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ORIGINAL RESEARCH
published: 13 November 2018
doi: 10.3389/fenrg.2018.00107
Frontiers in Energy Research | www.frontiersin.org 1November 2018 | Volume 6 | Article 107
Edited by:
Sebastià Puig,
University of Girona, Spain
Reviewed by:
Ioannis Andrea Ieropoulos,
University of the West of England,
United Kingdom
Matteo Grattieri,
University of Utah, United States
*Correspondence:
Daniel Puyol
daniel.puyol@urjc.es
Abraham Esteve-Nuñez
abraham.esteve@uah.es
Specialty section:
This article was submitted to
Bioenergy and Biofuels,
a section of the journal
Frontiers in Energy Research
Received: 15 May 2018
Accepted: 25 September 2018
Published: 13 November 2018
Citation:
Vasiliadou IA, Berná A, Manchon C,
Melero JA, Martinez F, Esteve-Nuñez A
and Puyol D (2018) Biological and
Bioelectrochemical Systems for
Hydrogen Production and Carbon
Fixation Using Purple Phototrophic
Bacteria. Front. Energy Res. 6:107.
doi: 10.3389/fenrg.2018.00107
Biological and Bioelectrochemical
Systems for Hydrogen Production
and Carbon Fixation Using Purple
Phototrophic Bacteria
Ioanna A. Vasiliadou 1, Antonio Berná 2, Carlos Manchon 3, Juan A. Melero 1,
Fernando Martinez 1, Abraham Esteve-Nuñez 2,3
*and Daniel Puyol 1
*
1Department of Chemical and Environmental Technology, ESCET, Rey Juan Carlos University, Móstoles, Spain, 2IMDEA
Water, Parque Tecnológico de Alcalá, Alcalá de Henares, Spain, 3Department of Chemical Engineering, University of Alcalá,
Alcalá de Henares, Spain
Domestic and industrial wastewaters contain organic substrates and nutrients that
can be recovered instead of being dissipated by emerging efficient technologies.
The aim of this study was to promote bio-hydrogen production and carbon fixation
using a mixed culture of purple phototrophic bacteria (PPB) that use infrared radiation
in presence or absence of an electrode as electron donor. In order to evaluate
the hydrogen production under electrode-free conditions, batch experiments were
conducted using different nitrogen (NH4Cl, Na-glutamate, N2gas) and carbon sources
(malic-, butyric-, acetic- acids) under various COD:N ratios. Results suggested that the
efficiency of PPB to produce biogenic H2was highly dependent on the substrates
used. The maximum hydrogen production (H2_max, 423 mLH2/L) and production rate
(H2_rate, 2.71 mLH2/Lh) were achieved using malic acid and Na-glutamate at a COD:N
ratio of 100:15. Under these optimum conditions, a significant fixation of nitrogen in
form of single-cell proteins (874.4 mg/L) was also detected. Under bio-electrochemical
conditions using a H-cell bio-electrochemical device, the PPB were grown planktonic
in the bio-cathode chamber with the optimum substrate ratio of malic acid and
Na-glutamate. A redox potential of −0.5 V (vs. Ag/AgCl) under bio-electrochemical
conditions produced comparable amounts of bio-hydrogen but significantly negligible
traces of CO2as compared to the biological system (11.8 mLCO2/L). This suggests
that PPB can interact with the cathode to extract electrons for further CO2re-fixation
(coming from the Krebs cycle) into the Calvin cycle, thereby improving the C usage. It has
also been observed during cyclic voltammograms that a redox potential of −0.8 V favors
considerably the electrons consumption by the PPB culture, suggesting that the PPB can
use these electrons to increase the biohydrogen production. These results are expected
to prove the feasibility of stimulating PPB through bio-electrochemical processes in the
production of H2from wastewater resources, which is a field of special novelty and still
unexplored.
Keywords: purple phototrophic bacteria, biolectrochemical, high value-added products, bio-hydrogen, carbon
fixation, proteins
Vasiliadou et al. PPB Meet BES
INTRODUCTION
Typical wastewater systems entail the dissipation of the
contamination. However, the high content of organics and
nutrients in industrial and domestic wastewaters is a valuable
resource for energy and products recovery (Puyol et al., 2017a).
Hence, upgrading of existing WWTP as resource recovery
systems by implementing novel technologies, are mandatory
steps considering economic and environmental benefits and
recent policies within the circular economy.
Among the competing technologies, the biological
accumulation of nutrients and their subsequent recovery,
has received great attention as an environmental friendly
and certainly cost-effective process (Batstone et al., 2015).
Purple phototrophic bacteria (PPB) have shown significant
accumulation of organics and nutrients from wastewater
through assimilative processes (Batstone et al., 2015). PPB is a
group of anaerobic facultative microorganisms, which can utilize
infrared light (IR) as the main energy source. The use of PPB in
the Partition-Release-Recovery concept proved to be far superior
to other phototrophic organisms (as algae or cyanobacteria),
since they achieve high growth rates and are not inhibited by O2
(Muñoz and Guieysse, 2006).
PPB are extremely versatile organisms due to their complex
metabolic system, involving major C, N, S, P, and Fe pathways,
which absorbs the IR energy through their photosystem,
composed by carotenoids and bacteriochlorophyls (Hunter,
2008). Anoxygenic photosynthesis generates practically all the
energy required for growth via the so-called cyclic electron
flow (Klamt et al., 2008). In domestic wastewater treatment,
the main metabolism follows photoheterotrophic growth on
volatile fatty acids and sugars, although chemoheterotrophy
(e.g., fermentation and anaerobic oxidation) can provide the
necessary electrons for photoautotrophic growth (via hydrogen;
Hülsen et al., 2014, 2016; Puyol et al., 2017a,b). The internal
electron recycle, however, can be used for obtaining ammonium
through dinitrogen gas fixation or directly dissipating electrons
in the nitrogenase complex, which generates bio-hydrogen as
the electron acceptor (Koku et al., 2002), or for direct internal
accumulation of organic acids as poly-hydroxy-alcanoates (PHA)
(Fulop et al., 2012). Moreover, the assimilative partitioning of
wastewater macronutrients and organics through PPB leads to
the production of one solid bacterial stream rich in proteins.
