Ghosh 2017

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Review Article
A review on single stage integrated dark-photo

fermentative biohydrogen production: Insight into
salient strategies and scopes
Shiladitya Ghosh a, Ranjana Chowdhury a,*, Pinaki Bhattacharya b

a
Chemical Engineering Department, Jadavpur University, Kolkata, 700032, India
b
Chemical Engineering Department, Heritage Institute of Technology, Kolkata, 700107, India
article info abstract
Article history: In recent times, systematic integration of dark and photofermentation has attracted a lot of
Received 18 July 2017 attention due to the advantage of enhanced H2 yields and better substrate conversion ef-
Received in revised form ficiencies. This integration is done either in a sequential two stage or in a single stage
28 November 2017 manner, between which the single stage integration (SSI) seems more cost effective. This
Accepted 4 December 2017 article thoroughly reviews the salient operational strategies, key factors affecting the H2
Available online xxx yields and overall increment in H2 yields attained in the SSI biohydrogen processes. Se-
lection of more complementing pair of dark and photofermentative microbes, optimization
Keywords: of composition of common growth medium, better strategies for consistent pH control and
Biohydrogen facilitation of lignocellulosic feedstocks have been identified as major areas requiring in-
Dark fermentation depth focus and subsequent improvements. Based on the insightful discussions, the cur-
Photofermentation rent state-of-the-art of SSI bio-H2 technology has been evaluated and its potential to
Integrated biohydrogen production become a reliable hydrogen production technology has been factually assessed.
Single stage integration © 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
Amelioration strategies
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Key drivers of biohydrogen production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Main challenges of individual fermentative biohydrogen processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Biochemical basis of integration of dark and photofermentative metabolic routes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Representative stoichiometry of integrated biohydrogen production systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Two stage integrated (TSI) biohydrogen processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Single stage integrated (SSI) biohydrogen processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Basic techno-economic comparison of TSI and SSI biohydrogen systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Single stage operations: strategies and performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
* Corresponding author.
E-mail addresses: shiladityag18@gmail.com (S. Ghosh), ranjana.juchem@gmail.com (R. Chowdhury), pinaki_che@yahoo.com
(P. Bhattacharya).
https://doi.org/10.1016/j.ijhydene.2017.12.018
0360-3199/© 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Ghosh S, et al., A review on single stage integrated dark-photo fermentative biohydrogen production:
Insight into salient strategies and scopes, International Journal of Hydrogen Energy (2017), https://doi.org/10.1016/
j.ijhydene.2017.12.018
2 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 7 ) 1 e1 7
Feedstock usage pattern in SSI biohydrogen systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Ratio of dark and photo fermentative organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Lighting characteristics of the bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Hydrogen production performance of single stage integrated systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Generalities of SSI dark and photofermentative biohydrogen systems and probable upgradation strategies . . . . . . . . . . . . . . . 00
Common growth medium for the dark and photofermentative bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Preferred microorganisms of SSI biohydrogen production systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
pH shift and control in SSI systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Overall outlook: single stage integrated bio-H2 systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
with regard to - savings in energy expenditure, reutilization

Introduction of energy rich resources discarded as waste, combating
environmental decay etc. and needed to be strategized
Regular and excessive consumption of the conventional scientifically for ultimate betterment.
fossil fuels worldwide during the last few decades has led to Most of the previous research studies reported on bio-
continuous and rapid depletion of the limited reserves [1e3]. hydrogen production from complex wastes including starchy
In addition, concomitant emission of several noxious green and ligno-cellulosic feedstocks, focused majorly on either
house gases (GHG) from the combustion of these carbona- dark or photo-fermentative processes. The earlier researchers
ceous fuels have polluted the air to extremely alarming identified high production cost, low H2 yield and low energy
levels [4,5]. The consequences of these two issues coupled recovery from the feedstocks as the main disadvantages of
together will be a world with severe energy deficiency and these processes [28e30]. In case of photofermentation, low
degraded environment, if alternative measures are not light conversion efficiency (LCE) has been reported as a major
adopted right now with immediate effect. The current sce- drawback by many of the researchers [31,32]. Probably, due to
nario of global pollution demands severe curtailment of these issues encountered in individual fermentative pro-
GHG emissions from combustion of fossil fuels [6]. This cesses, the recent trend of research on biohydrogen produc-
cognizance has motivated the worldwide research quest for tion is directed towards the possibility of integration of
environmentally cleaner, energy efficient and completely heterotrophic dark fermentation with phototrophic light
renewable options of biofuels viz., bioethanol [7,8], bio- fermentation either by using sequence of two reactors or by
butanol [9], biodiesel [10], biogas/biomethane [11e13] and using a single reactor [28,33e35]. In the integrated systems the
biohydrogen [14e18]. Molecular biohydrogen, which is long carbon-rich by-products of dark fermentation is utilized by
been recognized as the future green-fuel is the most the photofermentative microorganisms. Overall, there is an
attractive alternative of the soon to be scarce pollution increase in carbon conversion efficiency and enhancement of
causing fossil fuels. Biohydrogen as a fuel has several ad- hydrogen yield over the conventional systems either using
vantageous attributes, including; carbon-neutral or carbon- dark or photofermentative bacteria [28,33e35]. Based on the
zero nature, easy renewability, eco-efficient productivity stages of reactor operation the integrated bio-H2 systems are
(via biological routes), eco-friendly conversion and highest classified into two main classes; two stage integration (TSI)
energy content among all existing fuels [14,19]. Undoubt- and single stage integration (SSI). In the single stage integrated
edly, hydrogen is the most reliable future biofuel in terms of system, both the dark and photofermentation processes occur
energy efficiency as well as being environmentally benign. in the same reactor. Therefore, the initial investment and
Biological production of fuel grade hydrogen from various running cost of energy for pumping of the by-product of dark
waste feedstocks has emerged as a very promising tech- fermentation step from 1st to 2nd stage, as required in TSI, are
nology in the recent years [16,17,20e23]. This globally up- eliminated. Hence, the SSI system is cost-effective and is
rising research field is continually exploring the newer becoming more attractive in comparison to TSI for the user-
avenues in search for low cost and economically feasible friendly revamping of the biohydrogen production. The pur-
biohydrogen production processes with higher hydrogen pose of this article is to review the recent advances particu-
yields to make the employability of biohydrogen as the larly made in SSI biohydrogen production from either simple
efficient future fuel a reality soon. Currently, biohydrogen carbon sources or complex waste streams from both applied
production processes are being combined with waste recy- and fundamental aspects. Special emphasis has been given on
cling through utilization of abundant and cheap phyto- information related to integrated dark and photo fermentative
biomass and wastewaters as feedstock materials to reduce processes and on principles of arrangement used for
the high cost of production along with reduction of augmentation of overall hydrogen productivity. Based on a
pollutant load on environment [20,23e27]. Multivariate fea- simple techno-economic comparison between the TSI and SSI
tures of these integrated bioprocesses are indeed alluring systems, the superiority of SSI over the TSI has been
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established. Moreover, the operational detailing of several biohydrogen processes the following issues appear to be the
reported SSI bio-H2 systems have been provided to highlight most challenging ones:
the increment in H2 yields achieved in these systems. The
factors influencing the performance of biohydrogen produc- Enhancement of hydrogen yield and energy efficiency
tion of SSI dark-photo fermentation systems and key funda- Utilization of lignocellulosic wastes
mentals of those processes have also been elucidated and Enhancement of substrate conversion and energy recovery
discussed on scientific basis. Based on the elaborate discus-
sions of reported literatures the current state-of-the-art of SSI The biomass or biomass waste originated from a phyto or
systems has been presented, existing bioprocess challenges plant source (particularly, cellulosic, lignocellulosic and star-
have been underlined and scopes for the amelioration of chy waste) is reported as renewable resources in all bioenergy
existing SSI systems have been recommended in the article. research studies [40,56e60]. Undoubtedly, they serve as
excellent sources of feedstocks for biohydrogen production.
