2_Articulo_2 (2) (1).pdf_is La electrohidrogénesis se llevó a cabo en una celda electroquímica microbiana (CEM) de doble cámara, típica de tipo H, compuesta por dos botellas de vidrio ScottDuran (volumen total/de trabajo: 65/50 ml) separadas por una membr

is
La electrohidrogénesis se llevó a cabo en una celda electroquímica microbiana
(CEM) de doble cámara, típica de tipo H, compuesta por dos botellas de vidrio ScottDuran (volumen total/de trabajo: 65/50 ml) separadas por una membrana de intercambio
de protones (PEM, Nafion 117). Se utilizó una varilla de grafito revestida con tela de
carbón activado (ACC) (superficie proyectada de 67 cm²) como contraelectrodo, mientras
que la varilla de grafito revestida con malla de acero inoxidable (49 cm² ) se utilizó como
electrodo de trabajo. Ambos electrodos se conectaron mediante un cable de acero
inoxidable (colector de corriente) al electrodo de potencia.

1. ARTICLE INFO ABSTRACT
T
and economic viability of the process [9–13]. Similarly, due to the low
photo-biological H2 production or electrohydrogenesis (MEC) or me-
thanogenesis or with both can result in additional H2 and CH4 pro-duction
respectively, thereby increasing the overall energy recovery
minimal residence time. Various studies have documented the im-provement of energy
efficiency from 20% (hydrogen production
fuels with relatively higher energy density (H2 ÿ 122 MJ/kg,
efficiency only) to volatile fatty acids (VFAs), acidogenic fermentation
and H2 production) as well as with electrohydrogenesis for effluent
terms of total biogas yield (H2 + CH4) and substrate degradation rate in
has certain pitfalls that need to be overcome for bioprocess enhancement-
ment. Low hydrogen yield and inefficient substrate degradation questions the
economic and practical viability of the acidogenic fermentation process. Due
to incomplete oxidation of substrate (33% conversion
Contents lists available at ScienceDirect
1. Introduction
The rising concerns regarding the detrimental environmental effects
can only deliver a theoretical maximum hydrogen yield of 4 mol H2/
lowers the system pH, thereby inhibiting the further hydrogen pro-duction [8].
Effective utilization of these residual VFAs present in
containing VFAs cannot be further used by microbes and this also
(HRT) of anaerobic digestion technology, its implementation on a large
CH4 ÿ 55 MJ/kg) than that of hydrocarbon fuels and burns clean substrate conversion efficiency and higher hydraulic retention time
acidogenic fermentation effluent in second stage by coupling it with
of fossil fuels and sustainable energy security encouraged the re-searchers
around the globe to explore alternative renewable energy
energy intensive as compared to other physical and chemical methods
process integration strategies viz. acidogenic fermentation (HAF), me-
thanogenesis (MAD) and electrohydrogenesis (HMEC) in diverse combi-
nations as two-stage and third-stage steps to find an effective integrated
mole glucose. Further, the reported experimental yield of hydrogen is
scale requires huge capital investment [14]. However, improved energy
polishing along with additional H2 recovery. In this regard, a comprehensive
effort has been made in the present study to examine various
without emitting harmful greenhouse gases [3–6]. Biological production of
hydrogen and methane by acidogenic fermentation and anae-robic digestion
respectively is considered more economical and less
approach that enables to achieve higher energy conversion efficiency in
restricted only to 2–3 mol/mole glucose as the residual organic matter
journal homepage: www.elsevier.com/locate/enconman
resources [1,2]. Biohydrogen and biomethane are two such sustainable
recovery and shortened HRT could be effectively achieved by coupling
anaerobic digestion step with a pre-acidification stage (for hydrolysis
https://doi.org/10.1016/j.enconman.2018.10.106
[7]. However, individual implementation of each of these approaches
0196-8904/ © 2018 Published by Elsevier Ltd.
Energy Conversion and Management 180 (2019) 25–35
Energy Conversion and Management
An integrated approach for efficient conversion of Lemna minor to biogas
b
to
SK Purib, Manoj Kumarb,ÿ, Sandipam SrikanthbManpreet Kaura Sarita Sachdevaa, ,
HAF ÿ MAD ÿ HMEC (38.77 mol biogas/kg TOCR; 25,415 KJ/kg TOCR) as compared to HAF ÿ HMEC ÿ MAD
in case of HMEC. Among, three-stage integrations, higher biogas yield and energy recovery was observed in
(HMEC) and methanogenesis (MAD), were evaluated in single stage as well as in different combinations of two
Aquatic weed, Lemna minor was evaluated for its potential as a feedstock for gaseous fuel production (bio-
hythane) in an integrated strategy. Three approaches viz., acidogenic fermentation (HAF), electrohydrogenesis
feasible energy. Compared to single and two-stage operations, three-stage operation evidenced higher biogas
acids (VFA) along with H2 production. Similarly, integration of MAD in second stage showed the possibility of
tapping higher energy rather than HMEC due to higher carbon loss as CO2 coupled to more H2 fraction in biogas
(H2 + CH4) yield with remarkable total organic carbon (TOC) reduction. Irrespective of the integration sequence,
HAF in first stage represented the possibility of harnessing higher energy by accumulation of volatile fatty acids
(37.79 mol biogas/kg TOCR; 15,416 KJ/kg TOCR). Along similar lines, analysis of organic carbon flow exhibited
and first stage (39.7–56.5%).
significant substrate degradation in three stage integrations (72.5–81.4%) as compared to second (66.2–70%)
ÿ Corresponding author.
E-mail address: [email protected] (M. Kumar).
Received 17 September 2018; Received in revised form October 29, 2018; Accepted 31 October 2018
stage (HAF ÿ HMEC, HAF ÿ MAD) and three stages (HAF ÿ HMEC ÿ MAD, HAF ÿ MAD ÿ HMEC) to tap the maximum
Acidogenic fermentation
Keywords:
Manav Rachna International Institute of Research and Studies (MRIIRS), Sector 43, Faridabad, Haryana 121004, India
Indian Oil Corporation Limited (IOCL), R&D Centre, Sector 13, Faridabad 121007, Haryana, India
Methanogenesis
Electrohydrogenesis
Aquatic weed
Biohythane
Machine Translated by Google

2. bioethanol production but more efforts are required to develop an array
So far, the most extensively studied plant biomass for biogas pro-
duction is lignocellulosic biomass including corn stover, wheat straw,
and up to 90% in an integrated acidogenic fermentation and electro-
hydogenesis [15–18]. However, the integrated approach adopted for
mild pretreatment and subsequently used as feedstock for maximizing
enhanced biohydrogen and biomethane recovery in a biorefinery con-
cept needs a more detailed study for understanding its significance in
rice straw and sugarcane bagasse [2,19]. Neverthless, the substantial
cell wall structure and low lignin content (< 10%) of an aquatic weed,
about the conversion of harvested Lemna biomass to commercially
viable products [23]. In this respect, the idea of using this aquatic waste
protein content (15–45 wt% dry basis) and starch content (30%)
comparable to that of soybean, and corn respectively, L. minor is seen as a
Lemna has been intensively studied for wastewater treatment for
decades [22]. Although Lemna-based wastewater treatment system has
be an eco-friendly and profitable option for coupled wastewater treatment-
ment and bioenergy production. Few studies have elucidated its role for
In this study, L. minor, harvested from wastewater was subjected to
benefits (nutrient recovery, removal of excess biomass) and turns out to
process) to about 60% in combined hydrogen and methane production
the production of gaseous biofuels through various integrated ap-
proaches. Further, by integrating biohydrogen and biomethane
capital and operating costs associated with the extensive pretreatment
upgrading the total energy conversion efficiency of the overall process.
of Lemna-derived products that are commercially viable.
Lemna minor, its potential as an alternate feedstock for bioenergy pro-
duction was evaluated in the present study [20]. Owing to its high
promising substrate for bioenergy production [21]. Because of its pro-
filic growth in wastewater and extraordinary nutrient uptake ability,
weed for biofuel production would bring significant environmental
not been implemented on large scale due to the lack of knowledge
of the feedstock makes the conversion bioprocesses economically in-
feasible. In this context, considering the higher productivity, different
Fig. 1. Different process configurations namely (A) Single-stage process (B) Two-stage integration (C) Three stage integration for enhanced biogas (H2 + CH4)
production from Lemna biomass.