In this sense, PPB can be used for the extraction of high
value-added products from waste sources, such as biofuels
like bio-hydrogen, bioplastics as PHA and single-cell proteins.
The metabolic pathways to obtain the valuable bioproducts
are catalyzed by variant enzymes (McKinlay and Harwood,
2010). Monitoring the functionality of the involved bacteria
and following-up their activity, can be of added value toward
maximizing the bioproducts’ formation. To add to the complexity
of the above system, the end product depends greatly on
the environmental conditions (IR light intensity, temperature,
nutrients concentration, etc.). Thus, wastes rich in nitrogen are
good sources for PPB growth producing biomass with high
protein content (Verstraete et al., 2009), which can subsequently
be used as additive animal food. In organic media lacking
nutrients, PPB can accumulate high quantities of PHA, achieving
up to 70–90% w/w (Mas and Van Gemerden, 1995). They are
therefore an interesting alternative to fossil-fuels for plastics
production. When the organic matter composition is quite high
and is more reduced than biomass (i.e., butyrate), the excess of
electrons (in absence of ammonium) are driven toward hydrogen
production that can be used as a clean and renewable biofuel.
Understanding the factors of importance and unraveling their
relationship with the desired end bioproduct, remains one of the
most important challenges in the ongoing research.
Finally, the internal electron recycling of PPB is a key
issue, and an active modification of the electronic fluxes by
means of artificial addition of electrons could drive toward
different targeted bioproducts (Varfolomeyev, 1992). In this way,
the concepts supporting microbial electrochemical technologies
(METs) could be used to enhance the biochemical reactions
of PPB by supplying electric current to microorganisms using
electrodes as electron donors. In this context, METs have received
great attention due to their potential applications in nitrate
reduction (Pous et al., 2013; Tejedor et al., 2016), methanogenesis
(Cheng et al., 2009) and microbial electrosynthesis (Logan
and Rabaey, 2012). Likewise, the wise use of electricity to
enhance PPB activity toward high value-added compounds
(i.e., biohydrogen) through a bio-electrochemical system is
undoubtedly an attractive challenge. PPB are highly electroactive
organisms with high ability to generate bioelectricity through
MFCs (Xing et al., 2008; Park et al., 2014). However, the use
of electricity to enhance the PPBs metabolic activity aiming to
produce high value-added bioproducts is an unexplored field
with high growth potential in the short-term.
Based on the above-mentioned grounds, the aim of the present
work was the assessment of PPB to enhance the formation of
valuable bioproducts, such as biohydrogen, using electric and
light energy as the driving forces. This was accomplished by
identifying the biological and electrochemical conditions that
influence the process of bio-hydrogen production from PPB. The
wise use of electric energy to decontaminate wastewater and to
produce bio-hydrogen is undoubtedly an attractive and novel
challenge, yielding substantial ecological and economic benefits.
MATERIALS AND METHODS
Chemical Compounds and Growth Media
All the chemicals compounds used were purchased from Sigma-
Aldrich. The organic compounds that were used were: L-malic
acid (C4H6O5), butyric acid (C4H8O2), acetic acid (C2H4O2),
propionic acid (C3H6O2) and ethanol (C2H6O). Stock solutions
of individual organic compounds (20 gCOD/L) were prepared
in ultra-pure water and stored at 4◦C. The nitrogen sources
used were: ammonium chloride (NH4Cl) as inorganic N-source,
L-glutamic acid monosodium salt monohydrate (Na-glutamate,
C5H8NNaO4·H2O) as organic N-source and nitrogen gas (N2)
as external gaseous source. Stock solutions of both organic and
inorganic nitrogen sources (5 gN/L) were prepared in ultra-pure
water and stored at 4◦C.
Finally, macro- and micro-nutrient solutions were prepared
following the recipe proposed by Ormerod et al. (1961). The
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Vasiliadou et al. PPB Meet BES
macro-nutrient solution contained 10.86 g K2HPO4·3H2O;
6.66 g KH2PO4; 2 g MgSO4·7H2O, 0.75 g CaCl2·2H2O; 69 mg
FeCl2·4H2O and 0.2 g EDTA in 1 L ultra-pure water. The
micro-nutrient solution contained 1.4 g H3BO3; 1.013 g
MnCl2·4H2O; 274 mg (NH4)6Mo7O24 ·4H2O; 57 mg ZnCl2;
14 mg CuCl2·2H2O; 7.5 mg biotin and 1 g EDTA in 0.5 L
ultra-pure water. The pH in all solutions was adjusted to 7.
Purple Phototrophic Bacteria (PPB)
Enrichment
All experimental tests were inoculated with a mixed culture of
PPB. These bacteria were enriched from a wastewater influent
taken from the pilot-scale WWTP located at the Rey Juan
Carlos University (Mostoles, Madrid, Spain). Enrichment was
performed by inoculating a 1 L suspended growth reactor (SGR)
with sludge liquor, and subsequent incubation under near infra-
red (NIR) light illumination and anaerobic conditions using
a synthetic wastewater (SW) as growth medium. The SW
(prepared with tap water) contained the 5 different organic
carbon sources (acetic acid, malic acid, propionic acid, butyric
acid and ethanol) with a total COD concentration of 2 gCOD/L,
0.26 gN/L as NH4Cl and 1 and 100 mL/L of micro- and
macro-nutrient solutions, respectively. After the addition of SW
the bioreactor liquor was flushed with argon gas in order to
remove any presence of oxygen. The bioreactor was illuminated
with LED lamps (850 nm) as IR light source. The reactor’s
surface was covered with UV-VIS absorbing foil (ND 1.2 299,
Transformation Tubes, Banstead, UK). The foil absorbed around
90% of the wavelength below 750 nm. The average light intensity
measured on the outside reactor’s surface was 13 W/m2. The
PPB mixed culture was continuously stirred and incubated at
room temperature (25 ±1◦C). The liquor of the reactor was
refreshed every week with fresh SW (99% volume exchange) to
achieve final concentrations of 2 gCOD/L and 0.26 gNH4-N/L.