Unfortunately in many cases the energy recovery from these
Key drivers of biohydrogen production valuable resource materials is not complete and a consider-
able amount of energy of feedstock remains unconverted [61].
As reported by IPCC, 2015 [36], the worldwide rise in atmo- Although lignocellulosic and starchy wastes have sometimes
spheric temperature aroused interdependent devastating been utilized for hydrogen production through individual
impacts like changes in rainfall pattern, agriculture and crop bioprocess, namely, dark fermentation, the level of energy
cycles, shortages in food, increased vulnerability towards recovery and overall conversion are very low [33,62,63]. This
diseases and overall stagnation of the economic growth of the incomplete energy recovery from valuable feedstocks de-
nations [37]. Diversification of fuel usage from conventional mands immediate attention of the researchers worldwide.
carbon-based to renewable low-carbon or no-carbon fuels is This problem often faced in single stage dark fermentation
one wise strategy that can provide immediate solution to processes should be addressed through process innovation
these lingering problems with long-lasting effect [38,39]. In approach. One promising solution is the integration of dark
this context, carbon-zero and most energy dense (122 kJ/g) fermentative processes with photofermentative hydrogen
fuel hydrogen is a strong contender among the present genera production processes [30,64,65]. These hybrid processes
of renewable energy vectors [26]. From the analysis of goals ensure complete and higher energy recovery from the feed-
and motivations of several reports, it is clear that the energy stocks being used with increased hydrogen yields [66,67]. The
security and mitigation of global climate change are the key integration may be done by two stage reactors in which the
drivers behind biohydrogen generation [40e43]. Presently, first one is a dark fermenter and the second one is a photo-
over 90% of hydrogen is produced through catalytic reforming fermenter which uses the effluent from the first stage [65,66].
of natural gas or fossil fuels, which emits huge amounts of CO2 Alternatively, the integration of dark and photofermentation
as by-product into the atmosphere [19,44,45]. Contrary to this, processes can be achieved in a single reactor through the
hydrogen generation through biophotolytic (direct and indi- combination of the dark and photofermentative microbes. As
rect) and fermentative (dark and photo) routes is much already mentioned in an earlier section, the former process in
eco-friendly and less energy intensive than the chemical known as TSI and the later is SSI. The basics of both TSI and
or physico-chemical processes [14,19,34,35]. Additionally, SSI bio-H2 processes have been provided in the subsequent
fermentative production of H2 from complex biomass wastes sections to facilitate better understanding of biochemical as
provide feasible means for waste management and disables well as operational aspects of the two distinctly different
usage of edible pure carbohydrates as feedstocks boosting the modes of integration.
“Food Vs Fuel” conflict [19e22,26].
Biochemical basis of integration of dark and

Main challenges of individual fermentative photofermentative metabolic routes
biohydrogen processes
The microorganisms of dark and photo-fermentative H2 pro-
The main fermentative processes which are used for H2 production systems follow completely different cellular meta-
duction are individual dark fermentation and photo- bolic pathways catalyzed by unique enzyme systems
fermentative conversion of organic carbon substrates to (metallo-biocatalysts). As scientifically demonstrated in Fig. 1,
hydrogen [19,46,47]. Metabolic routes and the enzymes an integrated dark-photo fermentative H2 production scheme
involved in H2 generation are very well studied in individual is viable only because of the metabolic end products of the
dark and photofermentative systems [19,48e50]. In individual dark fermentation process serving as the nutrient for the
dark fermentation, carbohydrate molecules are converted to microorganisms of the succeeding photofermentative pro-
hydrogen and an array of organic acids and their salts, e.g., cess. The integration of the diverse metabolic pathways of
acetate, butyrate, propionate, malate, lactate etc. and a small dark and photofermentative microorganisms through the
amount of alcohols, e.g., ethanol [51e54]. Purple non-sulfur VFAs acting as the intermediate linking metabolite has been
bacteria (PNSB) photosynthetically produce hydrogen using vividly illustrated in Fig. 1.
organic molecules as carbon source and light as the energy Fig. 1 clearly illustrates the generation of VFAs as extra-
source [26,55]. From the analysis of the literature data on cellular metabolic end products of microorganisms carrying
the performance of individual dark and photofermentative out dark fermentative pathway and its eventual entrance in
Fig. 1 e Integration of dark fermentative and photofermentative hydrogen production pathways via VFAs acting as
intermediate metabolic linker. (* Double column fitting image).