26
Energy Conversion and Management 180 (2019) 25–35
M. Kaur et al.
Machine Translated by Google

3. Pretreatment of the biomass is one of the crucial steps in effective
valorization of the biomass to enhance the enzymatic hydrolysis step and to
release the fermentable sugars from biomass [26,27]. In this study, the
pretreatment was carried out through acid hydrolysis using dilute sulfuric acid.
Thermo-chemical pretreatment was optimized on dried Lemna biomass (6%
w/v) using different H2SO4 concentrations (0.5–4% v/v) at 121 °C for 15 min.
The acid hydrolysed slurry was neutralized with NH3 (10% v/v) and filtered
using Whatman filter paper discs. The collected filterate was analyzed for
reducing sugars by dinitrosalicylic acid assay against glucose standard.
Among all con-centrations, higher reducing sugar was observed at 1% (v/v)
acid concentration and hence pretreatment step of further experiments was
carried out at 6% (w/v) biomass loading under similar conditions.
producing bioprocesses in one system, the motivation is to produce desired
H2/CH4 ratio optimal for biohythane generation, which is considered as a
high value gas fuel for vehicles due to the combined advantages of hydrogen
and methane that contributes to its higher combustion effi-ciency [24,25]. To
the best of our knowledge, the use of Lemna for bioenergy production by an
integrated process of acidogenic fermentation, electro-hydrogenesis and
methanogenesis has been reported for the first time in this study.
The whole content was mixed thoroughly followed by the pH ad-justment
to 6.0 using 1 M ortho-phosphoric acid. The reactors were sealed properly to
avoid gaseous exchange and purged with N2 to create an anaerobic
environment. Experiments were carried out at 37 °C in an incubator shaker at
120 rpm for 7 days during single stage, while it was restricted to 48 h (2 days)
in two and three stage operations. The bio-process parameters viz., H2
production, TOC, pH and VFA were analyzed at regular time intervals to
monitor the process performance.
2.4.1. Acidogenic fermentation
Acidogenic fermentation for biohydrogen production was carried out in
anaerobic reactors (Total/Working volume, 125/100 ml).
CH4 production was carried out at 37 °C for 7 days in the single stage, while
it was restricted to 48 h in two and three stage integrations.
Pretreated Lemna biomass at 6% (w/v) loading was inoculated with 5 ml of heat-treated mixed
culture (5% v/v) under anaerobic conditions.
2.4.2. Electrohydrogenesis
2.5.1. Characterization of Lemna biomass as biofuel feedstock
To develop exoelectrogenic biofilm that can deliver higher current density,
the counter electrode of MEC was inoculated with selectively enriched
exoelectrogenic inoculum (10% v/v) from an operating MFC treating synthetic
wastewater along with 10 mM of acetate in 50 mM phosphate buffer and was
set to +0.2 V against Ag/AgCl (3 M KCl).
After the biofilm stabilization phase, the counter electrode chamber was finally
fed with pretreated biomass slurry during single stage or the effluent of
acidogenic fermentation/methanogenesis during integrated approaches. On
the other hand, the working electrode chamber was filled with PBS added
with 1% (w/v) NaCl solution to maintain electrical conductivity. Experiment
was carried out under poised potential of ÿ1 V to the working electrode against
Ag/AgCl (3 M KCl, +0.195 V vs SHE) reference electrode, placed near to the
working electrode.
Electrohydrogenesis was carried out in dual chambered typical H-type
microbial electrochemical cell (MEC), made up of two Scott-Duran glass
bottles (Total/working volume, 65/50 ml) separated by proton exchange
membrane (PEM, Nafion 117). Activated carbon cloth (ACC) wrapped graphite
rod (projected surface area of 67 cm2 ) was used as counter electrode while
the stainless steel mesh wrapped graphite rod (49 cm2 ) was used as working
electrode. Both the electrodes were connected through stainless steel wire
(current collector) to the po-
Each set of experiment was conducted by transferring the whole content
(effluent and residual biomass slurry) of the first approach to the second and
there on. Single stage experiments were carried out for 7 days but based on
the results obtained, each experiment was re-stricted to 48 h during
integrations.
The objective of the experiment is to integrate different energy producing
approaches in sequence to maximize the energy yields (Fig. 1). In this context,
feasibility of coupling acidogenic fermentation (HAF), electrohydrogenesis
(HMEC) and methanogenesis (MAD) was identified by integrating them in
various sequences to maximize the energy recovery from Lemna biomass.
Initially, individual approaches viz., HAF, MAD and HMEC were tested for
their ability to produce biogas (H2 and CH4) along with substrate degradation.
Further, the experiments were carried out in different two and three-stage
combinations to increase the energy recovery as well as carbon conversion
efficiencies (Table 1).
2.2. Pretreatment
2.1. Feedstock and inoculum
Effluents of the acidogenic stage were fed to the subsequent stages of
electrohydrogenesis or methanogenesis as designated in experimental
combinations (Table 1).
2.4.3. Methanogenesis
Methanogenesis experiments were carried out exactly similar to the
acidogenic fermentation experiments, except that the inoculum added was
untreated anaerobic mixed culture, instead of heat-treated mixed culture and
the pH of the reactor was maintained at 7.0 instead of 6.0.
2.4. Operational details
2.3. Experimental design
Experiments were monitored both qualitatively and quantitatively based on
the composition and yield of biogas along with TOC and VFA.
2. Materials and methods
Characterization of biomass includes proximate analysis, ultimate analysis
and compositional analysis. Proximate analysis is one of the most important
characterization for determining the suitability of bio-mass as biofuel
feedstock. It includes moisture content, ash, volatile
tentiostat.
Experiment was carried out under anaerobic conditions at room temperature for 7 days
during single stage while for 48 h in different in-integrated approaches. The current
consumption, TOC, VFAs and H2 production were monitored at regular intervals to assess
the performance of MEC.
2.5. Analytical methods
Lemna minor harvested from a local wastewater pond was sun-dried and
powdered to 0.4 mm mesh size to be used as a feedstock in all the
experiments. Prior to experiment, the characterization of Lemna was done
through proximal, ultimate and compositional analysis. Specific biocatalyst
was used for each process during the experiment. Anaerobic mixed culture
obtained from laboratory scale anaerobic digester was used for
methanogenesis. Electroactive bacteria (EB) obtained from an operating
microbial fuel cell (MFC) was used in electrohydrogenesis experiment. Heat
treated (80 °C; 2 h) anaerobic mixed culture was used for biohydrogen
production after being enriched for several cycles in media containing (g/
L,NaCl2, 10; NH4CL, 1; K2HPO4, 0.3; KH2PO4, 0.3; MgCl2, 0.2; CaCl2·2H2O,
0.1; KCl, 0.1; MnO4·7H2O, 0.01 ; ZnSO4 · 7H2O, 0.05 ; H3BO3, 0.01 ;
MgCl2·6H2O, 00.2; FeCl3, 0.1; CuCl2·6H2O, 0.05) and vitamin solution (g/L,
riboflavin, 0.025; citric acid, 0.02; folic acid, 0.01; para-amino benzoic acid,
0.01) under acidic pH (5–6).
Energy Conversion and Management 180 (2019) 25–35
27
M. Kaur et al.
Machine Translated by Google

4. (HAF ÿ HMEC)
(MAD)
(HAF)
(HMEC)
(HAF ÿ HMEC ÿ MAD)
(HAF ÿ MAD ÿ HMEC)
(HAF ÿ MAD)
Experimental design including the operational conditions adapted.
Table 1
28
Energy Conversion and Management 180 (2019) 25–35
HRT (h)
Anaerobic
culture ÿ Exoelectrogens
chamber MEC
pH Temp. (°C)
48
Anaerobic
Heat treated anaerobic mixed
96 (48 + 48)
reactor ÿ Dual
37
Acidogenic Fermentation ÿ Electrohydrogenesis
Heat treated anaerobic mixed
MEC
6 ÿ 7 ÿ 6 37 ÿ 37 ÿ RT 144 (48 + 48 + 48)
reactor ÿ Dual
chamber MEC
mixed culture
M. Kaur et al.
7
Dual chamber
culture ÿ Untreated mixed
reactor
chamber MEC
96 (48 + 48)
culture ÿ Exoelectrogens
Acidogenic
48
37
Exoelectrogens
bottle ÿ Dual
6 ÿ 7
Anaerobic
6
Operational conditions
6 ÿ 6 ÿ 7 37 ÿ RT ÿ 37 144 (48 + 48 + 48)
Untreated mixed culture
Heat treated anaerobic mixed
Anaerobic
reactor
Catalyst
6 ÿ 6
Fermentation ÿ Electrohydrogenesis ÿ Methanogenesis
37
37 ÿ RT
Electrohydrogenesis
Anaerobic
culture ÿ Untreated mixed culture
37
Reactor design
culture ÿ Exoelectrogens ÿ Untreated
Experimental combinations
Heat treated anaerobic mixed culture
Acidogenic fermentation
reactor
Methanogenesis
6
Fermentation ÿ Methanogenesis ÿ Electrohydrogenesis
Heat treated anaerobic mixed
Anaerobic
48
Acidogenic
Acidogenic Fermentation ÿ Methanogenesis
Ultimate analysis includes the estimation of important chemicals
[30]. Briefly, lipid was extracted from biomass with a mixture of
Total sugar content after acid pretreatment was estimated by DNS
chloroform, methanol and water in the ratio of 2:2:1.8 which was later
and methane content using Gas Chromatograph (NAT GAS-B analyzer)
method [33], where 50g dried biomass was boiled with HCl at different
Biogas obtained during each experiment was analyzed for hydrogen
The total amount of biogas was recorded using water-displacement
quantities were calculated based on the percentage obtained in GC,
2.5.3. Electrochemical analysis
content of Lemna was determined using moisture analyzer (Hal.