The pH was weekly adjusted to 6.8 ±0.1. The enrichment of PPB
was evaluated by the detection of Bacteriochlorophylls (BChl)
and carotenoids accumulation by performing VIS-NIR spectra
analyses of the culture.
Biological Experiments
The ability of the PPB enriched culture to produce bio-hydrogen
using different carbon and nitrogen sources was evaluated in
batch assays. Initially, the capacity of the PPB culture to produce
hydrogen using different organic and inorganic nitrogen sources
was examined. The first set of experiments were conducted by
using 2 gCOD/L of L-malic acid as the carbon source. Malic acid
was chosen as a suitable carbon source that could favor hydrogen
production by PPB (Assawamongkholsiri and Reungsang, 2015).
Batch experiments were performed using: inorganic (NH4Cl) and
organic (Na-glutamate) nitrogen, both with concentrations of 75,
150, and 300 mgN/L, and finally dinitrogen gas (60 mL of N2
in the headspace). Thereafter, two additional experiments were
conducted using different carbon sources (butyric- and acetic-
acid) at a concentration of 2 gCOD/L each, with 300 mgN/L
of Na-glutamate as organic nitrogen source. A summary of the
experimental conditions of the batch assays is shown in Table 1.
All the experiments were conducted in 160 mL serum bottles
with a working volume of 100 mL. The reactors contained
99 mL of SW medium (prepared as described above) with the
corresponding COD and N contents and were inoculated with
PPB enriched culture (1% v/v inoculum). The initial pH of the
medium was adjusted to 6.8 ±0.1 using NaOH or H2SO4. The
liquid medium of each reactor was flushed with argon for 10 min.
Thereafter, the bottles were closed with rubber stoppers and
capped with aluminum seals. Subsequently, the headspace of the
reactors was flushed again with argon for 2 min except from the
reactors where nitrogen gas was used as nitrogen source that
were flushed with N2gas. The bottles were continuously shaken
horizontally at 120 rpm at 25 ±1◦C (Orbital shaker, optic ivymen
system) and illuminated at an average light intensity of 20 W/m2
using LED lamps for 7 days. The performance for H2production
using identical conditions but without PPB enriched culture
was studied by conducting control experiments under sterilized
conditions (all the glassware and media used were autoclaved).
During these control experiments, no biomass growth as well as
no H2production or acid assimilation were detected. Both the
liquid and the gas media were sampled periodically to evaluate,
the carbon and nitrogen assimilation, the PPB growth and the
hydrogen production. All the experiments were conducted in
duplicate.
Bio-Electrochemical Experiments
Bio-electrochemical experiments were performed in an H-cell
device as shown in Figure 1. The device was consisted of two
Duran glass bottles (8.6 cm diameter ×18.1 cm height) serving
as two chambers. Each cell (cathode and anode) had a working
volume of 500 mL. The cathode chamber was equipped with a
working electrode of graphite of 10 ×100 mm and a reference
electrode RE-5B Ag/AgCl. All potentials are quoted vs. Ag/AgCl.
The anode chamber was equipped with a counter electrode of
Ti/Pt (2.5 micro-m) 100 ×20 mm in a 10 ×5 mesh. The
cathode and anode chambers were separated with a cationic
membrane (RALEX, MEGA a.s., Straz pod Ralskem, Czechia).
The working, counter and reference electrodes were connected
to a potentiostat NEV4-V2 (Nanoelectra S.L., Alcala de Henares,
Spain) with maximum current of ±100 mA and compliance
voltage of ±10 V. A computer processed by specialized software
(Potentiostat NEV4 software, Alcala de Henares, Spain) was used
for the automatic recording of data.
As shown in Figure 1, the cathode chamber from the H-cell
was employed as bio-cathode containing SW (495 ml). The bio-
cathode was inoculated with PPB enriched culture (5 mL, 1% v/v
inoculum). Malic acid (2 gCOD/L) and Na-glutamate at COD:N
ratio of 100:15 were respectively used as carbon and nitrogen
sources. The anode chamber was filled with 495 mL of tap water
and 5 mL of the macro-nutrients solution. The initial pH in
both chambers was adjusted to 6.8 ±0.1. The bio-cathode was
illuminated with LED lamps as NIR light source with an average
light intensity of 20 W/m2. Bio-electrochemical experiments
were performed at 25 ±1◦C and the cell of bio-cathode was
continuously stirred at a speed of 200 rpm. The media in both
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Vasiliadou et al. PPB Meet BES
TABLE 1 | Experimental runs of PPB biological experiments under different nitrogen and carbon sources.
ID Carbon source Organic acid concentration (mgCOD/L) Nitrogen source N concentration (mgN/L) COD:N ratio
1 Malic acid 2,000 NH4Cl 75 100:3.75
2 Malic acid NH4Cl 150 100:7.5
3 Malic acid NH4Cl 300 100:15
4 Malic acid 2,000 Na-glutamate 75 100:3.75
5 Malic acid Na-glutamate 150 100:7.5
6 Malic acid Na-glutamate 300 100:15
7 Malic acid 2,000 N2gas 8.8* –
8 Butyric acid 2,000 Na-glutamate 300 100:15
9 Acetic acid 2,000 Na-glutamate 300 100:15
*Based on Henry’s Law and the solubility of gases.
FIGURE 1 | Experimental set-up of the foto-bio-electrochemical H-cell device.
cells were flushed with argon for 20 min. Subsequently, the cells
were closed with butyl septa and capped with GL45 Duran caps.
The headspaces of the cells were flushed again with argon for
3 min.
Experiments were conducted by setting the potential of
bio-cathode at −0.5 V in order to force the PPB culture to
be adapted to the electrochemical conditions. The reaction
period among the PPB culture and the bio-cathode was chosen
to be 1 week, similar to the biological experiments. Control
electrochemical (abiotic) experiments were conducted using the
same experimental conditions without PPB biomass. In order to
determine whether the PPB culture interacted with the cathode or
not by means of electron acceptance from PPB, cyclic voltametry
(CV) in the range of −0.8 to 0.8 V was performed during the
weekly reaction process.