the succeeding photofermentative pathway as nutrients for Representative stoichiometry of integrated biohydrogen
PNSBs. The resulting metabolic network so formed through production systems
the combination of dark and photofermentative metabolic
pathways permits higher energy recovery from the feed- From the literature survey it is clear that through dark
stocks. The individual hydrogen yields of the two metabolic fermentative route production of 4 mol of hydrogen per mole
pathways contribute to the higher value of cumulative of glucose is theoretically feasible. However the production of
hydrogen yield obtainable from the integrated dark-photo 12 mol of H2 per mole of glucose is possible if the same dark
system [68,69]. In Fig. 1 production of two major VFAs fermentative system producing only acetic acid as a by-
namely, acetate and butyrate have been shown [28], whereas product is integrated with a photofermentative process
other VFAs i.e., lactate, formate, propionate etc. may also be [73,74]. The stoichiometric equations for the integrated pro-
produced as the end products of dark fermentation depending cess may be as follows (1e3):
on the feedstock being used and other micro-environmental
parameters [57,70]. PNSBs can efficiently assimilate all of C6 H12 O6 þ 2H2 O/2CH3 COOH þ 4H2
these VFAs for biomass growth and H2 production with vari- þ 2CO2 ðDark fermentation stageÞ (1)
able yields [26,71]. While, in dual stage integrated systems the
VFAs are supplied to a separate stage of photofermentation Light
2CH3 COOH þ 4H2 O ! 8H2 þ 4CO2 ðPhotofermentation stageÞ
after the completion of the first stage of dark fermentation, in
(2)
single stage integrated scheme dark and photofermentation
occurs simultaneously [28,64,72]. Due to this difference be-
C6 H12 O6 þ 6H2 O/12H2 þ 6CO2 ðOverall ReactionÞ (3)
tween the two schemes of integration, operational strategies
and performance of both schemes differ remarkably from Production of 12 mol of hydrogen per mole of glucose in-
each other. dicates complete conversion, which can never be attained
through individual dark fermentation process due to the Numerous research articles report that this mode of inte-
constraint of cell energetics requiring formation of ATP mol- grated biohydrogen production has been adapted by several
ecules from the carbon substrates [73,75,76]. However, inte- groups of researchers and studied in detail [65e69,73e77]. In
grated dark-photo fermentation processes hold the potential this hybrid scheme the dark fermentation process is sepa-
to overcome the major obstacle of low hydrogen yield of in- rately conducted in bioreactors utilizing pure bacterial culture
dividual dark or photofermentation. or mixed anaerobic consortia producing carbon dioxide,
The mode of integration of dark to photofermentation hydrogen and soluble metabolic by-products either from
systems is an important factor which affects both the complex organic feedstocks or from pure sugars like glucose,
hydrogen production efficiency and operational cost of the sucrose etc. [65e69,73e85]. It is notable that the earlier groups
entire system [64]. In these hybrid systems the dark and photo of researchers have mainly used the simple sugar glucose as
phases should be given equal importance to obtain overall primary substrate for the dark fermentation phase [76,77],
maximum H2 yield from the entire bioprocess operation. The while the complex feedstock materials gained importance
dark and photo systems are combined and operated jointly via recently [78e80]. In TSI systems, the dark fermentation may
two different modes, namely TSI and SSI, as mentioned in the be conducted either by a thermophilic or a mesophilic
preceding section. Although, both modes of integration have microbe. Use of thermophilic bacteria is very well docu-
been researched in the recent past, the discussion of the up- mented in TSI bio-H2 systems [73,78,80]. The hydrogen
coming sections will focus more on the single stage integrated generated in dark fermentation stage is collected from the
(SSI) bio-H2 processes. Characteristic features of single stage reactor headspace volume as gas mixture and purified. The
mode of integration have been discussed in the subsequent VFA rich dark fermentation effluent (DFE) is fed to a second
sections emphasizing on the effect of several important pa- stage of photofermentation occurring in a photobioreactor
rameters on the bioprocess operation. These include; initial after some modifications or adjustments. The second stage is
substrate or feedstocks used, ratio of dark and photo- mediated by potent photosynthetic organisms, preferably
fermentative microorganisms, lighting characteristics, PNSBs, for the light driven production of hydrogen from the
hydrogen production potential of the system and existing DFE of the preceding dark fermentation system. The overall
obstacles barring process implementation. hydrogen yield obtained from the entire operation of two re-
actors is calculated as the summative value of the individual
hydrogen yields of the dark and light fermentations [81,82].
Two stage integrated (TSI) biohydrogen
processes
Single stage integrated (SSI) biohydrogen
The most common mode of integration is the hybrid hydrogen processes
production system constructed through the serial connection
of dark fermentative process to the light fermentative In this mode of integrated operation the dark and photo-
process. A simple schematic diagram of hybrid Dark- fermentation are allowed to occur simultaneously in a single
Photofermentation system operating in this sequentially bioreactor/photobioreactor by combining the dark fermenta-
connected two reactor mode is represented in Fig. 2. tion microbes with the photofermenting microbes together in
Fig. 2 e Reactor operation scheme of two stage integrated (TSI) mode of biohydrogen production. (* Double column fitting
image).
the single reactor environment. The dark fermentation is PNSB reactor, as it may suppress the hydrogen evolving ac-
conducted by either a pure culture or mixed anaerobic con- tivity of nitrogenase) used for the maintenance of anaerobic
sortia from the simple sugars or pre-hydrolysed complex condition are costly [83,89]. Hence, the operating cost in-
feedstocks fed to the reactor producing hydrogen and VFAs, creases directly with the increase in the number of reactors
which are assimilated as carbon substrate for hydrogen pro- being used and is lowest for SSI systems. However, from a
duction by the photosynthetic microorganisms as soon as process engineering viewpoint it seems much easier to sys-
they are formed [86e88]. Since dark and photo fermentative tematically integrate dark and light fermentation processes in
reactions are occurring simultaneously in the same reactor, it a sequential manner than a single stage manner due to sub-
is often termed as combined fermentation. The cumulative stantial differences in optimum operating conditions of the
amount of hydrogen produced from the simultaneous dark two types of microorganisms. On the other hand, single stage
and photofermentation in a single stage integrated system is integration is a much less energy consuming process due to
higher than that obtained from the individual processes avoidance of pumping energy and sterilization (autoclaving,
(Table 2). A schematic diagram of single stage hybrid Dark- centrifugation etc.) energy required for DFE in case of two
Photo fermentation system operating in this one stage mode stage integrated system [65,78,84]. Thus from simple under-
is depicted in Fig. 3. standing it is clear to recommend that SSI of dark and pho-
tofermentation is way more cost effective than the TSI
process. Therefore, based on these facts, the SSI system has
Basic techno-economic comparison of TSI and been considered as the more fitting contender to become a
SSI biohydrogen systems future bio-H2 technology in terms of economic sustainability
and selected as the focal area of emphasis for the present
It is important to judge the extent of practical applicability of a article.