(VS) of Lemna were estimated using method described in ASTM D3175
furnace at 550 °C for 6 h and TVS was measured by subtracting the
respectively. Helium was used as carrier gas at flow rate of 30 ml/min.
residual weight from the initial biomass weight. Ash content of biomass
National Renewable Energy Laboratory (NREL) [29]. The lipid content
evaporated out of the micelle with a vacuum evaporator (Rotavapor R-14, Buchi
AG, Flawil, Switzerland). The protein content of Lemna was
Moisture Analyzer HE53 (230 V), Mettlar Toledo). The volatile solids
mV, Mettlar Toledo).
boiling water bath with 4 ml anthrone reagent and taking the absorber-bance at
630 nm. The pectin content was determined using gravimetric
TOC (total organic carbon) was determined using TOC analyzer (Ana-lytik Jena
multi N/C 2100 S). The carrier gas (air) flow was set to
through a pre-weighed Whatman filter paper. The filter paper with the
method at room temperature and pressure. The hydrogen and methane
[28]. Briefly, 1 g of sample weighed in a crucible, was ignited in muffle
matter and fixed carbon contents of the raw biomass. Moisture
Briefly, 0.1 mg of biomass was homogenized with 80% hot ethanol to
remove sugars and the residue was added with 6.5 ml of 52% perchloric
was increased from 0 °C to 190 °C with a rate of 15 °C/min. The tem-peratures of
injection port and detector were 200 and 150 °C, respectively. Nitrogen was used
as the carrier gas at a flow rate of 10 ml/min.
considering the head-space available and displaced water.
having the protein extract was later analyzed by Lowry method. Similarly, the
starch content was analyzed by anthrone method [32].
(0.01, 0.05 and 0.3 N) in sequential steps with the retention concentration of
filtrate at each step. The filtrate was later pooled together,
calcium chloride. After boiling for 1 to 2 min, the mixture was filtered
acid to extract the starch by centrifuging at 0 °C for 20 min. starch
30m × 0.53mm × 1.0mm). The temperature program for the column
150 ml/min and the working temperature was 800 °C. The pH of the
The pectin content was calculated as % calcium pectate:
weighing dish, and weighed thereafter.
working electrode potential was maintained at ÿ1 V vs Ag/AgCl
(Hewlett Packard, HP 5890 series II) equipped with a flame ionization
elements that make up the biomass, namely carbon, hydrogen, ni-trogen and
sulfur. The ultimate analysis for elemental content of
this manuscript were against Ag/AgCl reference electrode (3 M KCl,
% calcium pectate = Wt. of calcium pectate × 500 × 100/ml of
(1)
Potentiostat-Galvanostat system (Ivium n-Stat, The Netherlands). The
Lemna biomass was done in a CHNS/O analyzer (Perkin-Elmer 2400
sample was determined using pH meter (S400 SevenExcellence™ pH/
(dinitrosalicylic acid) assay [34]. Volatile fatty acids (VFAs) in the
(ÿ795 mV Vs SHE). Unless stated otherwise, all potentials provided in
detector (FID) and HP FFAP column (dimensions
determined by Lowry's assay after extraction of protein using hot Tri-chloroacetic
acid (TCA) extraction method [31]. The protein was pre-cipitated from biomass
with 24% (w/v) TCA and the supernatant
content was determined by incubating the collected supernatant in a
were measured on cyclic voltammetry (CV) between ÿ0.8 and +0.4 V
precipitate (calcium pectate) was dried overnight at 100 °C in a
of the injector, detector and column were kept at 100, 110 and 60 °C,
Collected samples were analyzed using Gas Chromatograph (GC)
in Lemna was analyzed according to Bligh and Dyer (1959) method
was estimated based on procedure adopted from the National Renewable Energy
Laboratory (NREL) [29]. The fixed carbon content was
neutralized and was mixed with 5 ml of 1 N acetic acid and 25 ml of 1 N
computed using Eq. (1)
Series).
where PVM denotes percentage volatile matter and PAC denotes per-centage
ash content.
equipped with a thermal conductivity detector (TCD). The temperature
Current consumption during MEC experiments was measured on
chronoamperometry (CA) under designated applied potential using
+0.195 V vs SHE; ALS, Japan). Change in redox activities of the system
Composition of Lemna was estimated in terms of cellulose, hemi-celluloses
and lignin according to the procedures established by
2.5.4. Gas chromatography
filtered taken × Wt. of sample for estimation
of vertex potentials at a scan rate of 0.5 mV/s.
2.5.2. Biochemical analysis
Fixed Carbon 100% (PVM PAC) =ÿ ÿ
Machine Translated by Google

5. - 2I ESpecific Hydrogen Yield (moles H )/(TOC TOC )
Knock [(Knock Knock )/Knock ] 100
=ÿ ×
2 4I F)Total Biogas yield (moles H moles CH )/(TOC TOC =+ ÿ
=
4% (0.090 g/g) resulted in decreased reducing sugar release. This might
Initial studies pertaining to single stage approaches viz., acidogenic
The conversion efficiency of individual processes into an integrated
and standard deviations were calculated using descriptive statistics.
Specific H2 yield (mol H2/kg TOCR) and specific CH4 yield (mol
3.1. Characterization of Lemna as biofuel feedstock
acid from 0.5% (0.360 g/g) to 1% (0.426 g/g). However, further in-crement in
acid concentration to 2% (0.221 g/g), 3% (0.095 g/g) and
concentrations, thereby impacting the overall reducing sugar yield. The
concentration of Lemna biomass (mg/l) and TOCF represents the final
suitability for biogas production [35]. Similarly, the ultimate analysis
while lower C/N ratio suggests high nitrogen content resulting in high
variance (ANOVA) was used to establish differences among the means of
Hydrogen Yield (SHY) of 5.53 mol/kg TOCR followed by the production
ammonium content that is toxic to the microbes. The C/N ratio of
3. Results and discussion
of 0.47 mmol in next 24 h accounting for 4.09 mol/kg TOCR (Fig. 2).
The ability of pretreatment was comparatively determined at
be due to the degradation of reducing sugars into more simpler mole-cules
like furfural and 5-hydroxymethyl furfural, etc., under high acid
average results were presented and discussed. A one-way analysis of
which suggests the possibility of higher biogas conversion efficiency.
approach was assessed based on H2 and CH4 recovery from degradation
in Eq. (2)
1% acid concentration which is about 59% of the theoretical yield.
39.72% after 48 hours. Few other works have also reported lower TOC re-
moval efficiency (17%–32%) in single stage acidogenic fermentation
3.2. Pretreatment of Lemna biomass
represents the TOC concentration (mg/l) in the effluent.
different H2SO4 concentrations (0.5–4%) through quantifying the re-ducing
sugar content produced from 1 g Lemna biomass against theo-retical possible
reducing sugar content of 72.5% (w/w) [38].