Analytical Methods
All parameters except total chemical oxygen demand (COD) and
total Kjeldahl Nitrogen (TKN) were determined after filtering
with a 0.45 µm nylon filter (Chrodisc filter/syringe, CHMLab,
Barcelona, Spain). Total and soluble COD were determined
using a dichromate-reflux colorimetric method (APHA, 2005).
The nitrogen contents of filtered and non-filtered samples
were determined by the standard Kjeldahl procedure (Gerhardt
TNK, Vapodest 450, Königswinter, Germany) using 20 mL of
concentrated H2SO4and K2SO4-CuSO4as catalyst. Organic
nitrogen content of PPB culture was determined as the difference
between Kjeldahl nitrogen of filtered and non-filtered sample.
The single cell protein (SCP) content of cell dry weight (CDW)
was obtained by multiplying the obtained nitrogen value with
a conversion factor of 5.33 (Salo-Vaananen and Koivistoinen,
1996). The inorganic nitrogen was analyzed as NH4Cl using
Spectroquant Ammonium Test (Merck, Darmstadt, Germany).
The optical density of PPB biomass was measured at 590 nm by
UV-VIS spectrophotometer (V-630, Jasco, Madrid, Spain) and
the concentration of biomass was calibrated using a standard
curve of PPB optical density on the basis of volatile suspended
solids (gVSS/L) concentration (Vasiliadou et al., 2008). The VSS
concentration (gVSS/L) was measured according to standard
methods (APHA, 2005). The detection of BChl and carotenoids
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Vasiliadou et al. PPB Meet BES
of PPB was performed by determining the VIS-NIR spectra
(400–950 nm) using a UV-vis spectrophotometer (V-630, Jasco,
Madrid, Spain). The pH was measured using a pH meter (Crison
GLP22, Hach Lange, Loveland, CO, USA). Illuminance was
measured with a VIS-NIR spectroradiometer (STN-Bluewave-V,
MTB, Madrid, Spain). The concentrations of VFAs (malic, acetic
and butyric acids) in the liquid samples were analyzed using
high performance liquid chromatography (HPLC) (Varian 356-
LC, Agilent Technologies, Santa Clara, CA, USA), employing
refractive index (RI) detector with a MetaCarb 67H 300 ×
6.5 mm column (Agilent Technologies, Santa Clara, CA, USA).
The oven temperature was 65◦C. The mobile phase was 0.25 mM
H2SO4at a flow rate of 0.8 mL/min. The volume of the gas was
determined by releasing pressure from the reactors headspace
using a Boyle-Mariotte Apparatus (3B Scientific S.L., Hamburg,
Germany). The composition of each reactors head-space was
analyzed using a 7820A GC system equipped with a 3Ft 1/8
2 mm Poropak Q 80/100 SS column, a 6Ft 1/8 2 mm Poropak
Q 80/100 SS column and a 6Ft 1/8 2 mm MolSieve 5A 60/80 SS
column, a fitting external Luer lock and a thermal conductivity
detector (TCD) (Agilent Technologies, Santa Clara, CA, USA).
The mobile phase was Argon at a flow rate of 5 mL/min. The
temperature of the oven and the detector were 45 and 220◦C,
respectively.
RESULTS AND DISCUSSION
The following section include all results generated after exploring
the physiology of PPB for selecting those culture conditions,
including nitrogen and carbon sources, for achieving an optimal
conversion of an extracellular source of electrons into hydrogen
production and CO2fixation.
Enrichment of a PPB Mixed Culture From
Domestic Wastewater
The enrichment process was performed aiming to enhance the
growth and acclimation of a mixed PPB culture from domestic
wastewater, using a specific environment of NIR radiation. The
organic mixture used for the enrichment was chosen on the
basis that PPB can efficiently produce hydrogen from wastes that
contain mixed VFAs (Wu et al., 2012). It should be noted that
the optimum COD:N ratio for efficient C and N assimilation
from domestic wastewater by PPB was reported to be 100:7.1
(Puyol et al., 2017b). However, during conventional DWW
treatment operation, nutrients, such as N and P are usually
in excess (Puyol et al., 2017b). Therefore, a COD:N ratio of
100:13.1 was chosen for the enrichment and acclimation process.
Following a 2-weeks enrichment period, PPB biomass growth
was evidenced through the BChl aaccumulation as detected from
the peaks with maximum absorbance at 590, 805, and 860 nm.
This clearly indicated that the enrichment under anaerobic
conditions and NIR light source could selectively enrich PPB
from wastewater and express their photosynthetic apparatus via
bacteriochlorophylls (Melnicki et al., 2008).
Figure 2 shows an example of PPB culture performance
during a weekly operating cycle, after a 2-months acclimation
period. Figure 2A shows the absorbance spectra of PPB culture
aliquots that were taken at different time intervals during
the weekly cycle (day 0–7). The PPB culture produced and
accumulated with time BChl aas well as carotenoids that
are naturally synthesized by photosynthetic organisms. The
absorption spectrum of BChl aappeared in the spectral
range between 560 and 930 nm, with maximum peaks at
590, 805, and 860 nm, respectively, while the spectrum of
carotenoids appeared in the range between 400 and 550 nm.
It has been previously reported that PPB produce molecules
referred to as open-chain carotenoids and incorporate them
into their photosynthetic system, such as light-harvesting
complexes and the bacteriopheophytin-quinone type reaction
center (Niedzwiedzki et al., 2017).
As shown in Figure 2B, the PPB concentration reached 750
mg/L at the end of the weekly cycle, giving a growth yield of 0.75
±0.05 gCODPPB/gCODVFA. Moreover, the removal of COD and
N by PPB culture over the whole acclimation period resulted to
an average COD:N of 100:10, which was higher than the optimum
ratio (100:7.1) previously reported for domestic wastewater. This
high ratio suggested that the PPB enriched culture may have
potential for enhancing nitrogen removal in order to achieve the
discharge limits for total nitrogen (Hülsen et al., 2014).
Enriched PPB biomass was used for inoculum purposes in
order to study the hydrogen formation from wastewater in
presence and absence of an electrode as electron donor.