process before advocating its implementation as a large scale
enduring technology. After emphasizing on the basic modal-
ities of TSI and SSI biohydrogen production processes a Single stage operations: strategies and
comparative analysis is urgent to assess the suitability of the performance
processes for long term sustainable hydrogen production. A
general techno-economic comparison of TSI and SSI systems Feedstock usage pattern in SSI biohydrogen systems
is being provided here to recommend the better scheme based
on analysis of operational cost of both systems (Table 1). The majority of reported research studies on SSI, dark-photo
From the overall estimation of total energy requirements to fermentative hydrogen production were conducted by the
operate bioreactors/photobioreactors for hydrogen produc- research group of Kargi and co-workers. Notably, hydrolysed
tion it is clear that energy expenditure increases directly with wheat starch was used as the substrate material in most of the
increase in the number of reactors being used (Figs. 2 and 3). In research studies reported so far on the one stage combined
case of the TSI systems using a thermophilic microbe in the dark-photofermentation (Table 2). The feeding rate of sub-
dark fermentation stage the energy requirement is clearly strate in the combined dark-photo fermentation highly in-
much higher due to the high optimum temperature (70e72 C) fluences the overall productivity of the system. It has been
requirement of the thermophile [73,78,80]. In this respect, SSI realized that the rate of substrate utilization and product
system of dark and photofermentation is advantageous due to formation by dark fermentative bacteria is much faster than
its low energy requirement compared to TSI using more than that of photo bacteria present in the same reactor [86]. This
one reactor. The SSI system is also less costly due to the may lead to the accumulation of VFAs in the media towards
avoidance of prerequisite processing of DFEs in TSI systems. their inhibitory concentration level for the photofermentative
Extensive adjustments of DFEs, such as dilution of metabo- bacteria present in the system as observed by Argun and Kargi
lites, addition of important co-factors, removal of inhibitory [87]. The operation of the reactor with periodic feeding of the
substances, buffering, autoclaving etc., before their introduc- substrate and effluent removal has been found to effectively
tion as feedstocks for photofermentation increases the oper- increase the hydrogen yield from combined dark-light
ating cost of the whole process significantly to several folds fermentation, Sagnak and kargi [90]. In case of continuous
[73,78,80,85]. Another important operating condition that reactor operation hydraulic residence time (HRT) is another
significantly increases the operating cost is the maintenance significant factor that directly influences the overall hydrogen
of strictly anaerobic or anoxic environment inside the bio- generation of single stage combined dark-light fermentation
reactors during biohydrogen production. The inert gases systems. Increased hydrogen gas production has been ob-
namely; argon and nitrogen (nitrogen is not suggested for the tained at higher HRT values of 6 days and 8 days due to
Table 1 e Major cost factors associated with the operation of TSI and SSI biohydrogen systems.
Cost factors Two stage integration (TSI) Single stage integration (SSI)
Energy requirement for reactor operation For two reactors For one reactor
DFE sterilization Required Not required
DFE adjustment Required Not required
Maintenance of anoxic condition For two reactors For one reactor
Table 2 e Single stage integrated (SSI) dark and photo fermentative bioprocesses for biohydrogen production.
Feedstock/Carbon Dark fermentative microorganism/s Photofermentative microorganism/s used H2 yields (mol H2/mol glucose) Ref.
source/s used used
Ground wheat starch Clostridium beijerinkii DSMZ 791 Rhodobacter sphaeroides RV 0.6 mol H2/mol glucose (DP-Y) [86]
a
Ground wheat starch Heat treated anaerobic sludge Mixed phototrophic consortium (Rhodobacter sphaeroides NRRL 0.433 mol H2/mol glucose (DP-Y) [104]
B-1727 þ Rhodobacter sphaeroides DSMZ158 þ Rhodopseudomonas
palustris DSMZ-127)
a
Ground wheat starch Heat treated anaerobic sludge Rhodobacter sphaeroides RV 1.45 mol H2/mol glucose (DP-Y) [87]
b
Synthetic waste water Anaerobic consortia Mixed photosynthetic consortium 1.74 mol H2/mol glucose (D-Y) [98]
b
3.35 mol H2/mol glucose (DP-Y)
Acid hydrolysed wheat Heat treated anaerobic sludge Rhodobacter sphaeroides (NRRL-B 1727) 3.4 mol H2/mol glucose (DP-Y) [90]
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starch
Starch Heterotrophic Rhodobacter sphaeroides N7 0.6 ± 0.1 mol H2/mol hexose (D-Y) [91]
consortium (Dominant strain: Clostridium 2.6 ± 0.5 mol H2/mol hexose (DP-Y)
butyricum)
Glucose Clostridium butyricum Rhodopseudomonas faecalis RLD 53 5.374 mol-H2/mol-glucose (DP-Y) [94]
Dairy Wastewater (Da) Anaerobic consortia Mixed photosynthetic consortium N.A. [99]
Distillery wastewater (Di)
Glucose Clostridium butyricum Rhodopseudomonas faecalis RLD 53 1.8 mol H2/mol glucose (D-Y) [105]
Glucose Lactobacillus delbrueckii NBRC13953 Rhodobacter sphaeroides RV 7.1 mol H2/mol glucose (DP-Y) [93]
Glucose Clostridium Butyricum Rhodopseudomonas faecalis RLD 53 1.92 mol H2/mol glucose (D-Y) [106]
Sucrose Clostridium acidisoli Rhodobacter sphaeroides 5.08 mol H2/mol hexose (Y) [95]
Cellulose Clostridium cellulovorans 743B Rhodopseudomonas palustris CGA009 N.A. [88]
Distillery effluent Citrobacter freundii 01, Enterobacter Rhodopseudomonas palustris P2 1.30e1.49 mol H2/mol glucose (D-Y) [100]
aerogenes E10 1.62 mol H2/mol glucose (P-Y)
Starch Clostridium butyricum Rhodobacter sphaeroides 1.5 mol H2/mol hexose (D-Y) [92]
4.9 mol H2/mol hexose (DP-Y)
Glucose Clostridium acetobutylicum Rhodobacter sphaeroides 2.39 mol H2/mol glucose (D-Y) [97]
6.14 mol H2/mol glucose (P-Y)
Potato starch, Clostridium butyricum Rhodopseudomonas palustris 6.4 ± 1.3 mol H2/mol glucose (DP-Y) [101]
Glucose
Corn starch Clostridium acetobutylicum Rhodobacter sphaeroides 2.13 mol H2/mol hexose (D-Y) [102]
2.62 mol H2/mol hexose (DP-Y)
Starch Clostridium acetobutylicum Rhodobacter sphaeroides 2.09 mol H2/mol glucose (D-Y) [103]
Glucose Ethanoligenens harbinense B49 Rhodopseudomonas faecalis RLD-53 1.83 mol H2/mol glucose (D-Y) [96]
Abbreviations used in the table: (D-Y) ¼ yield of hydrogen from individual dark fermentation; (P-Y) ¼ yield of hydrogen from individual photofermentation; (DP-Y) ¼ Cumulative yield of hydrogen form
SSI dark-photo fermentation; N.A.: H2 yield cannot be calculated in terms of mol H2/mol glucose.
a
H2 yield calculated using the correlation 90 mL H2/g starch (DP-Y) ¼ 0.6 mol H2/mol glucose (DP-Y) [86].
b
H2 yield calculated using the details of the system provided [98].