VFA from the degradation of organics along with H2. With increase in
(4)
fermentation (HAF), electrohydrogenesis (HMEC) and methanogenesis
CH4/kg TOCR) depicts the H2 or CH4 generated in a given time with the
Maximum reducing sugar yield of 0.426 g/g Lemna was obtained at
2.6. Calculation of energy conversion efficiency
efficient, economical and alternate biofuel feedstock.
showed that Lemna has higher carbon content and low nitrogen content
Total biogas yield from Lemna (mol/kg TOCR) depicts the total
found to have a high VS content and low ash content representing its
(MAD) for H2/CH4 has resulted in significantly varied biogas production
VFA concentration, pH decreases from initial value of 6.0–5.18 in 48 h
biogas (H2 + CH4) generated with the function of total substrate re-moved in
all combinations, where, TOCI represents the initial TOC
Control pretreatment without acid addition yielded 0.064 g/g sugar
content, which increased with addition of increasing concentration of
to the other lignocellulosic biomass.
hoc analysis. Experimental data sets were analyzed with Graphpad
(2)
the reducing sugar degradation of about 29.53% was observed after
24 h and increased to 34.04% in 48 h (Fig. 2b). On the contrary, the VFA
biogas yield obtained in different experimental combinations, and significant
differences (p < 0.05) were identified using the Tukey post
Lemna showed lower lignin content along with higher cellulose, hemi-cellulose
and moderate starch content suggesting its utility as an energy
The total energy yield of the produced biogas was calculated by
as substrate degradation was recorded. In line with the TOC content,
Similar to H2 production, substrate degradation was also found to in-crease
with increase in time from 30.19% during first 24 h to only
determine its bioenergy conversion efficiency (Table 2). Lemna was
and further decreased there on. Acetate and butyrate were the main
(15:1), however, higher carbon content makes it competent for higher
Complete characterization of Lemna biomass was done in terms of
function of total substrate removed (TOC in kg) in that specific time in
Lemna was found to be comparatively lower than the ideal C/N ratio
Similar trend of secondary decomposition of carbohydrates into in-hibitors at
high acid concentrations was also reported in previous studies [39,40]. Lower
lignin content in Lemna biomass might have fa-voured the requirement of
milder pretreatment condition as compared
3.3. Single-stage approaches (HAF, HMEC, MAD) for H2/CH4 production
(5)
of Lemna biomass in each process. Substrate removal was evaluated by
2.7. Statistical analysis
The ideal C/N ratio is 20:1 to 30:1 for efficient biogas production
each process using Eq. (3) & (4).
All the experimental sets were performed in triplicates and the
proximate, ultimate and compositional analysis, which is essential to
and CH4 (55,530 kJ/kg of CH4).
biogas production. On the other hand, the compositional analysis of
estimating total organic carbon removal efficiency (TOCR) as described
along with degradation of organic fraction (Table 3, Fig. 2). During HAF,
about 0.48 mmol of H2 produced in first 24 h accounting for Specific
considering the theoretical energy values of H2 (141,790 kJ/kg of H2)
Prism (Graphpad Software, Inc., version 5, San Diego, USA). The means
concentration was found to increase with time of operation (from
652 mg/l at 24 h to 1456 mg/l at 48 h) due to the persistent acid-ogenesis
prevailing in the system which generates soluble metabolites/
TOC concentration (mg/l) as depicted in the following equation:
[41–43]. After 48 h, negligible increase in hydrogen production as well
[36,37]. Higher C/N ratio favors microbes for carbon utilization,
(3)
where TOCI represents the initial TOC concentration (mg/l) and TOCE
29
Energy Conversion and Management 180 (2019) 25–35
2.68
Cellulose
12.05
26.2
15.2
Pectin 1.9
Hemicellulose
Fixed Carbon
87.95
Extractives
Ash
Moisture
M. Kaur et al.
82.0
TS
Lignin
15
7.0
15.8
S
Protein
H
N
Content (%)
TVS
7.0
7.2
5.56
C/N ratio
15.6
Lipid
Starch
23.5
Proximate analysis
C
77
Compositional analysis
Ultimate analysis
5.53
3.5
I EIR
=
Specific Methane Yield (moles CH )/(TOC TOCÿ 4I E)
Table 2
Characterization of Lemna biomass.
Machine Translated by Google

6. HAF+MEC+MAD
MAD
HAF
HMEC
HAF+MAD+HMEC
40
2.5
3.0
100
120
20
0.5
24 168
80
480 72
1.5
2.0
1.0
0.0
0
96 192
60
120 144
Fig. 2. (a) Hydrogen and methane production rate (b) TOC and reducing sugar
Energy recovery and substrate removal efficiency of combined process integration studies.
Table 3
TOCR: TOC removal efficiency; RSR: Reducing sugar removal; SHY/SMY: Specific Hydrogen/Methane Yield (moles/kg TOCR).
removal efficiency against different experimental approaches.
30
Energy Conversion and Management 180 (2019) 25–35
–
M. Kaur et al.
23.69
3.96
62.3
7.62
Experimental combinations Biogas production (mmol)
0.96
–
–
4.48
70.0 20.54
3.52
2.21
7.0755.9
2.21
kg TOCR)
84.6
–
TOCR (%) RSR (%) SHY (moles/
0.36
0.96
– –
5.56
30.1665.2
33.31
–
(mmol)
62.5
5.48
92.0
–
–
5.45
Total biogas
–
0.36
68.9
1.6
kg TOCR)
0.89
95.8
–
–
9.62
–
77.9
9.62
34.0 9.62
0.89
3.52
7.07
SMY (moles/
–
0.96
3.0
9.62
37.78
0.96
kg TOCR)
9.62
3.0 20.54
Total biogas (H2 + CH4)
1.0
0.96
50.9
39.7
0.96
23.69
SHY (moles/
56.5
38.76
9.62
yield (moles/kg TOCR)
–
–
operation. The hydrogen yield in HMEC alone was 20.5 mol H2/kg TOCR
55.9% in 48 hours. However, negligible amount of VFAs content was observed
during the operation of HMEC. This may be due to the simulta-neous breakdown
of produced VFAs and soluble metabolites coupled to
reactor (5844 mg/l TOC) was fed into the cathode chamber of MEC
yield of 7.07 mol/kg TOCR in 48 h of operation. During MAD, in the first
168 h, methane production was not observed. After 192 hours, about
yield was noticed after 48 h. Overall in 48 h, 0.89 mmol H2 produced
was found to increase with time of operation (from 346 mg/l at 24 h to
energy. To increase the energy conversion efficiency, integration of
these bioprocesses in different sequential combinations were thus re-quired.
Acidogenic fermentation (HAF) was selected as the initial step in
with minor amounts of propionic and valeric acids. Previous reports
steps for harnessing additional energy [17,46].
substrate degradation efficiency (Table 3, Fig. 2). The effluent from HAF
CH4
TOCR followed by the production of 0.35 mmol in next 24 h accounting effluent [47]. Marone et al. [48] evaluated different industrial waste-waters and
by-products coming from fruit juice, paper, sugar and fruit
with higher H2 yield and these VFAs could be used in the further
To increase the sustainability and process economics of acidogenic
HAF ÿ MAD) to utilize the residual carbon source towards additional
which is equivalent to 0.46 dm3 /g TOCR, while the cumulative hydrogen yield
(HAF + HMEC) accounted for 30.16 mol H2/kg TOCR
suggested the similar VFA composition during acidogenic fermentation
also observed about 47.4% after 48 h and increased to 62.3% after
under constant voltage of ÿ1 V for 48 h. Integration of HMEC showed a
thereby stabilizing the pH to 7.0. However, the substrate degradation
192 h (Fig. 2a, b).
fermentation, two-stage integration was evaluated during the study. in
39.48% during first 24 h to 50.9% after 48 h. Similarly, the reducing
0.364 mmol methane was produced accounting for Specific Methane
subsequent studies because it produces maximum VFAs content along
H2 production at cathode. Neverthless, a minor increase in hydrogen
two-stage integration, the effluent/residual slurry of (HAF) were further
first 24 h accounting for Specific Hydrogen Yield (SHY) of 4.66 mol/kg
found to be insufficient in substrate degradation and its conversion to
VFAs observed accounting on average for over 85% of the total VFAs
system, after 144 h, due to the consumption of VFAs by methanogens
1286 mg/l at 96 h) due to the ongoing acidogenesis pathway in the
marked increase in H2 yield as compared to the single stage (HAF)
was only 36.70% in the first 48 h which increases to about 56.50% after
employed in methanogenesis or electrohydrogenesis (HAF ÿ HMEC or
from the degradation of about 4938 mg/l TOC which resulted in H2
In conclusion, individually all the three single stage processes were
sugar degradation was also observed to increase from 42% in 24 h to
H2/CH4 production. Both the integration strategies maximized the en-ergy
recovery in the form of H2 and CH4 production and also enhanced
processing for their potential to generate hydrogen by coupling dark
[44,45]. Overall, 0.96 mmol H2 produced from the degradation of
48 hours of operation. During HMEC, about 0.53 mmol of H2 produced in
Yield (SMY) of only 2.21 mol/kg TOCR. Initially, the VFA concentration
(0.68 dm3 /g TOCR). A similar study reported H2 yield of 0.33 dm3 /g
192 hrs. In line with the TOC content, the reducing sugar degradation was
for methane production, the VFA content decreased to 478 mg/l,
TOCR during MEC alone during treatment of acidogenic fermentation
about 3852 mg/l TOC and resulted in H2 yield of 9.624 mol/kg TOCR in
for 2.41 mol/kg TOCR. The substrate removal also increased from
to)
b)
HAF + HMEC
HAF
HMECHAF MAD HMEC MADHAF
MAD
HAF + MAD
HAF + HMEC + MAD
HAF + MAD + HMEC
HMEC
Biogas (H2/ CH4) production (mmol)
TOCR/ RSR (%)
Time (h)
Experimental combinations
HAF+HMEC
HAF+MAD
3.4. Two-stage integration (HAF ÿ HMEC, HAF ÿ MAD) for additional H2/
RSR
TOCR
HAF+MEC+MAD HAF+MAD+MEC
MECHAF
MAD
HAF+MECHAF+MAD
Machine Translated by Google

