Green Chemistry

PAPER

Low carbon hydrogen production from

Cite this: Green Chem., 2021, 23, 561
a waste-based biorefinery system and
environmental sustainability assessment†
Omprakash Sarkar,a,b Ranaprathap Katakojwalaa,b and S. Venkata Mohan *a,b

Received 9th September 2020,
Accepted 17th November 2020
DOI: 10.1039/d0gc03063e
rsc.li/greenchem
Production of low carbon biofuels and biochemicals from renewable feedstocks using a biological
process is considered a sustainable alternative to the fossil-fuel-based linear economy. This study
demonstrated the pilot-scale (10 m3) production of biohydrogen (bio-H2) and volatile fatty acids/car-
boxylic acids (VFA) through acidogenic fermentation (AF) using food waste (FW) as renewable feedstock.
Bio-H2 production of 54 288 L (155.10 LH2 per kg COD) along with 25.77 g L−1 of VFA composed of acetic
(HAc: 15.2 ± 1.98 g L−1), propionic (HPr: 4.89 ± 1.26 g L−1) and butyric (HBu 5.67 ± 0.96 g L−1) acids was
achieved within 48 h of the fermentation period. The further acidogenic process was integrated with a
biorefinery platform (methanogenesis + photosynthesis) in a circular loop strategy which helped derive
multiple biobased products (CH4, algal biomass, O2 and treated water for reuse) from fatty acid-rich
acidogenic effluent and untreated COD of AF. The whole bio-manufacturing unit (acidogenesis + metha-
nogenesis + photosynthesis) converted the renewable feedstock (waste/wastewater) into fuels and plat-
form chemicals, in analogy to a conventional oil refinery with a maximized resource recovery. The life
cycle assessment (LCA) tool was employed to study the environmental impact of both the bio-H2 (stan-
dalone, ST) and waste biorefinery (WB) processes, and the results depicted that the WB approach offered
a relatively low impact (approx. 3.5 fold less than ST). The approach helped determine the flow of carbon
and its conversion to products; this aided the reduction of carbon emissions as well as minimized the
burden on natural resources with the biosynthesis of green H2 and other value-added products, addres-
sing carbon neutrality with bioeconomy.

1. Introduction
Greater demand for energy/materials due to the finite avail-
ability of the fossil reserves associated with environmental and
societal issues creates the need for the development of new
and sustainable strategies that can result in renewable and
carbon-neutral energy/materials.1 A shift from conventional
fossil-based pathways to ecologically sustainable energy
systems and production methods is an essential prerequisite
for the anticipated global mission of climate neutrality.2,3
Hydrogen (H2) is the most promising clean energy carrier with
a
Bioengineering and Environmental Sciences Lab, Department of Energy and
Environmental Engineering (DEEE), CSIR-Indian Institute of Chemical Technology
(CSIR-IICT), Hyderabad-500007, India. E-mail: svmohan@iict.res.in,
vmohan_s@yahoo.com; Tel: +0091-40-27191765
b
Academy of Scientific and Innovative Research (AcSIR), Ghaziabad-201002, India
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0gc03063e
an energy density (LHV, 120 MJ kg−1) higher than the liquefied
natural gas (54.4 MJ kg−1), ethanol (29.6 MJ kg−1) and
methane (50 MJ kg−1).4–6 Hydrogen is being considered as a
future energy carrier and transition to the ‘Hydrogen
Economy’ is marching rapidly around the global energy portfo-
lio. At present, H2 is mostly produced from a wide source of
feedstock, such as natural gas, coal, biomass, and solar energy
(thermal and photovoltaic).7–11 Currently, fossil-based H2 pro-
duction is achieved through various physicochemical pro-
cesses, such as steam reforming of natural gas followed by
cracking oil products, coal gasification, and water
electrolysis.12,13 Requirement for non-renewable fossil
resources, excessive energy inputs for production, and signifi-
cant emissions of greenhouse gases (GHGs) during the pro-
duction life cycle are some of the inherent limitations of tra-
ditional H2 production, irrespective of the conversion techno-
logy used and the energy source employed.
In the sustainability context, H2 production should be
renewable in nature with low embodied carbon by adopting a
biological process for its synthesis. Biohydrogen (bio-H2) pro-