7
Fig. 3 e Reactor operation scheme of single stage integrated (SSI) mode of biohydrogen production. (* Single column fitting
image).
simultaneous dark and light fermentation [86,90]. At lower systems, acetate and butyrate were formed as dominant VFAs
HRTs the photofermentative organisms tend to have a longer and served as feedstocks for the PNSB present in the same
lag period for adaptation to VFAs resulting in an overall reactor [94]. Enhanced biohydrogen production from glucose
decrease in H2 production. Another interesting fact reported through combined dark-photo fermentation was reported by
by Argun and Kargi, is that when dark and photo bacteria them (Table 2). Sun et al. conducted a statistical analysis to
simultaneously use the glucose released from the hydrolysis design a SSI with optimally enhanced biohydrogen production
of starch the cumulative hydrogen production is less. This by combined dark-photo fermentation using pure sucrose as
may be due to the possible catabolite repression of glucose the initial feedstock [95]. Xie et al., 2010, studied a SSI system
imposed upon the PNSB, blocking the metabolism of the VFAs using glucose as the carbon source for the dark fermentative
[86]. After adapting to glucose as the preferred substrate for microbe [96]. In that system the PNSB has been used in
fermentation PNSB could not switch over to the metabolism of immobilized form and the pH of the system was controlled
VFAs easily. As a consequence, the VFAs accumulate in the using phosphate buffer, which enhanced the H2 production by
reactor and eventually lead to the cessation of the growth of the co-culture [96]. Zagrodnik and Laniecki, 2015, also used
the photosynthetic bacteria. Pre-adapting the PNSBs to VFAs glucose as the carbon substrate for individual dark (Clostridium
by growing them in glucose free maintenance medium acetobutylicum) and photofermentative (Rhodobacter sphaer-
amended with VFAs may help in resolving this problem. oides) bacteria and their co-culture for H2 production [97].
Laurinavichene et al. used pure starch for combined bio- Butyric acid was produced as the major VFA followed by acetic
hydrogen production by the consortium of fermentative dark acid by the dark fermentative bacteria upon the utilization of
and light bacteria cultured in a single reactor system [91]. In a glucose. Consumption of the produced VFAs by the PNSB
later study by Laurinavichene and Tsygankov, starch was showed an opposite trend, where the acetic acid was
successfully fermented by the co-culture of dark and photo consumed at faster rate than the butyric acid [97]. Application
bacteria to H2 [92]. of pH control strategies in co-culture SSI systems for
According to our literature survey, a pioneering research enhancement of H2 yields has also been highlighted in this
study was conducted on SSI by Asada et al., they reported study [97].
enhanced biohydrogen production from combined dark- Chandra and Venkata Mohan used synthetic organic
photo fermentation using glucose as initial substrate [93]. wastewater as the feedstock in the SSI system they have
The interesting fact to be noticed in this study is that, a lactic constructed and termed as augmented dark-photo fermen-
acid producing bacteria, namely, Lactobacillus delbrueckii has tation system (ADPFS) [98]. Co-cultures of dark and photo
been used as the dark fermentative bacteria which itself is a bacteria have been used to conduct combined bio-
non-producer of hydrogen. The lactic acid produced by this hydrogenesis along with COD removal of the wastewater. The
bacterium served efficiently as the feedstock for hydrogen acidogenic dark bacteria efficiently fermented the organic
production by the PNSB, namely, Rhodobacter sphaeroides pre- substrates to VFAs (acetate, butyrate and propionate) along
sent in the system [93]. Liu et al. utilized simple sugar glucose with evolution of H2, these VFAs were subsequently taken up
to study biohydrogen production in SSI systems. In these by the photofermentative bacteria for a second simultaneous
set of H2 production. The combined fermentation of ADPFS Ratio of dark and photo fermentative organisms
exhibited overall increased H2 production with excellent VFA
reduction, which lowered the problem of accumulated VFA In one stage dark-light combined fermentation the initial
inhibition [98]. In another study recently performed on ADPFS biomass ratio of the dark to photo bacteria (D/L ratio) in the
by Chandra et al., organic compound rich distillery waste- inoculum plays very important role regarding the achieve-
water and protein rich dairy wastewater have been used as ment of highest possible hydrogen yield from the system.
the feedstock for combined dark and photo fermentation to Kargi and Ozmihci investigated the impact of the D/L ratio on
assess their potential as feedstock materials [99]. From the hydrogen productivity and yield by varying the ratio of the
findings of this study it was reported that distillery waste- dark and light bacteria in the inoculum from 1:1 to 1:10 [104].
water served as a more suitable feedstock for single stage H2 The optimum D/L ratio resulting in maximum combined
production than dairy wastewater, probably due to its higher hydrogen production rate and hydrogen yield reported was 1:2
organic fraction content. Vatsala et al., 2008, constructed a (D:L) [104]. Optimization of the biomass ratio of dark and
ternary co-culture comprising two dark fermentative bacte- photofermentative microorganisms in the seeding inoculum
ria, namely; Citrobacter freundii 01 and Enterobacter aerogenes is of utmost importance to obtain maximised hydrogen yield
E10 and a PNSB, Rhodopseudomonas palustris P2 for production from the system as reconfirmed by some other studies. Liu
of H2 from fresh distillery effluent [100]. The characterisation et al. studied the effect of the ratio of dark and photo fer-
of the fresh distillery effluent revealed initial COD, BOD, menting bacteria on the hydrogen production of the single
reducing sugar and VFA contents to be 101.2 g/L, 58.8 g/L, stage combined system of dark-photo fermentation [105]. The
7.5 g/L and 3.52 g/L, respectively. After 40 h fermentation by concentration of dark bacteria was maintained at a constant
the dark-photo co-culture, reducing sugar was completely value and the concentration of photo fermentative bacteria
consumed. COD and BOD levels have been substantially was varied to obtain dark-photo ratio of 1:1, 1:2, 1:3, 1:4 and 1:5
reduced to 43 g/L and 0.67 g/L [100]. The VFA content of the in the inoculum respectively. They have reported that use of
fermentation effluent was increased to 5.78 g/L at the end of mixed culture inoculum with ratio of dark-photo bacteria at
fermentation due to the acidogenic activity of the dark 1:2 facilitated attainment of maximum value of hydrogen
fermentative microbes [100]. In their pilot scale study, the yield from the system [105]. In a separate study also conducted
organic compound laden distillery effluent served suitably as by Liu et al. 1:600 ratio of dark-photo bacteria resulted in the
the feedstock for H2 production by the co-culture. Only one achievement of maximum hydrogen yield of 122.4 ml H2/
recent study reported the use of pure cellulose in a single vessel [94]. Ding et al. obtained maximum hydrogen yield of
stage combined dark-photo bio-H2 system conducted by a 4.134 mol H2/mol glucose by using the 1:10 ratio of dark to
syntrophic co-culture of dark and photo fermentative bacte- photo fermentative bacteria [106]. Chandra and Venkata
ria [88]. Hitit et al., 2017, very recently formulated a starchy Mohan used the dark-photo bacteria in 1:1 ratio in their ADPFS
potato juice and glucose based complex medium for the SSI system and obtained maximised cumulative biohydrogen
bio-H2 system comprising a pair of dark and photoproduction [98]. The statistical study performed by Sun et al.