Effect of Nitrogen Source on Biological
Hydrogen Production by PPB
Hydrogen production under nitrogen fixation conditions is
described by Equation (1) where molecular nitrogen (N2) is
converted to ammonia (NH3) and protons (H+) to hydrogen
(H2) (Rey et al., 2007).
N2+8e−+H++16ATP →2NH3+H2+16ADP +16Pi
(1)
Biological experiments were conducted in order to extract
the optimum biological conditions to maximize hydrogen
production while minimizing CO2emission. Our first approach
was to analysis how biohydrogen production depended on
nitrogen substrate at different concentrations by using three
different N sources (ammonium, glutamate and nitrogen gas)
and malic acid as a model substrate of organic carbon.
Interestingly, glutamate increased PPB growth rate by 2-fold
in comparison with ammonium or nitrogen gas (see Figure S1
in Supplementary Information). This also is shown in Table 2,
where the kinetic parameters of PPB metabolism are included.
Biohydrogen analysis revealed an interesting correlation of
hydrogen with the ratio COD:N. So, hydrogen production was
enhanced (451 ±2.1 mLH2/L) when NH4Cl was used as
inorganic nitrogen at a COD:N ratio of 100:3.75. In contrast,
very low amount of hydrogen was produced when higher
concentrations of NH4Cl (COD:N of 100:7.5 and 100:15)
were tested (Figure 3A;Table 2), with 95% confidence values
concurring with the zero value. This, in fact, indicates that
zero hydrogen production cannot be statistically discarded under
these conditions. This is in agreement with the results previously
Frontiers in Energy Research | www.frontiersin.org 5November 2018 | Volume 6 | Article 107
Vasiliadou et al. PPB Meet BES
FIGURE 2 | Performance of PPB culture during a weekly operating cycle: (A) BChl aand carotenoids absorption spectra due to PPB growth, (B) COD and NH4-N
assimilation and PPB concentration as VSS.
TABLE 2 | Comparison of H2production under different nitrogen and carbon sources.
No Sources COD:N Ra
PPB (mgVSS/Lh) Rb
acid (mMacid/Lh) Hc
2_max (mLH2/L) Hd
2_rate
(mLH2/Lh)
Ye
H2
(LH2/g_acid)
Yf
molH2
(molH2/mol_acid)
1 Malic/NH4Cl 100:3.75 3.67 ±0.82 0.21 ±0.05 451.0 ±2.1 2.63 ±0.13 0.13 ±0.01 0.70 ±0.04
2 Malic/NH4Cl 100:7.5 4.74 ±0.84 0.19 ±0.04 2.2 ±2.3 (1.35 ±1.1)
×10−2
(0.59 ±0.58)
×10−3
(3.15 ±3.18)
×10−3
3 Malic/NH4Cl 100:15 5.47 ±1.01 0.20 ±0.04 13.7 ±14.5 (1.30 ±0.7)
×10−2
(0.50 ±0.63)
×10−3
(2.55 ±2.76)
×10−3
4 Malic/Na-glutamate 100:3.75 3.39 ±0.97 0.21 ±0.06 300.2 ±85.0 2.06 ±0.90 (8.75 ±2.89)
×10−2
0.47 ±0.16
5 Malic/Na-glutamate 100:7.5 6.26 ±2.07 0.20 ±0.06 416.1 ±148.2 2.57 ±1.03 0.12 ±0.05 0.66 ±0.27
6 Malic/Na-glutamate 100:15 7.56 ±1.89 0.19 ±0.04 423.0 ±40.9 2.71 ±0.27 0.12 ±0.01 0.67 ±0.05
7 Malic/N2gas – 5.57 ±1.68 0.21 ±0.04 12.2 ±11.3 0.12 ±0.05 (0.36 ±0.39)
×10−2
(1.97 ±2.12)
×10−2
8 Butyric/Na-glutamate 100:15 3.23 ±0.18 0.06 ±0.01 214.2 ±7.2 1.21 ±0.12 0.22 ±0.02 0.79 ±0.08
9 Acetic/Na-glutamate 100:15 3.96 ±0.09 0.16 ±0.0 320.4 ±82.5 2.49 ±0.36 0.21 ±0.05 0.50 ±0.13
10 Bio-electrochemical
Malic/Na-glutamate*
100:15 5.91 0.17 390 2.32 0.11 0.60
aPPB growth rate, borganic substrate assimilation rate, cmaximum H2production, dH2production rate, e,fH2yield, *In the Bio-electrochemical experiments there were no replicates,
so no error analysis was able to be conducted.
reported, stating that high NH4Cl concentration inhibits the
function of the enzyme nitrogenase of PPB resulting in lower
hydrogen production (Kim et al., 2012a). Alternatively, N2gas
as a nitrogen source was used to enhance hydrogen production
under nitrogen fixation conditions without repressing the
expression of nitrogenase genes.
However, it was observed that the use of N2gas as nitrogen
source did not efficiently produce H2(Figure 3A;Table 2). The
low H2production rate (0.12 ±0.05 mLH2/Lh) observed in
this experiment was probably attributed to a higher energy
requirement for the process (16 ATP per mol of hydrogen
produced in the nitrogen fixation case vs. 4 ATP per mol of
hydrogen produced in the case of the hydrogen production
with no nitrogen fixation in the nitrogenase; McKinlay and
Harwood, 2010). Also, the extra consumption of reductants
to conduct nitrogen fixation for the heterotrophic growth
may be counteracted by an increase of the consumption of
the produced H2in autotrophic growth mode. The absence
of CO2evolution in the N2experiments confirmed such a
hypothesis. The PPB culture may use the H2produced during
the N2fixation to re-fixate, in the Calvin-Benson-Bassham
cycle (Calvin cycle, CBB), the CO2produced during the malic
acid assimilation. The analysis of the effect of an external
electron source (e.g., from the cathode) would give light
to this unsolved question and open possibilities for further
research.
Finally, the results indicated that PPB culture produced
large amounts of hydrogen when Na-glutamate was used
as an organic nitrogen source (300–423 mLH2/L, Table 2).