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Paper
duction through a dark-fermentation route (acidogenic fer-
mentation) using biogenic waste/wastewater as a feedstock is a
promising pathway.14,15 Along with bio-H2, the acidogenic
process concomitantly produces short-chain fatty acids/volatile
fatty acids (VFA) as main by-products.15,16 One mole of glucose
can produce 4 moles of bio-H2 along with 2 moles of acetic
acid through dark-fermentation (eqn (3)). When the process
proceeds by the butyric acid pathway, two moles of bio-H2 are
produced (eqn (4)). In contrast, the propionic acid (eqn 5) and
lactic acid pathways consume two and one moles of bio-H2,
respectively.
VFAs are platform chemicals that are normally used as
building blocks for the production of polymers, acidulants,
preservatives and flavouring agents or as precursors for the
synthesis of chemicals.16–18 Waste is a global issue with signifi-
cant availability; therefore, bio-H2 production from renewable
waste as feedstock reduces the burden on the environment
through remediation and avoids emissions from direct and
indirect use of fossil-based feedstock.3,19 In recent years, the
biorefinery concept has emerged as an alternative to address
the fossil-based resource depletion and associated environ-
mental impacts in addition to valorising a multitude of bio-
based products.14,20–22 A biorefinery, when designed systemati-
cally, may address the great prevailing challenges to environ-
mental sustainability through efficient utilization of resources
with lower emissions and switching to a circular economy
model from linear economy models. These biobased products
(low carbon) positively minimize the dependency on fossil-
derived resources and therefore reduce the respective environ-
mental issues to establish low carbon economy and systems.23
Due to its renewable and carbon neutral nature, bio-H2 is
gaining attention in the framework of sustainability.24 Over
the last two decades, researchers have performed numerous
studies in order to improve bio-H2 productivity from different
feedstocks on lab and semi-pilot scales.25–30 This communi-
cation presents and discusses bio-H2 production at the pilot
scale (10 m3) integrated with a waste biorefinery facility to
maximize resource recovery in a circular loop. The impact of
waste-derived bio-H2 on the environment was studied using
life cycle analysis (LCA; ISO 14040:2006) tools considering all
the inputs and outputs in the form of materials and energy to
and from the system to assess its relative environmental
sustainability.31
2. Materials and methods
2.1. Food waste
Composite food waste (FW) was collected from the institute
canteen. The collected FW was composed of cooked rice (47 ±
4%), boiled vegetables (16 ± 2%), vegetable peels (18 ± 3%),
spoiled fruits and vegetables (6 ± 2%), and egg/meat waste (13 ±
1%). The FW was masticated using a blender and then filtered
through a stainless-steel mesh (1.1 mm) to remove the coarse
materials. Oil was separated using a gravity separating system.
The homogenized FW was characterized and found to have
562 | Green Chem., 2021, 23, 561–574
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230 ± 4 g L−1 COD, 0.7 ± 0.2 g L−1 volatile fatty acids (VFA),
37 ± 1.2 mg L−1 nitrates, 13 ± 1.6 mg L−1 sulphates and
19 ± 2.4 mg L−1 phosphates. The oil-free filtrate was then used as a
substrate by adjusting the organic load of feed to 50 g COD per L.
2.2. Scaling up of the hydrogen production process – pilot
plant (10 m3)
2.2.1. Standalone acidogenic process. The acidogenic bio-
reactor (total/working volume 10/8 m3; L/D ratio 2) was fabri-
cated with SS316L to operate in up-flow mode. The major unit
operations of the pilot plant system consisted of a waste shred-
der, waste/wastewater feeder, multi-utility buffer tank/pre-fer-
mentation tank (2 m3), inoculum preparation tank (0.2 m3),
acid–base holding tank, H2 gas collector (0.2 m3), automated H2
flare and water storage tank. All individual unit operations of
the pilot plant are shown in Fig. 1. Accessories, namely an air
compressor, online gas flow meter, moisture trap and online
sensors (pH and temperature), were also integrated into the pro-
cesses at appropriate places. The reactor liquid volume was
filled with moving bed media (void fraction ∼0.18) to support
the growth of acidogenic consortia. The pilot plant was
designed to have control and process flexibility. Every unit in
the plant was systematically placed to minimize energy input.
2.2.2. Waste biorefinery-integrated process. To make the
process sustainable, acidogenic bio-H2 (AB) production was
integrated with a waste biorefinery facility to harness multiple
biobased products from fatty acid-rich acidogenic outlet (AO).
In addition to converting AO to multiple biobased products,
this integrated process also treats wastewater simultaneously.
The first bioprocess in the integrated biorefinery is methano-
genesis (MB; total volume/working volume of 4.0/3.5 m3 × 2
no), which converts the AO to biomethane at pH 7. The metha-
nogenic outlet was then transferred to algal raceway ponds
(ARP; 3.0 m3 × 2) for the mixotrophic cultivation of microalgal
biomass along with the utilization of CO2. The photosynthesis
unit reduces the residual carbon load in the wastewater and
then finally passes through an advanced ecological engineered
system (EES; 1 m3 × 3). EES was designed to mimic the natural
cleansing functions of wetlands to achieve wastewater treat-
ment. The EES system consisted of three tanks with diverse
biota, viz., aquatic macrophytes, submerged plants, emergent
plants and filter feeders, connected in series. The treated
water discharged from EES was re-circulated to the water
storage tank to be used in the process.
2.3. Experimental details
Anaerobic sludge from STP-HWSSB, Hyderabad, was used as a
parent culture after filtering with stainless steel mesh. The
thick filtered inoculum was subjected to heat-shock pretreat-
ment for 120 min at 80 °C–90 °C to suppress the methanogens
in the mixed inoculums.30,32 Before inoculation, the inoculum
was enriched with designed synthetic media providing 3 g
COD per L (glucose) for 48 h at pH 6. A heat-shock pretreated
enriched consortium from the inoculum tank was transferred
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Green Chemistry Paper

Fig. 1 Aerial view ( photographs) of pilot plant (10 m3) with its internal components integrated with a waste biorefinery installed at CSIR-IICT
(above). Process flow diagram of acidogenic bio-H2 production integrated with methanogenesis and photosynthesis in a closed loop biorefinery to
produce multiple biobased products from biogenic waste (below).

to the acidogenic bioreactor (ABT) before starting the reactor.
Initially, 110 kg of food waste (FW) was masticated (stage-I)
using a blender. The masticated FW was then transferred to
the buffer tank (BT) (stage-II) and mixed with water (left for
12 h under anaerobic conditions). A top mixer was used for
homogenous mixing of the content. Periodically, air was
sparged to the BT to suppress methanogens. The partially
degraded FW in the BT was then transferred to the main ABT
(stage-III) using a 1 HP motor. Fluid recirculation in the bio-
reactor was driven by a 1 HP non-flammable motor with a
capacity of 5 m3 h−1. NaOH was dosed into the ABT from the
acid/base tank to adjust the pH to 6.5 using a pH sensor. A
recirculation pump (1 HP) in an up-flow mode was used for
homogenization of the reactor content during the operation.