fermentative bacteria [101]. In that study the substrate con- reported that dark to photo bacterial inoculum ratio of 0.83
centration has been optimized for the production of resulted in maximum substrate conversion efficiency and
maximum amount of hydrogen. In another recent study on highest H2 yield [95]. Sagnak and Kargi used a seed culture
SSI, by Zagrodnik and Laniecki, 2017, used raw non- with biomass ratio of 3 (light bacteria/dark bacteria) to inoc-
pretreated corn starch as the carbon feedstock for produc- ulate the single stage reactor for biohydrogen production [90].
tion of H2 [102]. The dark fermentative organism hydrolysed In the studies on SSI system performed by Laurinavichene and
the starch and used the produced simple sugars for produc- Tsygankov, it was noticed that the H2 yield was much lower
tion of H2 and VFAs. The PNSB present in the mixed culture when a lower percentage (0.25) of PNSB was used, compared to
produced H2 utilizing the VFAs, increasing the total H2 yield to the experiments using a threefold higher percentage (1.25) of
higher values than the dark fermentation alone (Table 2) PNSB in the inoculum [92]. Hitit et al., 2017, found that
[102]. They reported that the repeated fed-batch mode oper- hydrogen yield maximizes when an optimum dark/photo
ation of the SSI enhanced the performance of the system and ratio of 3 is used in the SSI system [101]. These facts clearly
the H2 production was influenced by the organic loading rate indicate that the H2 yields of SSI systems can be maximized
(OLR) of the starch into the system. The same group per- only by using an optimized ratio of the dark and photo
formed another recent study on SSI using starch as feedstock fermentative bacteria.
and the same pair or dark and photo bacteria [103]. In this
study, the effect of pH control on the H2 production by the Lighting characteristics of the bioreactor
used co-culture has been thoroughly investigated (discus-
sions provided in a later section). From the foregoing dis- Along with the D/L ratio the type of light source and light
cussions on the trends of substrate usage in SSI systems, it is intensity used to illuminate the reactor also affects the cu-
apparent that mostly pure carbohydrates and in some cases, mulative hydrogen production performance of the SSI sys-
starchy materials have been conventionally used over the tem, as indicated by Argun and Kargi [87]. They have used
years (Table 2). As already mentioned in a previous section, tungsten, fluorescent, infrared (IR), tungsten with infrared,
this straightway leads to ‘Food vs. Fuel’ conflict. Therefore, halogen and sunlight separately to illuminate the reactor
development of more complex biomass derived feedstock conducting combined fermentation to detect the most suit-
base is a prerequisite for the further progress of the SSI sys- able source of light. Halogen lamps were found to be the best
tems towards attainment of larger scale H2 production. source of light for combined fermentation giving highest
amount of cumulative hydrogen yield (139 ml H2 g_1starch) at H2/L was obtained respectively [105]. Ding et al. achieved
optimum light intensity of 10 klux [87]. The light intensity maximum hydrogen yield of 4.134 mol H2/mol glucose from
was varied from 1 to 10 klux for halogen lamps. It was found SSI system they studied [106]. Sun et al. statistically opti-
that hydrogen production increased with increase in light mized the values of operating parameters of a single reactor
intensity. Yield of hydrogen obtained using solar light was biohydrogen system of dark and photo fermentation and
lower compared to halogen lamps probably due to uncon- experimentally obtained 10.16 mol H2/mol sucrose or
trolled change in light intensity [87]. Effect of alternate cycles 5.08 mol H2/mol hexose [95].
of dark and lighting conditions on the hydrogen production Vatsala et al., 2008, reported considerable increment in H2
by combined fermentation has been investigated by Argun yield obtained using the ternary mixed culture compared to
and Kargi. Use of dark/light cycles yielded lesser amount of the individual dark and photo fermentations of glucose,
hydrogen than continuous lighting conditions and hence re- Table 2. A total 21.38 kg of H2 was produced from a co-culture
ported as not beneficial for combined dark-photo fermenta- fermentation period of 40 h from the pilot scale co-culture
tion [87]. Ding et al., used incandescent lamps to maintain fermentation of distillery effluent [100]. Notable increment
light intensity of 8000 lux and obtained maximum hydrogen in H2 yield was obtained by Laurinavichene and Tsygankov
yield [106]. Liu et al. obtained maximum production volume form starch using the dark-photo co-culture over the dark
of 5892 mL H2/L culture using light intensity of 10.25 W/m2 fermentation alone, Table 2 [92]. High H2 yield of 7.02 mol H2/
illuminated by incandescent lamps [105]. Another interesting mol glucose was achieved from a co-culture SSI system,
fact reported by Liu et al. from this study is that highest H2 while the H2 yields of the individual dark and photo-
yield and productivity were achieved during continuous fermentation were 2.39 and 6.14 mol H2/mol glucose,
illumination of 24 h, whereas maximum LCE was obtained respectively [97]. Quite recently, Lu and Lee, observed that
when the reactor was operated under 16 h light/8 h dark cycle the co-culture of dark-photo fermentative bacteria produced
[94]. H2 at a higher rate (12.2 ± 1.2 mL H2/d) from cellulose than
that obtained with the mono-culture of the dark bacteria
Hydrogen production performance of single stage integrated only (7.5 ± 1.3 mL H2/d) [88]. Hitit et al., 2017, successfully
systems obtained maximized H2 yield (6.4 ± 1.3 mol H2/mol glucose)
from the SSI system using optimized values of substrate
Hydrogen production performance of single stage integrated concentration (15 g/L), buffer concentration (50 mM) and
systems is influenced by the above described operating pa- ratio of dark/photo bacteria (3) in the inoculum [101].