Na-glutamate enhanced the PPB growth and hydrogen
production rate (2.1–2.7 mLH2/Lh). The results of this
study are in agreement with those reported by other
Frontiers in Energy Research | www.frontiersin.org 6November 2018 | Volume 6 | Article 107
Vasiliadou et al. PPB Meet BES
FIGURE 3 | Hydrogen (A) and CO2(B) production by the PPB biological
activity after 1 week of reaction with different nitrogen substrates and malic
acid as organic carbon source. Error bars are standard deviations from
triplicate experiments.
researchers, who have shown that Na-glutamate enhances
hydrogen production without inhibiting the nitrogenase
enzyme (Melnicki et al., 2008; Assawamongkholsiri and
Reungsang, 2015). This is due to the fact that organic
nitrogen can be directly assimilated into proteins and a less
complex metabolic activity is required for the production of
amino acids compared to inorganic sources (Merugu et al.,
2010).
Considering Na-glutamate concentration, results showed that
H2production (mLH2/L) as well as its production rate was
increased as the organic nitrogen concentration increased,
achieving a maximum H2production at a COD:N ratio of 100:15
(Figure 3A;Table 2). Moreover, it is noteworthy to mention
that the CO2production was reduced as the Na-glutamate
concentration increased (Figure 3B). Therefore, the use of Na-
glutamate at COD:N ratios of 100:3.75, 100:7.5, and 100:15
resulted to the production of 74.2 ±11.6, 35.7 ±30.7, and 11.8 ±
16.0 mLCO2/L, respectively. PPB that grow on oxidized organic
substrates (as malic acid) produce CO2due to the oxidation of
these substrates. The released CO2can then be fixed through the
Calvin-Benson-Bassham cycle (Calvin cycle) into cell material as
an electron accepting process (McKinlay and Harwood, 2010).
This CO2fixation via the Calvin cycle enabled PPB to accept
excess of electrons and to maintain redox balance and to dispose
extra electrons that are generated during use of extra carbon
included in Na-glutamate. Therefore, the higher Na-glutamate
concentration in the medium could result in a greater CO2
fixation and lower emission.
It should be highlighted that reducing the NH4Cl, levels
(100:3.75 ratio) resulted in a high hydrogen production
(Figure 3A) by minimizing the inhibitory effect of this
compound on the activity of nitrogenase. In contrast, same
conditions resulted in a significant emission of CO2(56.7 ±
1.9 mLCO2/L). In addition, Na-glutamate in the ratio 100:15
could favor the PPB activity toward nitrogen assimilation. In
conclusion, based on the above, Na-glutamate at a COD:N ratio
of 100:15 was selected as the optimum culturing conditions for
maximized H2production with minimized CO2emission.
PPB Can be Cultured Under
Bio-Electrochemical Conditions
Considering the importance of the internal electron recycling,
an active modification of the electron fluxes through artificial
addition of electrons by applying electrochemical technology
may potentially enhance the PPB activity and drive toward
an optimum H2production process. In this sense, the bio-
electrochemical capability of interaction between PPB and
graphite-electrode has been explored, specially emphasizing the
situation when graphite electrode behaves as an electron donor
to PPB (setting graphite-electrode potential at −0.5 V) aiming
to increase PPB metabolic paths activity by supplying electric
current. Therefore, this study focused on the analysis of a
bio-electrochemical device based on PPB, using malic acid as
organic source and Na-glutamate as nitrogen source, compared
to electrode-free biological systems.
Figure 4 presents the cyclic voltammograms (CV) of bio-
electrochemical system as well as the bare graphite-electrode
as a control electrochemical process, at different time intervals
during a weekly operation. The electrochemical behavior for
the bare graphite-electrode in the culture media do not exhibit
any electrocatalytic behavior in the whole potential range of
study (−0.8 to 0.8 V). As it can be seen in Figure 4, the cyclic
voltammograms for bare graphite-electrode (electrochemical
abiotic control) has no reductive currents that can be assigned
to tentative hydrogen evolution or the malic acid reduction. As
expected, only capacitive currents typical from bare graphite-
electrode (ideally polarizable electrode, Bard and Faulkner, 2001)
were detected in absence of PPB. Only small positive currents
were observed at the more positive potentials explored, 0.8 V (vs.
Ag/AgCl), probably due to slight water oxidation and graphite
surface oxidation. Figure 4 shows the cyclic voltammograms for
the bio-electrochemical system during the first 24 h, which are
very similar to the bare graphite electrode. Only changes in
the capacitive currents were observed, exhibiting a higher value
for the interfacial pseudocapacitance under bio-electrochemical
conditions, so suggesting an electrode surface modification
by bacteria attachment. Just after inoculation no significant
electroactive biofilm formed but the presence of bacteria in the
interface increases the interfacial pseudocapacitance. This was
Frontiers in Energy Research | www.frontiersin.org 7November 2018 | Volume 6 | Article 107
Vasiliadou et al. PPB Meet BES
FIGURE 4 | Cyclic voltammograms at different time intervals during bio-electrochemical (red line) and control electrochemical operation (blue line).
probably due to the very low amount of PPB biomass existed at
the beginning (Time 0 h) of the experiment (0.01 gVSS/L).
After 48 h of polarizing the electrode at −0.5 V (vs. Ag/AgCl),
the cyclic voltammograms revealed the electroactivity of the PPB
biofilm interacting with the graphite-electrode surface. These
results suggested that PPB started to interact with the working
electrode when sufficient amount of biomass (0.1 gVSS/L) and
malic acid as carbon source were present in the cathode chamber
(Figure 5A). It can be observed in Figure 4C, how the current
was increased in correlation with a potential increase above
0.4 V. This result indicates that PPB biofilm used the electrode
as an electron acceptor, probably for malic acid oxidation. This
remarkably result indicates the use of PPB for anodic-based
oxidations in MET applications. A less noticeable change in
current in the negative potential region (between −0.2 and
−0.8 V) is starting to develop after 48 h. In Figure 4D the
negative currents in the potential region between −0.2 and
−0.8 V results in a clear negative feature indicating processes
related to the interaction of PPB with the graphite electrode as an
electron donor. A detailed analysis of the cyclic voltammograms
at 72 and 96 h suggests the presence of two processes responsible
for the negative feature between −0.2 and −0.8 V. Two processes
were may be identified: (a) between −0.2 and −0.4 V, there was
a steady increase in the negative current (in absolute value),
and (b) around −0.6 V there is a steep change in the slope
of the negative current indicating the occurrence of a second
process. In our experiments, the electrode was polarized at
−0.5 V, a potential able to explore the first reductive process
from electroactive PPB. Finally, it is noteworthy to mention
that after the depletion of malic acid in the medium at 170 h
(Figure 5A) the magnitude of redox reactions was changed
(Figure 4F), showing an electrochemical behavior similar to
this of Time 0 h, and suggesting low electroactivity of the PPB
culture.