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2.4. Analysis
The performance of the pilot plant system was assessed by
analysing the chemical oxygen demand (COD; closed reflux
titrimetric method), total volatile fatty acid (VFA), and pH
profile. Quantification of the H2 gas produced during the fer-
mentation of FW was carried out by withdrawing 1 mL of gas
from the headspace with a gas-tight syringe (Hamilton) fol-
lowed by injection into a calibrated gas chromatograph (GC:
Agilent7890) equipped with a thermal conductivity detector
(TCD; OD:1/8, ID: 2 mm, SS Heysep Q column) and argon as a
carrier gas (4 mL min−1). The injector and detector were both
maintained at 80 °C, and the oven was operated at 100 °C iso-
thermally. The volatile fatty acid profile (acetate, propionate,
and butyrate) was analyzed using HPLC (Shimadzu LC20A)
employing an RI detector (RID20A; Shimadzu) and a Rezex
monosaccharide column (Phenomenex India) by injecting a
filtered sample of 20 μl. For elution, a flow rate of 0.5 mL
min−1 was used in an isocratic method with filtered ultrapure
water as the mobile phase, and the column temperature was
maintained constantly at 60 °C in a column oven (RA20A).
Dehydrogenase (DH) enzyme activity was estimated by a col-
orimetric procedure based on the reduction of 2,3,5-triphenyl
tetrazolium chloride (TTC).25 Buffering capacity (βmol ) was
estimated based on acid–base titrations employing an auto-
titrator (Mettler Toledo DL50). β was calculated using eqn (1),
where C is the concentration of acid or base (N), Vs is the
volume of the sample (mL), and m is the slope of the tangent
of the curve.
C followed the avoided-products approach, where acceptable
βmol ¼ ð1Þ
Vs  m
The hydrogen conversion efficiency (HCE) was calculated by
considering the cumulative hydrogen production (CHP, L),
organic loading of the substrate (OL, g COD per L), substrate
conversion efficiency (CODC, %) and substrate feeding volume
(V, L), as depicted in eqn (2).
Hydrogen conversion efficiencyðHCE%Þ
CHP  10 000 ð2Þ
¼
OL  CODC  V  THY
THY is the theoretical H2 yield (466 L H2 per kg COD) based
on the acetate pathway from glucose (C6H12O6 + 2H2O → 4H2 +
2CH3COOH + 2CO2; 667.24 g COD CH3COOH per kg COD +
332.29 g COD H2 per kg COD; 466 L H2 per kg COD).
2.5. Metagenomic analysis
Microbial diversity analysis of parent inoculum (untreated)
and acidogenic consortia (heat-shock pretreated) at the end
of the experiment was studied. Genomic DNA (gDNA) extrac-
tion was performed by using a NucleoSpin® Soil kit
(Macherey-Nagel GmbH & Co, Germany). Agarose gel electro-
phoresis (1%) was used to check the presence of gDNA.
Universal primers [forward primer, 341FGC, 5′ – AGG CCT
AAC ACA TGC AAG TC – 3′; reverse primer 517R, 5′ – ATT
ACC GCG GCT GCT GG – 3′; GC clamp was added to the
564 | Green Chem., 2021, 23, 561–574
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forward primer related to the conserved region (V3) of the
16S rRNA gene] were used for PCR amplification using a
thermal cycler (Eppendorf, Germany). Further amplified PCR
products were checked for concentration using Nanodrop
(Thermo) and purified by a PCR purification kit (QIAquick,
Qiagen, USA). Denaturing gradient gel electrophoresis
(DGGE) with SYBR stain was used with polyacrylamide gel.
Each DGGE band was considered as an OTU for analysis. The
amplified bands were partially sequenced and compared with
the NCBI GenBank database using the BLAST server (NCBI
BLAST).
2.6. Life cycle analysis – environmental sustainability
Life cycle analysis (LCA; ISO 14040:2006) was employed to
study the environmental impact of both the standalone and
integrated biorefinery processes separately with defined
system boundaries (Fig. 2) using SimaPro9.0.0.49 software.
The primary data, i.e., the data pertaining to the raw
materials, chemicals, water, and energy, were used as direct
input, whereas the secondary data of processes such as pro-
duction of reactants, nutrients and water were available in
the Ecoinvent 3.4 database provided with the SimaPro soft-
ware.33 The influence of both processes was evaluated on
the major environmental impact categories such as global
warming potential, ozone depletion, health factors, resource
depletion, and ecotoxicity, along with other impacts. A gate-
to-gate LCA approach was considered in this study to evalu-
ate the respective environmental impacts.23,36 The analysis
allocations were granted to the by-products of both pro-
cesses. This approach facilitates the sharing of impact
burdens among the product and the by-products (H2 and
VFA, respectively). The allocations and the inventory data
employed for conducting LCA are provided in Table 1. The
functional unit for the study was set as one kilogram (kg) of
H2 production. The lifecycle impact assessment (LCIA) was
performed using the Impact 2002 + method considering
various mid-point impact categories and endpoint impact
categories.35 Uncertainty analysis was performed to compare
the actual and anticipated results, which ultimately is
crucial in accounting the LCA results. Monte Carlo simu-
lation was employed in the uncertainty analysis with
SimaPro software. A thousand runs were carried out to
obtain the analysis at a 98% confidence interval by using
the IMPACT 2002 + V2.15/IMPACT 2002 + method for both
the processes (standalone and integrated approaches).
Furthermore, a comparative study was conducted for standa-
lone and integrated biorefinery approaches with established
hydrogen production technologies such as ammonia crack-
ing (M1), steam reforming of methane (M2), the sodium
amalgam (mercury cell electrolysis; decomposer cell)
method (M3), and diaphragm technologies (M4). The com-
parative analysis was performed in the confines of the
obtainable data of processes M1 to M4 and features available
in the Simapro LCA software.
This journal is © The Royal Society of Chemistry 2021
Green Chemistry
Fig. 2 (a) System boundary for standalone bio-H2 and (b) the integrated biorefinery consisting of multiple unit operations for biobased product
recovery and wastewater treatment.
3. Results and discussion
3.1. Pilot plant (10 m3) performance
The pilot plant was initially operated at a lower organic load
(FW: 5 g COD per L) for adaptation of the biocatalyst and for
biofilm formation. Subsequently, the system was operated at a
higher organic load (50 g COD per L). A cumulative bio-H2 pro-
duction (CHP) of 54 288 L (H2: 4.5 kg) with a retention time of
48 h was observed with an organic load of 50 g COD per L,
accounting for an H2 composition of 61% along with 39% CO2
co-generation (Fig. 3). In this study, CH4 evolution was not
observed, confirming the suppression of methanogenic
activity. Pretreatment of inoculum is crucial prior to startup of
the acidogenic process to suppress the methanogens in the
mixed culture.18,32 Heat-shock pretreatment was employed for
terminating H2 consumers in the mixed culture. However, a
complete suppression of methanogenic activity is difficult
despite pretreatment because acetoclastic and methanoclastic
This journal is © The Royal Society of Chemistry 2021
Paper
methanogenic bacteria (MB) survive on weak acids/H2. Thus
air was bubbled into the reactor for a period of 5 minutes
(once in 12 h) in order to inactivate the functional activity of
the MB.30
Short-chain carboxylic acid production accounted for
271.33 kg COD by the end of the cycle operation, along with
bio-H2 production (Fig. 3). Compositional analysis revealed
the presence of acetic (C2H4O2; HAc), propionic (C3H6O2; HPr)
and butyric (C4H8O2; HBu) acids. HAc and HBu acid fermenta-
tion produced 4 mol H2 per mol glucose and 2 mol H2 per
mol glucose, respectively; these are the two main metabolic
routes by which molecular H2 is generated in dark fermenta-
tion (eqn 3 and 4). Production of HAc and HBu was higher
compared to HPr at both 24 h and 48 h of the cycle period.
Production of HAc increased to 8.15 g L−1 (24 h) from 0.68 g
L−1 (0 h), which was further maximized to 15.21 g L−1 (48th
h). Accumulation of HBu was higher during 8–24 h of oper-
ation, with the highest production of 4.89 g L−1 (24 h) and
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Paper Green Chemistry