rameters and varies greatly with changes in them and Zagrodnik and Laniecki, 2017, reported a twofold increase in
higher amount of cumulative hydrogen has been obtained cumulative hydrogen volume in the co-culture (3.23 L H2/L
from a single stage combined dark-photo system in com- medium) than that produced in the individual dark fermen-
parison to individual systems of dark or photofermentation tation (1.59 L H2/L medium) [102]. This led to a corresponding
by many researchers (Table 2). As reported by Kargi and higher H2 yield in the co-culture SSI system over the indi-
Ozmihci, considerably higher cumulative hydrogen forma- vidual dark fermentation process, as reported in Table 2. In
tion (CHF) of 1548 cm3 was obtained from a combined dark- the other study performed by the same group on same SSI
photo system compared to the lower CHF of 1000 cm3 and system, the co-culture produced 2.5 fold higher amount of H2
1268 cm3 obtained by them from individual dark and pho- (2.29 L H2/L medium) from starch than the amount (0.939 L
tofermentation stage respectively when they worked with H2/L medium) produced by the dark fermentative bacterium
2 dm3 fed batch reactors constantly fed with 20 g/dm3 alone. Upon the adjustment of pH of the system to the
wheat powder at a flow rate of 6.25 cm3/h [104]. Chandra preferable value of co-culture the H2 production further
and Venkata Mohan, obtained overall enhanced bio- increased, reaching a value of 2.67 L H2/L medium [103]. The
hydrogenesis, in terms of 40% increment in cumulative highest yield of H2 obtained in this study was 5.11mol H2/mol
hydrogen production from the augmented dark-photo glucose produced by the co-culture, which was much greater
fermentation system (ADPFS) proposed by them, compared than the yield of individual dark fermentation, Table 2. Xie
to a single dark fermentation system [98]. Sagnak and Kargi et al., 2010, achieved a maximum yield of 3.10 mol H2/mol
attained maximum hydrogen yield of 3.4 mol H2/mol glucose glucose from controlled operation of SSI regarding concen-
(470 ml H2/g total sugar) from a continuously operated SSI tration of glucose, phosphate buffer and initial pH of the
system when operated at high HRT values of 6e8 days [90]. system [96].
Investigation of a SSI system for hydrogen production con- From the analysis of the tabulated data (Table 2), it is
ducted by integrally mixed co-culture of dark and light evident that use of dark and photofermentative bacteria as co-
bacteria, described as light dependent consortium (LDC), has culture in the SSI systems effectively attained the higher H2
been reported by Laurinavichene et al. [91]. In this study, yields than the individual dark/photo fermentations. This is
almost 3 fold increased hydrogen production was obtained achieved through the complete utilization of the feedstocks by
when LDC (2.6 ± 0.5 mol H2/mol hexose) was used in place of the combined metabolic pathways of the heterotrophic dark
consortium of dark bacteria (0.6 ± 0.1 mol H2/mol hexose). bacteria and phototrophic PNSBs, as demonstrated in Fig. 1.
Liu et al., obtained maximum hydrogen volume of 5892 mL Understanding the effect of several parameters influencing
H2/L culture and total hydrogen yield of 5.374 mol H2/mol the hydrogen productivity and yields in SSI systems is of
glucose from single stage combined dark-light fermentation, utmost significance, which can substantially favour the
while in individual dark and light fermentation stage amelioration of the overall performance of these bioprocesses
maximum hydrogen volume of 1632 mL H2/L and 4260 mL in future.
research studies investigating single stage integrated dark-

Generalities of SSI dark and photofermentative photo fermentative bio-H2 production systems from starchy
biohydrogen systems and probable upgradation and cellulosic feedstocks (Table 2) [86e88,90e92,102e104]. On
strategies the other hand, the purple non-sulfur (PNS) photosynthetic
bacteria of the genera Rhodobacter and Rhodopseudomonas are
Common growth medium for the dark and the attractive photo-organisms that have been reported in
photofermentative bacteria numerous research studies dealing with the light induced
generation of bio-H2 utilizing produced VFAs as feedstocks in
For SSI systems, formulation of a suitable common growth the photofermentation stage of a single stage integrated dark-
medium is an a priori necessity to suffice the nutritional needs photo fermentation system (Table 2). It is simply under-
of the dark fermentative and the photofermentative bacteria. standable that, mesophilic dark fermentative organisms,
Dark fermentative bacteria are heterotrophic microorganisms, particularly different species of clostridium (temperature op-
which can grow using carbohydrates, either simple or com- tima 35e37 C, much closer to the optima of photo-
plex, as carbon and energy source [70,74e77,85]. Whereas, the fermentative bacteria) are also capable of breaking down
photofermentative microbes, particularly the PNSBs, are pho- complex substrates (cellulosic and starchy materials) for effi-
toheterotrophs, which uses light as the source of energy, while, cient hydrogen production [63,113,114]. Hence, they are more
the carbon source can either be a simple sugar or preferably suitable for less energy consuming single stage mode and can
VFAs [26,107e109]. This difference in carbon source utilization be successfully combined with the PNSBs for single stage
dissolves the occurrence of competition for limiting nutrients integration of dark to photofermentation processes. Thermo-
in SSI systems. Additionally, the PNSBs require several metal tolerant PNSB, e.g., H2 producing Rhodopseudomonas pentothe-
elements as micronutrients, including; Zn, Mn, Ca, Co, Ni, Fe, natexigens KKU-SN-1/1, recently isolated by Phankhamla et al.,
Cu, Na, Mo, V etc., as well as vitamins [31,80,93,100]. Although may be combined more easily with the dark fermentative
the basic nutrient requirements are same for all PNSBs, bacteria, e.g, Clostridial strains in a single stage integrated bio-
sometimes it varies depending on the species. These are H2 process [115]. This will lessen the difference in the tem-
necessary for the formation and activity of the complex perature optima of dark and photo counterparts allowing
photosynthetic machinery and nitrogenase of PNSBs, which is optimum performance by both.
