only dependent on local graphene
defects. We would elaborate on this in the following
subsections.
4.1. Graphene variation
Upon exposure to microwave, the GO is reduced as soon as
wrinkling in surface morphology sets in. The wrinkling
probably occurs due to differential exposure of outermost GO
layer to microwave (and induced heating) compared to inner
layers of non-exfoliated, bulk GO. If GO is exfoliated and
dispersed by ultra-sonication prior to microwaving such
wrinkling will not take place.
Electron transport properties on mwGO are expected to
change following microwaving. Raman scattering data indi-
cate C-C bond annealing in GO. The synthesized GO shows a
high defect density (see the chart in figure 2). The LD value
increases upon treating the GO sample with microwave
because of the annealing of the carbon - carbon bond up to
10 min but again the value decreases for 20 min treated GO
sample. The increase in LD (as calculated from Raman
spectra), which is obtained upon microwaving of GO, sup-
ports our hypothesis of C–C bond annealing upon microwave
treatment for 5–10 min.
A Bose et al
There are 2 competing processes when the GO solution is
microwaved. One is oxidation, the other is reduction and C–C
(3 ) bond annealing. For shorter microwaving times (less heating)
the re-annealing predominates. At greater optimum micro-
waving, oxidation sets in due to greater heating.
The observable point is here without any reducing (or
inert) environment, just treating the GO sample in microwave
for up to 10 min (and no more) would provide a less defective
GO. During the microwave treatment the oxidised functional
groups like –COOH, –C=O and epoxy and alkoxy groups get
reduced without any reducing agent which is evident from
FTIR spectra of the samples (figure 3). The microwave
induced differences are also evident from the X-ray diffrac-
tion patterns of the samples (see fig. S4 in supplementary
information). Therefore, microwave treatment enables an
alteration of both morphological and oxidative states of GO
sample. These different forms of GO samples interact in a
discrete manner with photosynthetic pigment complexes
(ePPCs) present in the LHCs of photosynthetic bac-
teria [30, 31].
4.2. Bacterial discrimination
The effect of surface defects and corresponding changes in
photoluminescence properties are reported for inorganic
semiconductor materials [32], we investigated similar effect
on photo-pigments and studied the photophysical properties
of the same. It seems evident from the observation made so
far in figure 4 that the two photosynthetic species of purple
non-sulfur bacteria - Rhodobacter sp and Rhodopseudomonas
sp induce different photo-physical responses with GO and its
reduced forms. Though both the strains show similar growth
and pigment formation in the presence of GO, (supplementary
figure S1), structural alteration of graphene surface (due to
microwave treatment) induces differential response from the
two strains. Δk dynamics (as shown in figure 4) corresponds
with carboxyl and other oxygenated groups (as shown by
FTIR spectra in figure 3). Thus lower oxygenation of gra-
phene nano-sheets leads to higher slope of amplification for
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Adv. Nat. Sci.: Nanosci. Nanotechnol. 11 (2020) 015004
Rhodobacter sp. and the opposite for Rhodopseudomonas. A
possible mechanism is given in supplementary figure S3.
Graphene acts as an electron sink. Charge transfer from
pigment to graphene is affected by the presence of point
defects and edge defects, both of which correspond to more
carboxyl, epoxide and other oxygen containing groups.
There are some important differences between the pho-
tosynthetic apparatus of the two organisms. The photo-
responsive extracellular complexes (ePPCs) secreted by
Rhodobacter sp are smaller in size than those from Rho-
dopseudomonas sp (figure 5). Hence, we may conclude that
the former consists of disaggregated moieties, while the latter
is composed of pre-formed aggregates. When the population
of disaggregated complexes are more, UV induced aggrega-
tion (hence fluorescence amplification occurs at a faster rate).
When the population of pre-formed aggregates is higher, the
rate of fluorescence amplification is lesser (see supplementary
figure S2).
Graphene surfaces provide the opportunity for π- stack-
ing of porphyrin class of materials on its surface. However for
graphene oxide, which is a highly oxidised form of graphene,
porphyrin-graphene interaction is mostly mediated by elec-
trostatic interaction [33]. Reduced graphene oxide promotes
higher amplification rates for Rhodobacter sp (compared to
control, see figure 4) since it pushes the molecular equilibrium
towards aggregation when exposed to UV light. RGO surface,
in essence, promotes faster aggregation dynamics of ePPCs
most likely due to seeding (pi stacking) on its surface. This
results in fluorescence amplification rates that are higher
compared to control.
From figure 5, it is apparent that for Rhodobacter sp GO
surfaces promote formation of pre-formed aggregates whereas
in Rhodopseudomonas sp GO promotes disaggregation of the
same. A Drastic increase in aggregate size in presence of GO
(figure 5) results in photo-bleaching upon UV exposure for
Rhodobacter sp whereas for Rhodopseudomonas sp sub-
stantial de-aggregation leads to fluorescence amplification
rates that are higher than control (figure 4).
We show how two closely associated photosynthetic
bacterial species could be differentiated on the introduction of
a graphene interface. In figure 6 one can distinctly find that
two photosynthetic bacteria as mentioned are negatively
correlated in the presence of graphene materials and the defect
density is better correlated with only Rhodobacter sp.
Another interesting aspect of this report is how a cost-effec-
tive, one step technique like simple microwave treatment for a
few minutes to GO can reduce the defect density. How sig-
nature of photosynthetic microbes is detected by fluorescence
enhancement as well as photon uptake efficiency by the
ePPCs (or supra-molecular assembly of LHCs) and how that
is modulated by GO varieties seem to be a promising field of
graphene-based photochemistry. This phenomenon could be
beneficial for developing light harvesting and energy gen-
eration devices since illumination would result in charge
separation and transfer of electron from ePPCs to graphene
which is known as a good electron sink. Effective charge
transport will be achieved through the cross-section of the
9
A Bose et al
material due to the presence of conductive surface of gra-
phene. This charge may be stored for power generation.
5. Conclusion
Photo-physical changes in pigment complexes derived from 2
purple non-sulphur bacterial species have been studied in the
context of their interaction with graphene oxide and its
reduced forms. The bacterial species show diametrically
opposite photo-physical responses to the series of graphene
varieties. The effects of graphene materials are explained on
the basis of their defect density, functionalisation and
wrinkling of the nanosheet. The differential bacterial
responses have been explained in terms of the size of their
pigment complexes and how this size is affected by being in
the vicinity of graphene surfaces. Thus graphene can act as a
tool for discriminating between photosynthetic organisms
preferentially used in the construction of effective solar cells
or solar powered devices.
Acknowledgments
The authors would like to thank DBT (BT/PR3957/NNT/
28/659/2013) and MEITY (13(10)/2016—CC & BT & 18/
7/17).
Anirban Bose carried out all UV-VIS spectroscopy,
fluorescence spectroscopy and dynamic light scattering,
scanning electron microscopy experiments and wrote the
paper. Sanhita Ray helped to analyse Raman, FTIR and SEM
data and wrote these portions and revised the paper. Abesh
Banerjee helped to carry out fluorescence spectroscopic
experiments. Chumki Nayak helped in Raman spectroscopic
experiment and Achintya Singha provided the facility.
Amartya Bhattacharyya, Dipankar Chattopadhyay provided
FTIR facility and performed the experiment. Vivek Singh and
Santanu Das provided synthesised GO and mwGO samples
and did all the optimisations regarding GO material proces-
sing and characterisations, carried out TEM experiment.
Anjan Kr Dasgupta conceived the idea and wrote the paper.
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