Table 1 Inventory data employed for performing LCA

Standalone Integrated biorefinery

Input to the system approach approach

Food waste 30 kg 30 kg
Sodium Hydroxide 0.07 kg 0.12 kg
Tap Water 1500 L 1500 L
Electricity 4 Kwh 7 Kwh
Anaerobic Sludge 25 kg 25 kg
Acetic acid — 15.47 kg
Butyric acid — 11.41 kg
Propionic acid — 9.94 kg
CO2 — 3.6 kg

Output from the system

Allocation (%) Allocation (%)

Biohydrogen 1 kg 45 1 kg 45
CO2 3.6 kg 14 — —
Acetic acid 15.47 kg 24 — —
Butyric acid 11.41 kg 12 — —
Propionic acid 9.94 kg 5 — —
Algal biomass — — 1 kg 13
Treated water — — 700 L 7
O2 — — 1 kg 35

5.6 g L−1 (48 h). In contrast, biosynthesis of HPr (1.5 g L−1)

was low up to 24 h of the cycle period compared to the other
two fatty acids. The HPr concentration was doubled in the
next 24 h of fermentation (3.12 g L−1), with a total accumu-
lation of 4.89 g L−1.

C6 H12 O6 þ 4H2 O ! 2CH3 COO þ 2HCO3 

ð3Þ
þ 4H2 þ 4Hþ ΔG° ¼ 48 kJ mol1

C6 H12 O6 þ 2H2 O ! CH3 CH2 CH2 COO þ 2HCO

3
ð4Þ
þ 2H2 þ 3Hþ ΔG° ¼ 137 kJ mol1

C6 H12 O6 þ 2H2 ! 2CH3 CH2 COO þ 2H2 O þ 2Hþ ð5Þ

H2 production showed a good correlation with VFA pro-

duction;16 interestingly, no consumption of VFA was seen
during pilot plant operation. The efficiency of a system
towards bioconversion of the substrate to VFAs was expressed
in terms of acidification potential (AP). The AP was derived
based on the COD equivalents of the individual fatty acids
(C2H4O2: 1.07 g COD per g; C3H6O2: 1.51 g COD per g;
C4H8O2: 1.82 g COD per g).15,16 With the production of
16.27 g COD per L of HAc followed by 10.26 g COD per L and
7.4 g COD per L of HBu and HPr, respectively, the acidification
potential reached 67.91%. With the distribution of fatty acids,
HAc alone contributed to 32.56% of the acidification, followed
by 20.54% and 14.88% by HBu and HPr, respectively. The Fig. 3 (a) Biohydrogen production during acidogenic fermentation of
pilot-scale operation showed >60% AP, which is important food waste at an organic load of 50 g COD per L; (b) total volatile fatty
in terms of production of renewable chemicals (HAc, HBu acid production; (c) composition of individual fatty acids in the total VFA
and HPr). derived from food waste at 50 g COD per L along with biohydrogen.
The pH of the influent was adjusted to 6.5 prior to
feeding. Initial mild acidic pH favors acidogenic function by genic fermentation (Fig. 4a). Controlled pH can be adopted
suppressing the methanogenic activity.32 The bio-H2 pro- throughout the process; however, it soon becomes economi-
duction correlated well with the pH changes during acido- cally unfeasible due to the large consumption of chemicals.

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Green Chemistry Paper

Biogenic

RT = retention time; HAc = acetic acid; HBu = butyric acid; HPr = propionic acid; OL = organic load; CODC = substrate conversion efficiency; HCE = hydrogen conversion efficiency; DH =
CO2 (L)

4531.8
10 374

10 200
Utilization of Biogenic

2980
Biogenic CO2 (L)
buffering
capacity

CO2 (L)
In situ

(βmol )

0.26
of toluene)

CODC (after treat-

DH (μg

ment, kg COD)
mL−1

57.38 4.68
MCE (%)

66.41

0.074
HCE

4.28
(%)
CODC

CODC

CODC

95.7
(%)

(%)
(%)

58

76

93
129.68 82.40 59.25

15.57 11.20

0.014
1.02
HPr

Consumption (kg

Consumption (kg

0.020
1.70
Composition

HBu
(kg COD)

COD)

COD)
24.51

0.032
1.57
HAc

271.33

56.98

0.074
Final

Final

Final

3.42
Production
Consolidated performance data pertaining to standalone and integrated biorefinery system

(kg COD)

237.41
Initial

Initial
Initial

56.98
0.002

3.42
VFA

Fig. 4 (a) Dehydrogenase activity and pH change recorded with

respect to time during food waste fermentation; (b) substrate conversion
(CODC) and biohydrogen conversion efficiency (HCE) recorded during

O2 (evolution,
acidogenic fermentation of food waste.

dehydrogenase; MCE = methane conversion efficiency; VFA = volatile fatty acid.