7. Lemna
slurry
Residual
HAF
HAF
HAF
HAF
MAD
HAF
MAD
M. Kaur et al.
31
Energy Conversion and Management 180 (2019) 25–35
Fig. 4. Organic flow, substrate degradation (TOCR) and biogas yield with respect to experimental combinations.
Fig. 3. (a) Current density (CD) profile with respect to time during enrichment of exoelectrogens on anode at polarized potential of ÿ0.1 V vs Ag/AgCl (3.5 M KCl).
(b) Variation in the cyclic voltammograms of the cell with respect to time of operation (anode and cathode as working and counter electrodes against Ag/AgCl (3.5
M KCl) reference electrode at scan rate of 0.5 mV/s) during growth of bioanode.
9.62
Organic carbon flux
MAD
MAD
43.5%
39.7%
60.3%
H2/CH4
yield (moles/kg TOCR)
22.1%
7.07
49.1%
2.21
38.76
31.1% 68.9%
30.1660.3%
62.5% 33.31
32.8%
TOC removal
efficiency
60.3%
60.3%
50.9%
43.5%
37.78
30.0%
70.0%
37.7%
56.5%
60.3%
77.9%
The combined hydrogen production process in the present study also facilitated
significant TOC removal, contributed to about 27.49% (2666 mg/l) alone in HMEC and
about 70.0% (6518 mg/l) cumulative of both the processes. Similar TOC removal efficiency
has been indicated by the literature studies employing acidogenic fermentation effluents
[49–52].
fermentation and microbial electrolysis in a two-step process. Total yield of 0.257 dm3 /g
TOCR and 0.396 dm3 /g TOCR in combined process (DF + MEC) was reported for paper
mill and sugar production waste-water, while a higher yield of 0.754 dm3 /g TOCR and
1.608 dm3 /g TOCR were observed for fruit processing wastewater and fruit juice production
wastewater respectively. This could be due to the composition of the effluent in terms of
easy degradable metabolites/sugars.
Similarly, the effluent generated during HAF (5844 mg COD/l) was also evaluated for
methanogenesis for 48 h. The integrated process (HAF ÿ MAD) showed additional
bioenergy generation with the me-thane production of 23.69 mol CH4/kg TOCR in 48 h
accounting for a total biogas yield of 33.3 mol/kg TOCR (Table 3). Enhanced biomethane
production in the combined system is possibly due to increased VFAs concentration in
acidogenic effluents that favored methane-producing species [46,53] The substrate
degradation was found to be low in MAD alone (1632 mg/l TOC; 16.83%) compared to
HMEC because MAD con-sumes the available VFAs and converts them to methane but
does not
involves additional VFAs production as in the case of HMEC. However, the total substrate
degradation (5484 mg/l TOC; 62.5%) was higher than the single stage operation (MAD)
(Fig. 2).
Machine Translated by Google

8. AF+MEC + AD
ADAF
MEC
AF + AD
AF + AD + MEC
AF + MEC
)H
25
80
30
(H
(
35
10
Total Biogas60
5
40
20
0
30
0
H
90
20
)
70
CH
15
10
50
Energy Conversion and Management 180 (2019) 25–35
32
M. Kaur et al.
TOCR kg/m3 reactor
Total Biogas/ H2/CH4 Yield (moles/ kg TOCR)
Experimental Combinations
4 (MAD )
2
2HAF
MEC
3.5. Three-stage integration for harnessing maximum possible H2/CH4
maximize the energy production. In first combination (HAF ÿ HMEC ÿ
MAD), the overall biogas yield was 37.78 mol biogas/kg TOCR with
substrate degradation of 7559 mg TOC/l (77.9%). In second combination
(HAF ÿ MAD ÿ HMEC), the overall biogas yield was 38.77 mol biogas/ kg
TOCR with substrate degradation 6682 mg TOC/l (68.9%)
However, the effluent of the integrated process (HAF ÿ HMEC) and (HAF
ÿ MAD) were still found to contain more organic content (3178 mg TOC/l
and 4212 mg TOC/l respectively) which could be further utilized for
harnessing energy.
Besides that, higher methane concentration (72%) was observed in
two-stage system as compared to MAD (65%) alone. The similar trend was
reported in previous studies [11,45,54]. The higher methane con-tent
obtained in coupled system would be profitable for biogas up-grading
processes as less CO2 would need to be separated. Therefore, employing
a two-stage system under suitable operating conditions could have an
added advantage of lower HRT with higher methane content in the biogas
(cheaper upgrading systems) when compared to single-stage systems.
In conclusion, the two-stage integration was found to be more ad-
vantageous than single-stage operation with respect to both substrate
removal and higher energy yield. Both two-stage combinations ex-hibited
a significant enhancement of biogas yield by 67.4% (HAF ÿ HMEC) and
70% (HAF ÿ MAD) compared to single stage HAF (p < 0.05).
(Table 3, Fig. 2). On the whole, third-stage integration was found to be
effective for both energy generation and substrate degradation where in
the first combination (HAF ÿ HMEC ÿ MAD) depicted to perform better in
increased substrate degradation while a slightly higher biogas yield was
observed in the second integration (HAF ÿ MAD ÿ HMEC). The maximal
experimental biogas yield was achieved for HAF ÿ MAD ÿ HMEC which
was 77% significantly higher (p < 0.05) compared to single stage HAF.
3.6. Electrochemical analysis
The performance of MEC reactor was determined by analyzing current
consumption on chronoamperometry and voltammogram during third-stage
integration approach. The current consumption on chronoamperometry
depicts the reduction reaction at working electrode. The redox equivalents
(H+ and eÿ) generated during substrate degradation at counter electrode
get transferred to the working electron via membrane (H+) and electric
circuit (eÿ).
The study was extended to evaluate the feasibility of reusing the
effluent of two-stage as substrates for third stage integration to
Fig. 5. A consolidated graph depicting the energy generation and TOC removal efficiency of each bioprocess during different process combinations.
Machine Translated by Google

9. Experimental combinations
Carbon footprint from biogas combustion
Total Energy Yield
M. Kaur et al. Energy Conversion and Management 180 (2019) 25–35
33
0
20000 400
600
500
30000
25000
300
10000 200
100
0
5000
15000
CV detects the redox signals and allows the elucidation of possible
oxidation and reduction reactions happening at the solution electrode
interface and the electrochemical reactions occurring at the electrode
surface [56]. Reducing equivalents [protons (H+) and electrons (eÿ)]
generated from the biocatalyst metabolic activities will move towards the
working electrode under applied potential generating a voltam-mogram.
The working electrode potential is ramped from ÿ0.8 V to +0.4 V at a scan
rate of 0.5 mV/s linearly and the current generated was recorded at each
potential. Voltammograms (vs. Ag/AgCl) visualized marked variation in
the electron discharge pattern and capacitance with the function of
substrate availability. Evaluation of electron discharge employing cyclic
voltammetry (CV) with respect to time of operation helps to understand the
growth of exoelectrogens on anode and electron carriers involved in their
transfer. Fig. 3b depicts the voltammetric response in the electron discharge
properties with respect to the time of operation. In both the integrations,
reduction current was more or less similar but the oxidation current varied.