Effect of Carbon Source on Biological
Hydrogen Production by PPB
The biological production of hydrogen by PPB was studied by
testing different organic carbon sources, as malic acid, butyric
acid and acetic acid, using Na-glutamate as optimum nitrogen
source at a COD:N ratio of 100:15. Results indicated that the PPB
culture was able to assimilate all the organic acids tested toward
biomass growth as well as hydrogen production. Maximum PPB
growth rate (7.56 mgVSS/Lh) was obtained when malic acid was
used as compared to butyric (3.23 mgVSS/Lh) and acetic acid
(3.96 mgVSS/Lh; Table 2). As a consequence of the higher C
assimilation, the N assimilation into bacteria (as proteins) was
also enhanced by using malic acid, giving a production of SCP
of 874 mg/L as compared to 621 and 346 mg/L obtained with
acetic and butyric acids, respectively. It was observed that the use
of malic acid as carbon source achieved the highest hydrogen
production (H2_max, mLH2/L) and the highest H2production
rate (H2_rate, mLH2/Lh; Table 2). Malic acid has widely used
as optimum carbon source for H2production, probably due
to its capacity to directly enter the tricarboxylic acid cycle
(Melnicki et al., 2008; Kim et al., 2012b; Assawamongkholsiri and
Reungsang, 2015). Other evidences supporting malic acid as the
optimum organic to conduct hydrogen production is shown in
Supporting Information.
The experimental results obtained from the biological study
(electrode-free) of hydrogen production indicated that the
combination of malic acid and Na-glutamate was the optimum
for maximizing the hydrogen production by PPB. The efficiency
of H2production from the PPB mixed culture enriched in this
Frontiers in Energy Research | www.frontiersin.org 8November 2018 | Volume 6 | Article 107
Vasiliadou et al. PPB Meet BES
FIGURE 5 | Culture of PPB under bio-electrochemical conditions at −0.5 V (vs. Ag/AgCl) (A) Malic acid removal and PPB growth and (B) production of H2, N2, and
CO2.
study is comparable to those of previous studies where pure
or mixed PPB cultures were used (Table 3). In conclusion, the
high H2production rates achieved in this study showed that the
PPB mixed culture could potentially be used for a feasible H2
production application during wastewater treatment processes.
This supports the use of malic acid and Na-glutamate as the C
and N sources for the bio-electrochemical production of H2.
Effect of Bio-Electrochemical Electron
Donor on PPB for Producing Hydrogen and
Fixing CO2
Results suggested that bio-electrochemical process of PPB
resulted to similar H2production rate and hydrogen yields
(Table 2) compared to the PPB biological process under the
same conditions. However, it was observed that after 1 week
of bio-electrochemical reaction PPB fixed all the amount of
CO2that was produced during the first 50 h (Figure 5B).
This resulted to zero CO2emission as compare to the PPB
biological process (see Supporting Information, Figure S2) that
produced an average of 11.8 ±16.0 mLCO2/L after 1 week of
biological process (Figure 3B). Subsequently, results suggested
that CO2fixation was the main mechanism of PPB metabolism
that was accepting electrons from the bio-cathode. This is in
agreement with the negative current values observed during the
bio-electrochemical process suggesting that there might be a
consumption of electrons due to the PPB activity (Figure 6).
Figure 5 shows the malic acid assimilation, the PPB growth
and the evolution of gas production during bio-electrochemical
process. The bio-electrochemical setup revealed that PPB
can effectively use the graphite-electrode as electron donor
(Figures 4C-48 h, D-72 h, and E-96 h) and, subsequently, reduce
the levels of CO2. Carbon dioxide fixation is not detected in such
a high extension when system was run in absence of electrode (see
Supporting Information, Figure S2). Carbon dioxide fixation
seems to occur at the origin of the first reductive process detected
between −0.2 and −0.6 V. The extra electron source provided
by the electrode promoted carbon dioxide fixation by PPB
beyond the standard activity of this bacterial genus in absence
of electrodes under limited electrons availability.
It is well-reported that graphite electrodes, and generally
carbon electrodes, exhibit a high overpotential for hydrogen
evolution and carbon dioxide reduction (Sullivan et al.,
1993; DuBois, 2006; Yang et al., 2016) and therefore poor
electrocatalytic properties. The standard electrode potential for
carbon dioxide reduction to formic acid and oxalic acid are
−0.199 and −0.590 V (Sullivan et al., 1993; Eggins et al., 1998;
DuBois, 2006; Yang et al., 2016), respectively. These values are
reported in the SHE scale, and taking into account that we
are working in pH =7 solutions and Ag/AgCl reference scale
(ca. 0.2 V vs. SHE), the standard electrode potential has to
be recalculated according to Nernst equation. Nernst equation
gives an equilibrium potential of −0.716 V for formate and
−0.790 V in the case of oxalate vs. Ag/AgCl. In any case,
these electrode potentials are more negative than −0.5 V vs.
Ag/AgCl, potential value used in this study. Furthermore,
these are the thermodynamic potential values, carbon dioxide
reduction has been reported on graphite electrodes taking place
at potentials more negative than −0.9 V vs. Ag/AgCl (Eggins
et al., 1998). Actually, this fact can be clearly observed in the
electrochemical control voltammograms reported in Figure 4.
The cyclic voltammogram corresponding to the bare graphite
electrode in the same solution but in absence of PPB, displays
the classical voltammogram of an ideally polarized electrode,
where there are no significant faradaic currents in the whole
potential range explored (0.8 to −0.8 V), as it would be expected.