CH4 (%)
H2 (%)

kg)
3.7
1.2
87
61

Further dehydrogenase enzyme (DH) activity during the oper-

ation was monitored at every 8 h of operation. DH is funda-
mental to the biological redox reactions supported by redox
Biomass
CH4 (L)

32 454
54 288
H2 (L)

mediators (NAD+, FAD+). At the 8th h, the DH activity was

(kg)
2.4
1.2

1.44 μg mL−1, which further increased to 2.88 μg mL−1 (24 h)

and 4.68 μg mL−1 (32 h). The dynamics of the fermentation
process were further assessed in terms of substrate conver-
Bioreactor

sion/degradation, which showed a significant increment with

fermentation time (Fig. 4b). The biohydrogen conversion
(m3)

10
8

efficiency (HCE) was calculated to assess the process

efficiency, which specifically represented the amount of bio-
120
RT
(h)

60

24
48

H2 being evolved from the substrate degraded. The HCE

Ecological Engineered System

based on waste-derived H2 was 57.38% by the end of the

fermentation.
Methanogenesis (MB)

Photosynthesis (ARP)

3.2. Biorefinery system

Acidogenesis (AB)

The acidogenic process left 271.33 kg of COD unutilized in the

acidogenic outlet (AO), which is fatty acid-rich (Table 2).
Bioprocess

Although the H2 produced in this study is of biological origin

Table 2

(EES)

from renewable feedstock, sole recovery and utilization of bio-

H2 leaving behind unutilized carbon as waste will be unsus-

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Paper Green Chemistry

tainable. There is a possibility of recovering fatty acids as plat-
form chemicals from AO, which will have an influence on
economics as well as on environmental sustainability.17 In this
study, fatty acids were not recovered; hence, disposal of the AO
as waste creates a substantially negative impact on the whole
process, and this scenario compelled us to give value addition
to AO by refining it in a waste biorefinery unit analogous to a
petro-refinery.3 A biorefinery facility integrated with bio-H2 was
designed with an aim to utilize the fatty acid-rich/unutilized
carbon in AO and CO2 for the production of multiple biobased
compounds in a carbon-neutral approach. Waste-based biorefi-
neries (WB) have been evolving into more resource-oriented
platforms.3,18,24,36–40 The concept of biorefinery was success-
fully documented in this study experimentally by integration
of various bioprocesses which accounted for efficient residual
carbon conversion (more than 95%) with resource recovery in
the form of methane (methanogenesis: MB), algal biomass
and oxygen (algal raceway pond: ARP) and treated water (eco-
logical engineering wetland systems: EES). The first integrated
methanogenesis was intended to facilitate the conversion of
organics to biomethane. The produced methane during
methanogenesis was 32 454 L, which can compensate the
majority of energy consumption of the pilot plant. The
methane yield from AO was due to the presence of high
amounts of end products (mostly HAc), which are simpler to
break down by methanogens compared to complex organic
waste.41–43 However, simpler substrate availability is an added
advantage, as bacteria skips a few steps (hydrolysis and acido-
genesis/acetogenesis) and proceeds to the subsequent step,
directly resulting in product formation (eqn (6)).44
effluent is then passed through an ecological engineered
system (EES) designed to mimic the natural cleansing func-
tions of wetlands to enable wastewater treatment.51–53 Three
tanks of the EES system consisted diverse biota, viz., aquatic
macrophytes, submerged plants, emergent plants and filter
feeders connected in series. The waste biorefinery unit finally
reduced the COD concentration of the water in the range of
0.074 kg COD (Table 2). The treated water from the EES is
again re-circulated to the water storage tank to use in the
process. The waste biorefinery has been a potential solution to
address energy, chemicals, materials and clean water needs in
a sustainable way, preserving the ecosystem and natural
capital.3,18
3.3. Metagenomic profile
At the end of the pilot plant operation, culture was collected
from the main outlet of the ABT to understand the diversity of
the microbiome in comparison with the parent culture. The
relative abundance of bacterial diversity at the phylum and
genus levels is depicted in Fig. 5. Among the total number of
18 operational taxonomic units (OTUs) observed, 15 were
present in the parent inoculum and 13 were present in the bio-
reactor outlet. From the number of OTUs, it can be inferred
that parent inoculum showed higher diversity. Both the
samples shared 10 OTUs in common. The parent inoculum

CH3 COO þ H2 O ! CH4 þ HCO3  ; ΔG°′

¼ 31 kJ per reaction ð6Þ

The higher CH4 fraction in the total biogas can be attribu-

ted to the direct conversion of VFA to CH4. Compared to bio-
H2 production by the acidogenesis processes, methanogenesis
is more efficient towards substrate conversion (>70%; final
COD of 56.98 kg COD) during this stage of operation. The
residual carbon of the methanogenic bioreactor is fed to
raceway ponds for the cultivation of microalgae. Microalgae in
mixotrophic mode convert both organic and inorganic carbon
in the presence of sunlight.45 Microalgae have a fast growth
rate and biomass productivity compared to other energy crops,
as this biomass is doubled in 24–30 h.46 The microalgae using
the residual carbon present in the methanogenic effluent pro-
duced 2–3 ± 1.2 kg biomass. Earlier studies showed that fatty
acid-rich acidogenic effluent from bio-H2 production can be a
potential feedstock for the valorisation of algal biomass.47 A
study on the utilization of acidogenic effluent as the main sub-
strate for mixotrophic algal cultivation showed carbon recov-
eries of more than 60%. Algae have the potential to capture
and convert atmospheric CO2 into edible biomass.48,49 The
Fig. 5 (a) Relative abundance of various OTUs in the heat-shock pre-
production of 1 kg of algal biomass requires about 1.83 kg of treated inoculum in the 10 m3 pilot plant and the parent inoculum; (b)
CO2.50 CO2 utilization by algae indicates its potential towards Venn diagram illustrating the unique and shared OTUs within the heat-
carbon neutrality. After harvesting the algal biomass, the shock pretreated and parent inoculum.