In case of HMEC in third integration, the oxidation current was much higher
than reduction current, while in the other integration, where HMEC was
integrated immediately after HAF, the reduction current dominated. This
can be attributed to the higher oxidation rates during HMEC operation after
MAD, due to the breakdown of simpler organics obtained subsequent to
methanogenesis [50,55]. However in the other case, most of the or-ganics
were in the form of VFA and available for the controlled oxi-dation along
with H2 production. When HMEC was integrated at 3rd
During the integration of three different bioprocesses, carbon flux analysis
was done to gain an insight into the performance of the in-dividual process
in substrate degradation (Fig. 4). Flow of TOC was analyzed sequentially
from one process to next process to identify the best combination with
proficient substrate removal efficiency. When individual processes were
compared, maximum flow of TOC to the subsequent process was observed
with HAF (60.3%) which resulted in only 39.7% of TOC removal. However,
individual operation of HMEC and MAD exhibited the TOC removal
efficiency of 50.9% and 56.5% respectively.
Substrate conversion efficiency, organic flux analysis, energy efficiency
analysis were employed for every approach to understand the process in
this direction. In anaerobic fermentation, the production of biogas (H2/CH4)
is directly proportional to the substrate consumption.
position after MAD, more number of redox peaks was identified, while in
the other case single redox peak was visible on the voltammetric sig-
nature. However, in both the cases, one redox peak was commonly
visualized which is relevant to NAD+/NADH indicating H2 evolution.
3.7. Analysis of organic flow
Between two-stage integration, maximum substrate removal (70.0%)
and less flux (30.0%) to the subsequent process was observed in HAF ÿ
HMEC whereas HAF ÿ MAD resulted in organic flow of 37.7%.
Subsequent three stage integration (HAF ÿ HMEC ÿ MAD) and (HAF ÿ
MAD ÿ HMEC) resulted in maximal substrate degradation. In HAF ÿ HMEC
ÿ MAD only 22.1% of organic matter was found in the residual slurry with
maximum TOC removal of 77.9%. On the other hand in HAF ÿ MAD ÿ
HMEC, the final slurry has higher organic matter (31.1%) with TOC removal
efficiency of 68.9%. Electrohydrogenesis at second position in integrated
processes showed comparatively lower organic matter in their effluent than
methanogenesis.
The redox equivalents get reduced at working electrode under ap-plied
potential to form H2. Hence, the current consumption at working electrode
is proportional to H2 production. In first integration, HAF ÿ HMEC ÿ MAD,
the current density (CD) followed an increasing trend with time and reached
maximum peak value of ÿ5.48 I/mA at 18th h, after which, a sharp drop
and subsequent decrease in CD was observed until the end of the
operation (48 h) (Fig. 3a). The remarkable increase of the CD with
increased substrate degradation rate (2666 mg/l) indicates high enrichment
of exoelectrogens resulting in the reproducible per-formance of the
bioanode [55]. However, an increased hydrogen yield of 2.09 mmol clearly
demonstrates that the higher VFA content of ef-fluent from acidogenic
fermentation might have contributed for H2 production at working electrode.
On the contrary, in the second three stage integration, HAF ÿ MAD ÿ
HMEC, only a minor increase in the CD was observed at the start of the
operation (0.33 h) after which the CD re-maintained constant at ÿ0.1 I/mA.
Similarly to CD, lower hydrogen yield (0.66 mol) and substrate removal
rate (591 mg/l) was recorded in this integration which can be correlated
with the absence of VFAs and breakdown of residual complex organics.
3.8. Comprehensive appreciation of biogas generation and substrate
utilization efficiency
Considering this, the specific hydrogen yield (SHY) and specific me-thane
yield (SMY) in single-phase and integrated processes were calculated.
On account of very low energy and substrate conversion efficiency, the
single stage processes were found incompetent to be employed at large
scale for gaseous biofuels production. However, significant improvement
in energy yield was recorded in different two-stage integrations wherein
the maximal biogas yield (33.31 mol/kg TOCR) was recorded in HAF ÿ
MAD followed by HAF ÿ HMEC (30.16 mol/kg TOCR). On the contrary,
higher TOC removal was observed in HAF ÿ HMEC (10.37 TOCR kg/m3
reactor) as compared to HAF ÿ MAD (9.33 TOCR kg/m3 reactor) (Table 3,
Fig. 5). This may probably because of simultaneous conversion of the
VFAs generated in MEC (via oxidation of residual organics) along with
VFAs already present in HAF ef-fluent to hydrogen. This also resulted in
carbon loss as CO2 along with H2 production in MEC. While in HAF ÿ
MAD, the VFAs from HAF effluent were rapidly utilized by microbes to
produce methane but no significant additional substrate oxidation occurred
accounting for lower substrate degradation efficiency. Further, HMEC also
resulted in faster utilization of VFAs as well as other simple organics
leaving no scope for the next integration. On the other hand, MAD after
HAF utilizes the VFAs towards methane generation through reductive
pathways resulting in gradual substrate degradation leaving the residual
simple organics for next stage integration. Therefore, placement of MEC
reactor in third stage yields more biogas coupled to effective substrate
degradation rather than placing it at second stage. Both the three-stage
integrations were found efficacious in exhibiting highest cumulative biogas
yield combined with the two-stage integrations. However, lower SMY (7.62
mol
Comparative evaluation of single and integrated bioprocesses for
gaseous biofuel production is an important criterion to validate the
importance of integration studies in maximizing the energy yield.
Energy (KJ/KgTOCR)
CO2 (l/biogas generated from kg TOCR)
HAF + MAD
HAF + HMEC
HAF+HMEC + MAD
HAF + MAD + HMEC
HAF MEC MAD
Fig. 6. Illustration of total energy efficiency and carbon footprint based on CO2
emission on biogas combustion.
Machine Translated by Google

10. Carbon footprint evaluation was done by calculating the carbon emission during
the biogas combustion produced in each process for energy applications. As H2 is
clean burning fuel, carbon emissions is considered zero in HAF, HMEC and HAF ÿ
HMEC experiments. Contrary to that, one mole of methane on combustion generates
one mole of CO2.
20911/IJBBT-101.
M. Kaur et al.
[2] Cherubini F. The biorefinery concept: Using biomass instead of oil for producing
[5] Billig E, Thraen D. Renewable methane—a technology evaluation by multi-criteria decision
making from a European perspective. Energy 2017;139:468–84.
[7] Chandrasekhar K, Lee YJ, Lee DW. Biohydrogen production: Strategies to improve process
efficiency through microbial routes. Int J Mol Sci 2015;16:8266–93. https://doi.org/
10.3390/ijms16048266.
[9] Srikanth S, Venkata Mohan S, Devi MP, Babu ML, Sarma PN. Effluents with soluble metabolites
generated from acidogenic and methanogenic processes as substrate for additional hydrogen
production through photo-biological process. Int J Hydrogen Energy 2009;34:1771–9.
[12] Khan MZ, Nizami AS, Rehan M, Ouda OKM, Sultana S, Ismail IM, et al. “Microbial electrolysis
cells for hydrogen production and urban wastewater treatment: a case study of Saudi Arabia.”
Appl Energy 2017;185:410–20.
[13] Dhar BR, Elbeshbishy E, Hafez H, Lee HS. Hydrogen production from sugar beet juice using
an integrated biohydrogen process of dark fermentation and microbial electrolysis cell.
Bioresour Technol 2015;198:223–30.
[8] Srikanth S, Mohan SV. Regulating feedback inhibition caused by the accumulated acid
intermediates during acidogenic hydrogen production through feed replacement . Int J
Hydrogen Energy 2014;39(19):10028–40.
[3] Kÿrtay E. Recent advances in production of hydrogen from biomass. Energy Convers Manage
2011;52:1778–89. https://doi.org/10.1016/j.enconman.2010.11.01.
Manage 2009;50:2239–49.
[4] Kim SH, Bhatia SK, Yoon JJ, Yang YH. Current status and strategies for second generation
biofuel production using microbial systems. Energy Convers Manage 2017;148:1142–56.
https://doi.org/10.1016/j.enconman.2017.06.073.
energy and chemicals. Energy Convers Manage 2010;51:1412–21. https://doi.org/ 10.1016/
j.enconman.2010.01.015.
[14] Patterson T, Esteves SRR, Dinsdale RM, Guwy AJ. An evaluation of the policy and techno-
economic factors affecting the potential for biogas upgrading for transport
[10] Srikanth S, Venkata Mohan S, Devi MP, Dinikar P, Sarma PN. Acetate and butyrate as
substrates for hydrogen production through photo-fermentation using mixed culture:
Optimization of process parameters and combined process evaluation. Int J Hydrogen Energy
2009;34:7513–22.