Only pseudocapacitive electrochemical behavior is detected in
the electrode interface in absence of PPB. Regarding the previous
arguments, electrochemical carbon dioxide reduction on bare
graphite electrode can be discarded, and the only contribution of
PPB metabolism can explain the consumption of carbon dioxide.
In contrast, at −0.5 V, the capability of hydrogen production
of the bio-electrochemical system was comparable to the
exhibited by PPB in absence of electrode, and no electrode
potential was observed in this process. The origin of the second
bio-electrochemical process developed below −0.6 V, that can be
tentatively assigned to hydrogen production, will require further
investigation beyond the scope of this work. The capability
of PPB for using graphite-electrode as electron donor was
demonstrated. The extra electron donor source can be used in
Frontiers in Energy Research | www.frontiersin.org 9November 2018 | Volume 6 | Article 107
Vasiliadou et al. PPB Meet BES
TABLE 3 | Comparison of hydrogen production rates by different cultures and systems with the one studied in the present work.
PPB culture IR Process
mode
Carbon/Nitrogen
sources
YH2
(LH2/gacid)
H2_rate
(mLH2/Lh)
YmolH2
(molH2/mol_acid)
References
Rhodospirillum rubrum
Rhodopseudomonas
palustris
60 W/m2Batch Succinate/glutamate – 21
4.3
–Melnicki et al., 2008
Rhodobacter
sphaeroides
100 W Batch Malate/glutamate
Malate/NH4Cl
Acetate/NH4Cl
0.541
0.224–0
0.467–0.135
5.1
4.6–0
5.8–3.3
–Akkose et al., 2009
Rhodobacter
sphaeroides
5 klux* Continuous Mixture of
VFAs**/(NH4)2SO4
0.185 1.125 – Ozmihci and Kargi,
2010
Rhodopseudomonas
acidophila
2,400
lux*
Batch Acetate/nitrate
Malate/nitrate
Succinate/nitrate
Succinate/N2
Succinate/NH4C
– 2.7
2.5
3.3
0.5
1.25
–Merugu et al., 2012
Mixed
culture–dominant
Rhodopseudomonas
palustris
190
W/m2
Continuous Mixture** of acetate,
lactate, butyrate,
propionate/NH4-N
0.97 121 – Tawfik et al., 2014
Rhodobacter
sphaeroides
10 W/m2Batch Succinate/(NH4)2SO4– 31 – Ryu et al., 2014
Rhodobacter sp.
KKU-PS1
2,500
lux*
Batch Malate/glutamate – 6.8 – Assawamongkholsiri
and Reungsang, 2015
Rhodopseudomonas
palustris
2,000
lux*
Batch Lactate/glutamate
Butyrate/glutamate
– 8.4
19.9
2.57
4.92
Hu et al., 2017
Mixed culture 20 W/m2Batch Malic acid/glutamate 0.12 2.71 0.67 This study
*Illumination intensity was calculated (1 lx =0.0161028 W/m2), **Dark fermentation effluent.
FIGURE 6 | Current evolution during bio-electrochemical and abiotic
electrochemical reactions.
more than one metabolic pathway. Polarizing the electrode at
−0.5 V, allows PPB to use electrode for carbon fixation reaching
almost no carbon dioxide accumulation in contrast to the
electrode-free biological system. This is the first study indicating
that electroactive capture of CO2by PPB is feasible.
Finally, the SCP production achieved by PPB during the
bio-electrochemical process (81% mgSCP/mgVSS) was similar
to this observed by PPB growing in absence of electrodes.
Therefore, the bio-electrochemical process did not seem
to affect proteins yields under the experimental conditions
tested.
CONCLUSIONS
This work analyzed the optimum culturing conditions for
maximizing the hydrogen production by a mixed culture
of purple phototrophic bacteria. In addition, the effect of
a negatively polarized bio-electrochemical device on the
modification of the behavior of the culture in terms of
metabolic shifts and current consumption was explored. The
main conclusions extracted from this work are shown below:
- Among all the conditions tested in absence of electrodes,
best results on the hydrogen production have been achieved
by using malic acid as a carbon source (instead of acetic
and butyric) and Na-glutamate as a N source (instead of
ammonium and dinitrogen gas), in a COD/N relationship of
100/15. Under these conditions, the production of CO2was
also minimized.
- Cyclic voltammograms of the bio-electrochemical system
shown the appearing of at least three potentials (two negative
and one positive) with clear interaction between the PPB
culture and the electrode. This makes evident the high
electroactivity of PPB cultures and their potential as a MET
microbial candidate.
Frontiers in Energy Research | www.frontiersin.org 10 November 2018 | Volume 6 | Article 107
Vasiliadou et al. PPB Meet BES
- Negative polarization of the electrode at −0.5 V caused
a detectable consumption of electrons associated with a
depletion of the produced carbon dioxide, which indicates
that the PPB culture was capable of using electrons from
the cathode to capture the excess of C released as CO2
during the CBB cycle. This behavior was not observed
before in an indigenous (non-genetically-modified) PPB
culture.
- Results presented herein have shown that further in-depth
research using different conditions (other polarization of the
cathode) will be of extreme benefit and may enhance the H2
production rate.
AUTHOR CONTRIBUTIONS
IV designed and performed the experiments and wrote the
manuscript, AB critically reviewed the manuscript, CM
helped in the experimental stage, JM critically reviewed
the manuscript, FM critically reviewed the manuscript
and supervised the work, AE-N and DP designed the
experiments, supervised the work, and corrected the
manuscript. Both DP and AE-N are corresponding
authors.
ACKNOWLEDGMENTS
IV thanks the International Excellence Campus Smart Energy
Program (CEISEP) for a Post-doctoral Fellowship. Financial
support of Regional Government of Madrid provided through
project REMTAVARES S2013/MAE-2716 and the European
Social Fund as well as Spanish Ministry of Economy are
acknowledged.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fenrg.
2018.00107/full#supplementary-material
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Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Copyright © 2018 Vasiliadou, Berná, Manchon, Melero, Martinez, Esteve-Nuñez
and Puyol. This is an open-access article distributed under the terms of the Creative
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