568 | Green Chem., 2021, 23, 561–574 This journal is © The Royal Society of Chemistry 2021
Green Chemistry Paper

had 5 unique OTUs compared to 3 OTUs of the ABT. The

WB (B1 + B2 +

−8.91 × 10−5
unique OTUs of the parent inoculums are related to non-acido-

1.82 × 10−9

2.58 × 10−5
B3 + B4)

−0.0004
0.00054
0.00246
0.00036
genic groups, whereas the unique OTUs of ABT are related to

−1.203
−19.22
0.7122

5.8392
1.3191
0.0330
0.0010
0.0039
acidogenic bacteria; this indicates the effectiveness of the
heat-shock treatment and reactor operation. The ABT diversity
showed the presence of firmicutes (54.55%) as the dominant

ST (A1 + A2 +

6.37 × 10−9

9.04 × 10−5
−0.000312
bacterial phylum, followed by Proteobacterium (25%),

A3 + A4)

−4.2110
−67.281
−0.0016
2.49271
Actinobacterium (9.1%), Bacteroides (6.2%), etc. as significant.

0.0019
0.0086
0.0012

20.437
4.6170
0.1155
0.0037
0.0137
Total
An abundance of firmicutes favours acidogenesis.32 The
enriched firmicutes are mostly acidogens, such as Clostridium

Impacts of processes (standalone; ST and integrated waste biorefinery; WB) on various environmental impact categories (IMPACT 2002+ LCIA method)

1.16 × 10−9
9.55 × 10−6

2.53 × 10−5
acetobutylicum, Clostridium propionicum, Clostridium beijer-

WB (B4)

0.00056

0.00022

0.00101
inckii, Clostridium aciditolerans, Bacillus amyloliquefaciens, and

0.1137
1.3852
0.0011
0.0022

5.5223

0.0015
0.4179

0.0005
1.262
Lactobacillus sp., which are well known for their ability to
produce fatty acids.54,55 Production and accumulation of high

(low voltage)

4.08 × 10−9
3.34 × 10−5

8.86 × 10−5
concentrations of short-chain fatty acids can be positively cor-

Electricity,

0.39813
0.00185
0.00802
0.00079

0.00356

0.00413
0.00162
related with the dominance of Clostridium sps.. Clostridia were

ST (A4)

19.328

0.0053
4.4203

4.848
1.462
previously reported to be a major part of the microbial consor-
tium, producing butyrate, acetate and propionate along with
bio-H2 under anaerobic conditions to support their

4.61 × 10−10
1.22 × 10−5
1.94 × 10−5
1.19 × 10−6

2.31 × 10−7

4.32 × 10−5
4.40 × 10−5
7.52 × 10−6
4.99 × 10−7

7.03 × 10−5
enrichment.56,57 The evolution of efficient acidogenic commu-

WB (B3)

0.0616
0.0012
0.0311
nities in the ABT demonstrated that the operating conditions

0.294

0.147
also have a strong selective and reproducible effect on the
structure and function of the microbial community. On the

4.26 × 10−5
6.80 × 10−5
4.16 × 10−6

1.61 × 10−9
8.07 × 10−7

2.63 × 10−5
1.75 × 10−6
1.0299985
Tap water
other side, Bacteroidetes play an important role in the hydro-

0.000246
0.00013

0.00449
0.00015
ST (A3)

0.2157
0.5149
0.1091
lysis and fermentation of macromolecular organic compounds
(e.g. proteins, carbohydrates).58 Production of propionic acid
can be directly related to the presence of Propionibacterium,

1.96 × 10−10
6.60 × 10−7
2.71 × 10−5
1.25 × 10−6

2.72 × 10−7

2.82 × 10−5

1.33 × 10−5
8.34 × 10−9
which is a Gram-positive bacterial group in the subdivision of WB (B2)

0.00144
0.00065
Actinomycetes. This anaerobic, rod-shaped bacterium is

0.023
0.160
known for its unique metabolic ability to synthesize propionic

0
0
acid using transcarboxylase enzyme.59

6.84 × 10−10
2.31 × 10−6
9.49 × 10−5
4.38 × 10−6

9.52 × 10−7

9.88 × 10−5

4.65 × 10−5
2.92 × 10−8
3.4. Life cycle analysis
ST (A2)

0.5601

0.0050
0.0831
0.0022
NaOH

3.4.1. Standalone process. Life cycle analysis (LCA) tools

will help quantify the impact of a process/product on the
0

0
0

environment throughout its life cycle; hence, it provides aware-

−9.92 × 10−5

ness to help inventors to take action to mitigate those impacts

0.000135
WB (B1)

−0.0012
0.00013

0.00344
by adopting environmentally sustainable processes.21 Table 3
−20.69
0.0097

−1.32
0.031
0.025

depicts the quantified impact of input materials (food waste,

0

0
0
0

NaOH, tap water and electricity) towards various environ-

Food waste

−0.000347

mental categories for the production of 1 kg of biohydrogen in

−0.0059
0.00044
0.00047
ST (A1)

−72.42

both standalone and integrated approaches. Among all the

−4.61
0.034

0.109

0.012
0.085

inputs of the standalone approach, electricity showed the

0

0
0
0

greatest contribution (around 95% of all inputs), mostly

kg CFC-11 eq.
kg C2H3Cl eq.
kg C2H3Cl eq.

towards impact categories such as aquatic ecotoxicity: 19.32

m2 org.arable
kg TEG water
kg PM2.5 eq.

kg PO4 P-lim
kg C2H4 eq.

kg TEG soil
Bq C-14 eq.

MJ primary
MJ surplus
kg CO2 eq.
kg SO2 eq.

kg SO2 eq.