[11] Guneratnam AJ, Xia A, Murphy JD. Comparative study of single- and two-stage fermentation
of the brown seaweed Laminaria digitata. Energy Convers Manage 2017;148:405–12.
[6] Soÿowski G. Bioprocessing and biotechniques biohydrogen production - sources and methods:
a review. Int J Bioprocess Biotech 2018;2018:1–22https://doi.org/10.
[1] Demirbas A. Biofuels securing the planet's future energy needs. Energy Converts
Energy Conversion and Management 180 (2019) 25–35
34
On the whole, the observed higher biogas yields in integrated bio-
processes suggests that Lemna has reasonably good amount of biode-
gradable organic content that can be utilized effectively in integrated
biorefinery approach for additional energy generation.
into energy cycle and its further combustion therefore results in delayed
CO2 release into the atmosphere. Utilization of CO2 generated from
biomethane combustion to enhance the buffering capacity of the acidogenic
fermentation [57] and sequestrating in the photosynthetic pathways [58,59]
as well as into microbial electrohydrogenesis [60] for biofuels generation,
are also being explored as coupled biological processes with a bio-refinery
perspective.
3.9. Assessment of energy efficiency and carbon footprint
This also implies that the placement of methanogenesis process in
biorefinery approach is predominant factor in enhancing the energy
recovery.
Remarkable increase in energy yield was observed in two-stage as well as
three-stage integration approaches. Among the two-stage process
integrations, comparatively higher energy efficiency was docu-mented in
HAF ÿ MAD (23856 KJ/kg TOCR) as compared to HAF ÿ HMEC (8621 KJ/
kg TOCR). Among three-stage integration approaches, in-tegration of
electrohydrogenesis as last process (HAF ÿ MAD ÿ HMEC) showed higher
energy recovery (25415 KJ/kg TOCR) compared to methanogenesis as
last process ( HAF ÿ HMEC ÿ MAD) (15416 KJ/kg TOCR). The significant
difference in energy yield apart from generating highest biogas yield (38.76
mol biogas/kg TOCR), the third-stage in-tegration where electrohydrogenesis
kept at last (HAF ÿ MAD ÿ HMEC) can be a better choice among different
integration strategies studied.
However, unlike fossil fuels use that increases the total amount of carbon
in the biosphere-atmosphere system, the CO2 emitted during
methanogenesis of Lemna would eventually become the part of the biogenic
carbon cycle that recycles renewable plant growth in a sustainable carbon
equilibrium thereby producing carbon neutral energy.
Additionally, as the whole process is less energy intensive which
consequently makes the approach environmentally benign. Further, in
comparison to the aerobic decomposition of Lemna in aquatic en-vironment
that causes instant CO2 emissions directly into the atmosphere, it is
proposed that the adapted biorefinery approach used in this study for the
conversion of Lemna biomass would bring the carbon first
When individual experiments (HAF, MAD and HMEC) were compared,
HAF depicted higher energy yield (2751 KJ/kg TOCR) followed by HMEC
operation (2020 KJ/kg TOCR) and MAD (1974 KJ/kg TOCR) (Fig. 6).
References
4. Conclusions
CH4/kg TOCR) and SHY (5.45 mol H2/kg TOCR) was registered in third-
stage integration of methanogenesis (HAF ÿ HMEC ÿ MAD) and electro-
hydrogenesis (HAF ÿ MAD ÿ HMEC) processes respectively, compared to
their integration in two-stage approach. Contrary to the biogas yield, HAF ÿ
HMEC ÿ MAD exhibited higher TOC removal efficiency (17.92 TOCR kg/
m3 reactor ) compared to HAF ÿ MAD ÿ HMEC (16.01 TOCR kg/m3 reactor)
(Fig. 5).
The study demonstrated the feasibility of coupling acidogenic fer-
mentation, methanogenesis and electrohydrogenesis for biohythane (H2 +
CH4) production from Lemna minor. This system provided an optimal
specific yield of hydrogen and methane corresponding to an overall energy
yield of (38.77 mol biogas/kg TOCR), which is sig-nificantly higher than the
values obtained in each single-stage process (2.21–9.62 mol biogas/ kg
TOCR) evaluated in the study. Furthermore, im-provement in the energy
conversion, an efficient reduction of the overall HRT (7 days) and organic
load (72.5%) was also evidenced via a three-stage system. The employment
of hydrogen rich fuel would pro-mote hydrogen fueling infrastructure and
also facilitates transition of the biofuel market towards a hydrogen economy.
The results obtained in this study clearly indicates that employing waste
aquatic weed Lemna as an alternative bioenergy feedstock would help in
overcoming major economic constraints associated with cellulosic biofuel
production viz., high-substrate cost and expensive biomass pretreatment
thereby making the overall process more economical. Further, this would
help developing a profitable system of combined Lemna cultivation, waste-
water treatment and biofuel production. Neverthless, considering the
novelty of the process, a complete techno-economic study with detailed
modeling and simulation is necessary for a precise determination of overall
economics of the proposed process to envisage its scale up.
Energy security and environmental benefits attributed by biofuels are
key criteria that need to be evaluated to determine their long term
sustainability for energy applications. In order to address the stated
measures, total energy efficiency (KJ/biogas produced from kg TOCR) of
each bioprocess was calculated. In similar lines, the carbon footprint in
terms of atmospheric carbon dioxide (dm3 /biogas gener-ated from kg
TOCR) was also computed. Both the energy conversion efficiency and the
carbon foot print analysis helps in assessing the economic and environmental
viability of all approaches adopted in this study.
Based on this, the maximum CO2 production (530.7 dm3 /biogas from kg
TOCR) was observed in HAF ÿ MAD/HAF ÿ MAD ÿ HMEC followed by HAF
ÿ HMEC ÿ MAD (171 dm3 /biogas from kg TOCR) (Fig. 6). Single stage
methanogenesis resulted in minimal CO2 emissions (49.61 dm3 / biogas
from kg TOCR). It means that the experimental combinations exhibiting
notable carbon footprint are also linked with higher energy production.
Considering the climate policy constraints, employing the processes with
smaller carbon footprint may be a more feasible option.
Machine Translated by Google

11. 35
Energy Conversion and Management 180 (2019) 25–35
fuel use in the UK. Energy Policy 2011;39:1806–16.
[52] Nam JY, Yates MD, Zaybak Z, Logan BE. Examination of protein degradation in continuous flow,
microbial electrolysis cells treating fermentation wastewater.
[23] Cheng JJ, Stomp AM. Growing duckweed to recover nutrients from wastewaters and for production of
fuel ethanol and animal feed. CLEAN-Soil Air Water 2009;37:17–26.
[32] Hodge JE, Hofreiter BT. Methods in carbohydrate chemistry. New York: Academic
[15] Xie B, Cheng J, Zhou J, Song W, Liu J, Cen K. Production of hydrogen and methane from potatoes by
two-phase anaerobic fermentation. Bioresour Technol 2008;99(13):5942–6.
chemical wastewater treatment by selectively enriched anaerobic mixed consortia in biofilm configured
reactor operated in periodic discontinuous batch mode. Wat Res 2007;41:2652–64.
mentation of wheat powder solution: effects of C/N and C/P ratio on hydrogen yield and formation rate.
Int J Hydrogen Energy 2008;33:1813–9.
[39] Hu R, Lin L, Liu T, Liu S. Dilute sulfuric acid hydrolysis of sugar maple wood extraction at atmospheric
pressure. Bioresour Technol 2010;101:3586–94. https://doi.org/10. 1016/j.biortech.2010.01.005.
[40] Jiang L, Wu N, Zheng A, Zhao Z, He F, Li H. The integration of dilute acid hydrolysis
[19] Cheng CL, Lo YC, Lee KS, Lee DJ, Lin CY, Chang JS. Biohydrogen production from lignocellulosic
feedstock. Bioresour Technol 2011;102:8514–23.
[37] Argun H, Kargi F, Kapdan IK, Oztekin R. Biohydrogen production by dark fer-
[41] Venkata Mohan S, Vijaya Bhaskar Y, Sarma PN. Biohydrogen production from
[16] Cheng S, Logan BE. Sustainable and efficient biohydrogen production via electro-
[26] Fu SF, Chen KQ, Zhu R, Sun WX, Zou H, Guo RB. Improved anaerobic digestion performance of
Miscanthus floridulus by different pretreatment methods and pre-liminary economic analysis. Energy
Convers Manage 2018;159:121–8. https://doi. org/10.1016/j.enconman.2018.01.014.