(kg TEG water) > non-renewable energy: 4.84 (MJ primary) >
terrestrial ecotoxicity: 4.42 (kg TEG soil) > global warming:
Unit

0.39 kg CO2 eq. (Table 3). The electricity was supplied from the
grid (Southern India), which is mainly produced from thermal
Aquatic eutrophication

power stations with non-renewable feedstocks. The electricity

Respiratory inorganics

Non-renewable energy
Ozone layer depletion

Terrestrial ecotoxicity
Terrestrial acid/nutri

Aquatic acidification
Respiratory organics

production in India is categorized by the highest share of

Aquatic ecotoxicity

Mineral extraction
Ionizing radiation
Non-carcinogens

Land occupation

Global warming
Impact category

fossil fuel-derived power generation, with 73% coal followed by

Carcinogens

4.5% natural gas and 0.5% diesel oil. In addition, nuclear,

hydroelectric and other renewable energy sources contribute
Table 3

3%, 10.5% and 8.5%, respectively.60 Combustion emissions

play a crucial role in affecting air quality, environmental

This journal is © The Royal Society of Chemistry 2021 Green Chem., 2021, 23, 561–574 | 569
Paper Green Chemistry

balance, climate change, and human health by the concen-

tration of anthropogenic carbon dioxide. However, using food
waste as the feedstock reduced the overall impacts of non-
renewable energy (−67.2 MJ primary) and global warming
(−4.21 kg CO2 eq.) by avoiding the utilization of conventional
fossil-based feedstock. This also contributed positively to the
mineral extraction category due to the utilization of a renew-
able resource as a feedstock (instead of non-renewable
sources).
The influence on impact categories, namely respiratory
organics, mineral extraction, non-renewable energy and global
warming, was represented with a negative sign, which indi-
cates the comprehensive and environmentally positive impact
of the process with respect to those impact categories.
However, the impact on aquatic ecotoxicity (20.43 kg TEG
water) and terrestrial ecotoxicity (4.61 kg TEG soil) was still
higher, possibly due to the substantial production of volatile
fatty acids (in the effluent) in the form of acetic, propionic and
butyric acids. These weak acids can be extracted from the fer-
mentation liquid and have their own application in the
market, thus lowering the impact on aquatic ecotoxicity and
terrestrial ecotoxicity. Despite that, the VFA mixture was used
as feedstock for the production of other biobased products
(methane, algal biomass, O2 and treated water) in the inte-
grated biorefinery approach.
3.4.2. Integrated biorefinery process. As discussed, the
standalone approach was upgraded to an integrated waste
biorefinery process and was compared with the standalone
approach and other existing hydrogen production techno-
logies. The system boundary included various unit operations
such as bio-methanation, algal cultivation and an ecological
engineered system (EES) to utilize maximum resources avail-
able in the system and to improve the process sustainability.
Fig. 6 Uncertainty analysis for the GWP impact categories of (a) bio-H2
Inputs such as VFA and CO2 in the integrated process were not
standalone and (b) an integrated (waste biorefinery) process using the
considered as an additional input to the system, as they were IMPACT 2002 + V2.15/IMPACT 2002 + method at confidence interval of
produced in the acidogenesis step and consumed in methano- 98%.

Table 4 Comparative LCA results of the standalone bio-H2 (ST), integrated biorefinery (WB), and conventional H2 production methods (IMPACT
2002+ LCIA method)

Bio-H2

Impact category Unit ST WB Method 1 (M1) Method 2 (M2) Method 3 (M3) Method 4 (M4)

Carcinogens kg C2H3Cl eq. 0.0019 0.00055 0.026 0.00085 0.00024 0.00029

Non-carcinogens kg C2H3Cl eq. 0.0086 0.0024 2.57 × 10−5 7.91 × 10−5 0.00178 0.0012
Respiratory inorganics kg PM2.5 eq. 0.0012 0.00036 0.00067 0.00570 0.00102 0.0008
Ionizing radiation Bq C-14 eq. 2.4927 0.7122 0 0 26.447 22.09
Ozone layer depletion kg CFC-11 eq. 6.37 × 10−9 1.82 × 10−9 0 0 2.40 × 10−7 2.01 × 10−7
Respiratory organics kg C2H4 eq. −0.00031 −8.91 × 10−5 0.00013 0.00085 0.00011 0.0001
Aquatic ecotoxicity kg TEG water 20.43 5.8392 5.5511 6.5603 3.26 0.3600
Terrestrial ecotoxicity kg TEG soil 4.617 1.3191 0.0087 0.01087 2.81 0.6435
Terrestrial acid/nutri kg SO2 eq. 0.115 0.0330 0.0136 0.1539 0.0166 0.01427
Land occupation m2 org.arable 0.0037 0.0011 0 0 0 0
Aquatic acidification kg SO2 eq. 0.014 0.0039 0.0035 0.02567 0.00721 0.0061
Aquatic eutrophication kg PO4 P-lim 9.04 × 10−5 2.58 × 10−5 3.75 × 10−6 4.19 × 10−7 1.34 × 10−5 1.21 × 10−5
Global warming kg CO2 eq. −4.211 −1.203 1.3797 5.3181 1.0307 0.8830
Non-renewable energy MJ primary −67.28 −19.22 73.786 89.530 19.094 16.212
Mineral extraction MJ surplus −0.0016 −0.0004 2.15 × 10−5 0.00072 0.00011 9.49 × 10−5