[20] Zhao X, Moates GK, Wellner N, Collins SRA, Coleman MJ, Waldron KW. Chemical characterization and
analysis of the cell wall polysaccharides of duckweed (Lemna minor). Carhohyd Polym 2014;111:410–
8.
[28] ASTM D3175. Standard test method for Volatile matter in the analysis sample of coal and coak. Annual
book of standards, Volume 05.06. West Conshohocken, Pennsylvania: ASTM International; 2013.
Bioresour Technol 2014;171:182–6.
hydrogenesis. PNAS 2007;104:18871–3.
potential of waste materials. BAE-1762. Oklahoma: Oklahoma Cooperative Extension Service; 2012.
[49] Escapa A, Lobato A, García DM, Morán A. Hydrogen production and COD elim-
[25] Fulton J, Marmaro R, Egan G. System for producing a hydrogen enriched fuel. US patent 7721682; 2010.
waste to biofuel by simultaneous dilute-acid hydrolysis of starch and pretreatment of lignocelluloses.
Energy Convers Manage 2018;166:569–78. https://doi.org/10. 1016/j.enconman.2018.04.067.
McGraw-Hill Publ. Co., Ltd.; 1979. p. 634.
[44] Akobi C, Yeo H, Hafez H, Nakhla G. Single-stage and two-stage anaerobic digestion of extruded
lignocellulosic biomass. Appl Energy 2016;184:548–59. https://doi. org/10.1016/
j.apenergy.2016.10.039.
[45] Wang Q, Liang Y, Zhao P, Li QX, Guo S, Chen C. Potential and optimization of two-phase anaerobic
digestion of oil refinery waste activated sludge and microbial community study. Sci Rep 2016;6:1–
10. https://doi.org/10.1038/srep38245.
[55] Chandra R, Venkata Subhash G, Venkata Mohan S. Mixotrophic operation of photo-bioelectrocatalytic
fuel cell under anoxygenic microenvironment enhances the light dependent bioelectrogenic activity.
Bioresour Technol 2012;109:46–56. https:// doi.org/10.1016/j.biortech.2011.12.135.
[35] Hamilton DW. Anaerobic digestion of animal manures: methane production po-
[48] Marone A, Ayala-Campos OR, Trably E, Carmona-Martínez AA, Moscoviz R, Latrille E, et al. Coupling
dark fermentation and microbial electrolysis to enhance bio-hy-drogen production from agro-industrial
wastewaters and by-products in a bio-re-finery framework. Int J Hydrogen Energy 2017;42:1609–
21. https://doi.org/10. 1016/j.ijhydene.2016.09.166.
[31] Slocombe SP, Ross M, Thomas N, McNeill S, Stanley MS. A rapid and general method for
measurement of protein in micro-algal biomass. Bioresour Technol 2013;129:51–7.
ination rate in a continuous microbial electrolysis cell: the influence of hydraulic retention time and
applied voltage. Environ Prog Sustain Energy 2013;32:263–8.
[34] Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar.
[50] Lenin Babu M, Venkata Subhash G, Sarma PN, Venkata Mohan S. Bio-electrolytic conversion of
acidogenic effluents to biohydrogen: an integration strategy for higher substrate conversion and
product recovery. Bioresour Technol 2013;133:322–31.
[24] Bauer CG, Forest TW. Effect of hydrogen addition on the performance of methane- fueled vehicles. Part
I: effect on SI engine performance. Int J Hydrogen Energy 2001;26:55–70.
[57] Devi MP, Venkata Mohan S, Mohanakrishna G, Sarma PN. Regulatory influence of CO2 supplementation
on fermentative hydrogen production process. Int J Hydrogen Energy 2010;35:10701–9.
of xylan and fast pyrolysis of glucan to obtain fermentable sugars. Biotechnol Biofuels 2016;9:1–
10. https://doi.org/10.1186/s13068-016-0612-0.
[53] Cheng J, Lin R, Ding L, Song W, Li Y, Zhou J, et al. Fermentative hydrogen and methane cogeneration
from cassava residues: effect of pretreatment on structural characterization and fermentation
performance. Bioresour Technol 2015;179:407–13.
[17] Bakonyi P, Kumar G, Koók L, Tóth G, Rózsenberszki T, Bélafi-Bakó K, et al.
[38] Cheng J, Xie B, Zhou J, Song W, Cen K. Cogeneration of H2 and CH4 from water hyacinth by two-step
anaerobic fermentation. Int J Hydrogen Energy 2010;35:3029–35.
[43] Paudel S, Kang Y, Yoo YS, Seo GT. Hydrogen production in the anaerobic treatment of domestic-grade
synthetic wastewater. Sustain 2015;7:16260–72. https://doi.org/ 10.3390/su71215814.
[22] Ozengin N, Elmaci A. Performance of duckweed (Lemna minor L.) on different types
[56] Patil SA, Hagerhall C, Gorton L. Electron transfer mechanisms between micro- organisms and
electrodes in bioelectrochemical systems. Bioanal Rev 2012;4:159–92.
[27] Mahmoodi P, Karimi K, Taherzadeh MJ. Efficient conversion of municipal solid
Methanogenic processes for simultaneous production of biohydrogen and methane from wastewater
treatment. Int J Hydrogen Energy 2008;33:2156–66.
electrolysis cells treating fermentation effluent. Int J Hydrogen Energy 2015;40:6782–
91. https://doi.org/10.1016/j.ijhydene.2015.03.101.
[58] Devi MP, Venkata Mohan S. CO2 supplementation to domestic wastewater enhances microalgae lipid
accumulation under mixotrophic microenvironment: effect of sparging period and interval.
Bioresour Technol 2012;112:116–23.
[29] Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D, et al. Determination of Structural
carbohydrates and lignin in biomass. Laboratory analytical procedure (LAP). NREL/TP-. Golden,
Colorado: National Renewable Energy Laboratory; 2008. p. 510–42618.
Anal Chem 1959;31:426–8.
[54] Schievano A, Pepé Sciarria T, Gao YC, Scaglia B, Salati S, Zanardo M, et al. Dark fermentation,
anaerobic digestion and microbial fuel cells: An integrated system to valorize swine manure and rice
bran. Waste Manage 2016;56:519–29. https://doi. org/10.1016/j.wasman.2016.07.001.
[18] Venkata Mohan S, Mohanakrishna G, Sarma PN. Integration of acidogenic and
[47] Ullery ML, Logan BE. Anode acclimation methods and their impact on microbial
Press; 1962.
[59] Mohedano RA, Costa RH, Belli Filho P. Effects of CO2 concentration on nutrient uptake and starch
accumulation by duckweed used for wastewater treatment and bioethanol production. Am J Environ
Biotech Algal 2016;7:30–41.
[42] Vijaya Bhaskar Y, Venkata Mohan S, Sarma PN. Effect of substrate loading rate of chemical wastewater
on fermentative biohydrogen production in biofilm configured sequencing batch reactor. Biores
Technol 2008;99:6941–8.
[46] Algapani DE, Qiao W, di Pumpo F, Bianchi D, Wandera SM, Adani F, et al. Long- term bio-H2 and bio-
CH4 production from food waste in a continuous two-stage system: energy efficiency and conversion
pathways. Bioresour Technol 2018;248:204–13.
Annu Rev. 2005;11:69–99.
[51] Modestra JA, Lenin Babu M, Venkata Mohan S. Electro-fermentation of realfield acidogenic spent
wash effluents for additional biohydrogen production with si- multaneous treatment in a microbial
electrolysis cell. Sep Purif Technol 2015;150:308–15.
[21] Stomp AM. The duckweeds: a valuable plant for biomanufacturing. Biotechnol
[36] Lin CY, Lay CH. Carbon/nitrogen-ratio effect on fermentative hydrogen production by mixed microflora.
Int J Hydrogen Energy 2004;29:41–5.
M. Kaur et al.
[60] Villano M, Aulenta F, Ciucci C, Ferri T, Giuliano A, Majone M. Bioelectrochemical reduction of CO2 to
CH4 via direct and indirect extracellular electron transfer by a hydrogenophilic methanogenic culture.
Bioresour Technol 2010;101:3085–90.
[30] Bligh EG, Dyer WJ. A lipid method of total lipid extraction and purification. Can J
[33] Ranganna S. Manual of analysis of fruit and vegetable products. New Delhi: Tata
Biochem 1959;37:911–7.
of wastewater treatment. J Environ Biol 2007;2:307–14.
Microbial electrohydrogenesis linked to dark fermentation as integrated application for enhanced
biohydrogen production: a review on process characteristics, experiences and lessons. Bioresour
Technol 2018;251:381–9.
Machine Translated by Google