570 | Green Chem., 2021, 23, 561–574 This journal is © The Royal Society of Chemistry 2021
Green Chemistry
Fig. 7 Comparative lifecycle impact assessment results of (a) the standalone biohydrogen process and (b) integrated waste biorefinery model with
respect to the endpoint (damage) categories.
genesis and algal cultivation steps, respectively ( produced and
consumed within the system). The electricity showed the
highest influence on impact categories, even in the integrated
process. The integrated approach resulted in −1.203 kg CO2
eq. of global warming potential (GWP), which is higher than
that of the standalone approach. Because the integrated waste
biorefinery approach includes additional unit operations,
where more energy is required, the net GWP was greater.
This journal is © The Royal Society of Chemistry 2021
Paper
However, the influence on the remaining impact categories
was considerably reduced. Owing to the utilization of CO2 and
VFA generated in acidogenesis for the subsequent biopro-
cesses, the impacts on various categories were reduced mark-
edly. The influence on non-carcinogens, terrestrial ecotoxicity,
aquatic eutrophication and aquatic ecotoxicity was reduced by
3.5 fold, which may be due to maximal resource utilization
with limited emissions. As an integrated approach, bio-seques-
Green Chem., 2021, 23, 561–574 | 571
Paper
tration of CO2 by algae as a unit operation also had an influ-
ence in reducing the net GWP. Integration of bioprocesses
favored a significant reduction in global warming, dependency
on non-renewable energy and mineral extraction. Based on the
results, it could be stated that implementing bio-H2 pro-
duction with a biorefinery have effectively reduced the load on
the environment along with multiple bio-based products.
3.4.3. Uncertainty analysis. Further, sensitivity analyses
(uncertainty analyses) were individually performed for the
standalone and integrated approaches. The uncertainty
graphs with respect to the GWP category are depicted in
Fig. 6. The uncertainty of the data was quantified according to
the guidelines of IPCC as the ratio of the standard deviation
and the mean value for an environmental impact category.34
The quantified uncertainty values of the aquatic acidification,
aquatic ecotoxicity, global warming, land occupation, mineral
extraction, non-renewable energy, ozone layer depletion, res-
piratory inorganics, respiratory organics, terrestrial acidifica-
tion/nitrification and terrestrial ecotoxicity impact categories
of both the standalone and integrated approaches were
observed to be less than or equal to 0.3; this demonstrates the
quality of the provided experimental data with good prob-
ability, which assists the repeatability as well as the reproduci-
bility of the results.
3.4.4. Comparative impact analysis on the damage/end-
point categories. Further, the comparative analysis of standa-
lone and integrated biorefinery approaches with the existing
conventional H2 technologies has shown interesting and sup-
porting results for biobased and low carbon products. The
integrated approach has shown the least influence on respirat-
ory organics, global warming, non-renewable energy and
mineral extraction; however, it showed a marginally higher
impact on the remaining midpoint categories (Table 4).
Specifically, aquatic acidification, aquatic eutrophication, ter-
restrial ecotoxicity, and respiratory inorganics showed higher
impact, which is mainly caused by the power utility in various
unit operations. Relative analysis depicted that the method
M2 has the highest effect on all the impact categories, except
the carcinogens and terrestrial ecotoxicity categories. For pro-
ducing 1 kg of H2, reformer technology (5.31 kg CO2 eq.) con-
tributed the most towards global warming. The M1 and
M2 methods showed no impact on ozone depletion and ioniz-
ing radiation. When the bio-H2 standalone process was com-
pared with the M1 and M2 methods, it showed a higher effect
on impact categories such as non-carcinogens, terrestrial eco-
toxicity and aquatic eutrophication, where the utilization of
chemicals, electricity and water was greater. The GWP was rela-
tively low with the standalone process operation compared to
all other production processes. The M4 method exhibited
much less impact on aquatic ecotoxicity, where the utilization/
exploitation of water was minimal.
In the impact 2002 + Lifecycle impact assessment method,
the related mid-point categories were consolidated as endpoint
or damage categories. The comparative impact analysis of stan-
dalone and integrated biorefinery approaches on the damage/
endpoint categories is depicted in Fig. 7. Towards the dama-
572 | Green Chem., 2021, 23, 561–574
Green Chemistry
ging categories, the standalone process showed a higher
impact on human health (9.21 × 10−7 DALY) and the ecosystem
(0.1617 PDF m2 year) owing to the utilization of chemicals for
buffering, electricity generation and the wastewater generated
in the process (Fig. 7). However, there was a positive influence
on climate change (−4.21 kg CO2 eq.) and resources (−67.28
MJ) owing to the use of food waste as renewable feedstock. In
comparison to the standalone process, the effect on the
human health-damaging category was significantly reduced
(2.63 × 10−7 DALY) in the integrated biorefinery approach,
which is the lowest effect compared with the existing methods.
The ecosystem quality of the integrated biorefinery approach
was slightly higher than that of all the existing methods except
M2; this may be due to the higher impacts in the corres-
ponding midpoint categories. Overall, the method M2 showed
higher impacts on all the damage categories. Among the con-
ventional H2 production technologies, the M4 method
depicted lower impacts on environmental factors. The present
study specifies that the production of H2 through biological
routes enables low embodied carbon, and eventually the inte-
grated systems facilitate sustainable platforms towards a circu-
lar bioeconomy.61
4. Conclusions
Upscaling of the acidogenic process from biogenic waste as
feedstock was documented for bioenergy and renewable
chemicals production, which in turn makes the whole process
environmentally sustainable when adopting the biorefinery
format. The 10 m3 pilot-scale operation showed an excellent
cumulative bio-H2 production of 54.2 m3 along with a total
VFA of 271.33 kg COD within 48 h of food waste fermentation.
Metagenomic data revealed that the enrichment of firmicutes
(55.4%) in the heat-shock pretreated mixed culture signifi-
cantly enhanced the biosynthesis of short-chain carboxylic
acids. Integration of the bioprocess in a circular loop assisted
the derivation of additional products in form of bio-CH4
(32 454 L), algal biomass (2.4 kg), oxygen (4.9 kg), and finally
treated water (3 m3), with significant utilization of both
organic and inorganic carbon produced during acidogenic and
methanogenic fermentation. As waste was consumed and
valorized during the process, LCA analysis showed a lower
impact with respect to global warming, non-renewable energy
resources, and mineral extraction. The biorefinery approach
facilitated maximum recovery with concomitant reduction in
environmental burdens. More significantly, the bio-H2 pro-
duction process projected a lower cumulative impact for
damage category in terms of human health, ecosystem quality,
climate change and resources than the traditional H2 pro-
duction technologies.
Conflicts of interest
There are no conflicts to declare.
This journal is © The Royal Society of Chemistry 2021
Green Chemistry
Acknowledgements
The authors acknowledge Ministry of New and Renewable
Energy (MNRE), Government of India for funding project (103/
131/2008-NT) for ‘Biohydrogen pilot plant’ and Council of
Scientific and Industrial Research (CSIR), Government of India
for funding ‘Waste biorefinery pilot plant’ as part of SETCA
Project (CSC-0113; 12th Five Year Plan Project; Chemical
Sciences Cluster). LCA part of the work was supported by
CSIR-INPROTICS Project (HCP-0011; MLP-0033). OS And RK
duly acknowledge the CSIR and UGC for providing fellowship,
respectively. The authors wish to thank Director, CSIR-IICT
(IICT/Pubs/2019/409) for supporting the research.
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