important for their growth and production of H2. It is worthy to In a very recent research study conducted by Laur-
mention that, some dark fermentative microbes as well inavichene et al. the PNSB - Rhodobacter sphaeroides has been
require some of the trace metals for H2 production by hy- incorporated integrally into a starch degrading consortium of
drogenases, which are metal co-factor requiring enzymes dark fermentative microbes (mainly Clostridium sp.) resulting
[110]. The nitrogen sources are more or less same for both dark in a light dependent consortium (LDC) [91]. It is interesting to
fermentative and photofermentative microbes. A common notice that this LDC successfully generated three times higher
growth medium for SSI system must incorporate all nutrients amount of hydrogen from starch/glucose than that can be
in adequate quantities, without compromising on the nutrient generated by dark fermentative consortium alone. The same
requirements of any of the microbes. Generally in the common study also reports that the substrate preference of LDC was
growth medium used for SSI systems the carbon source of the more dependent on VFAs (acetate and butyrate) than on
dark fermentative bacteria is initially added. The dark glucose, when all are available together [91]. No catabolite
fermentative microbe uses the carbon source and produces H2 repression of glucose or accumulation of VFAs was found in
coupled with production of different VFAs (mainly acetic acid this system for LDC. Chandra and Venkata Mohan, demon-
and butyric acid) [94,97,98]. As soon as the production of VFAs, strated a SSI scheme of ADPFS, in which co-culturing of dark
PNSBs start to consume them for growth and H2 production. acidogenic consortium along with photofermentative con-
Hence, optimization of the composition of the common growth sortium was observed to effectively overcome the persisting
medium is of extreme significance to boost up the hydrogen problem of induced end product inhibition imposed by the
productivity of both microbes simultaneously. VFAs upon the dark bacteria of the consortium [116]. Signifi-
cant reduction in VFAs along with notable increment (40%) in
Preferred microorganisms of SSI biohydrogen production cumulative hydrogen production and enhanced COD removal
systems (10%) efficiency of waste water were the major findings of this
research study using the co-culture of dark and photo mi-
Proper selection of suitable pair (or more) of dark and photo-
croorganisms [116]. From the success of these research
fermentative microorganisms for integrated biohydrogen
studies it is absolutely reasonable to expect that single staged
generation processes is of paramount importance, particu-
integrated biohydrogen production systems coherently uti-
larly when complex biomass is being used as feedstock. Only
lizing the dark and photofermentative microorganisms will
some potential microorganisms possessing unique enzyme
become the dependable technology of coming future for sus-
systems can efficiently hydrolyse the complex starchy and
tainable production of biohydrogen.
cellulosic (sometimes lignocellulosic) materials to simpler
However, some complexities may arise in this SSI mode of
metabolizable precursors and use them in their cellular
operation due to the possible interaction between the mixed
metabolic pathways for the eventual production of bio-H2
culture of dark fermentative and the photofermentative mi-
[111,112]. In this respect, mixed anaerobic consortia and
croorganisms and thus need to be studied elaborately for the
Clostridium sp. have been found as the popular candidate of
successful implementation of this type of integrated
choice to be used in the dark fermentation part of several
hydrogen production systems [117]. Establishment of a
positive or cooperative relationship like commensalism or H2/mol glucose under pH controlled condition [97]. The same
mutualism between the dark and light microbes is an co-culture generated maximum H2 yield of 2.28 mol H2/mol
extremely vital factor and is expected for the feasibility of SSI glucose when the pH was not controlled. However, use of pH
process of biohydrogen generation [117]. Apparently, the maintenance buffer may not seem feasible from an economic
commensal relationship between the dark and photo- viewpoint due to the high cost incurred by them. In this
fermentative bacteria in SSI systems prevails, since the car- context, use of base dosing to maintain and balance the pH of
bon sources (VFAs) of latter microbes (PNSBs) are the the SSI systems is more cost effective. In a latest study per-
metabolic by-products of the former (heterotrophic bacteria) formed by the same group it was observed that maintenance
[117]. This dependence becomes more prominent, when a of the system pH at 7 favoured the optimum H2 production by
complex carbohydrate like starch is used as feedstock in SSI the dark and photofermentative microbes [103]. Under the
systems, since the PNSBs absolutely lack the metabolic ca- controlled pH condition, the PNSB completely consumed the
pacity to hydrolyse starch to simple sugars [102,103]. In this VFAs (acetate and butyrate) produced by the dark fermenta-
case, the dark fermentative bacteria hydrolyse the starch to tive bacteria. Hence, H2 production in SSI systems can be
simple glucose units and consume it for production of significantly enhanced by the application of proper pH control
hydrogen and VFAs. Upon the production of the VFAs, PNSBs arrangements in optimized manner. Another important
consume them and produces H2. Hence, in SSI bio-H2 systems factor in this regard is the initial substrate concentration for
positive inter-microbial interactions play vital roles in the the dark fermentative bacteria of a co-culture SSI system. The
overall metabolic performance by the two distinct microbial VFA production by the dark microbe is directly related to
groups. Therefore, construction of dark-photo mixed cultures the availability of substrate in higher amounts [101]. There-
based on interaction study will ensure better performance fore, optimization of the initial substrate concentration or
and longer stability of the culture in SSI systems [117]. finding other substrate feeding strategies (fed-batch, semi-
Comparatively lower number of experimental studies per- continuous) may provide options for achieving non-hindered
formed on SSI hydrogen production indicates that there are H2 production in SSI systems for longer durations.
scopes for further research in this area and fulfilment of
which may provide a viable technology for large scale pro-
duction of biohydrogen. Overall outlook: single stage integrated bio-H2
systems
pH shift and control in SSI systems
Significant research has been conducted on SSI integrated
In SSI systems, pH is a factor of crucial importance having systems over the past decade. The findings of those studies
major influence on the H2 production by the dark and photo- have addressed several aspects of SSI systems and also
fermentative bacteria. Dark fermentative microbes, mainly identified various issues that need to be solved thorough
the Clostridia, prefer a neutral to mildly acidic pH lying in the process engineering and innovations. The continuing interest
range of 6e7.5 for optimum growth and H2 production. On the on SSI bio-H2 systems confirm that it has been well accepted
other hand, PNSBs prefer neutral pH of around 7 for H2 pro- as an attractive bioprocess which can generate H2 at higher
duction [97,118]. Generally the initial pH of the SSI systems is yields compared to the individual dark and photofermentative
kept at neutral. In a co-culture SSI system, the dark fermen- processes. However, some complexities still reside in SSI
tative microbes consume the carbohydrates provided in the systems due to the differences in the operating conditions of
common nutrient medium and produce H2 and several VFAs the candidate heterotrophic dark fermentative bacteria and
due to their ‘acidogenic’ nature. With the progression of the the photoheterotrophic PNSBs. Since the PNSBs are more
growth of dark microbe VFAs start to accumulate in the sys- sensitive to changes in environmental parameters the oper-
tem leading to a fall in the pH of the system towards acidic ating conditions of SSI systems are often kept sub-optimal for
range. When VFA production reaches the maximum level the the dark fermentative microbes. Despite this drawback,
pH may reach a very low level of 4.5e5.5 [97,106]. This highly increased H2 production has been obtained from all SSI sys-
acidic pH is detrimental for the PNSB as well as the dark tems (Table 2), probably due to the existence of positive
fermentative microbe itself, ceasing the H2 production metabolic interaction among the dark and photo fermentative
completely. Despite the fact that the PNSBs consume the VFAs bacteria. The hydrogen yields obtained from the reported SSI
after their immediate production in the SSI system the sharp systems are still much lower than the theoretical value of
fall in pH occurs. This is due to the much slower rate of con- 12 mol H2/mol glucose. It is expected that optimization of
sumption of VFAs by the PNSBs than their fast production by different operating parameters, selection of more com-
the dark fermentative microbes, causing the VFA accumula- plementing pair of dark/photo microbes and better bioprocess
tion in the system. This is an inherent problem encountered in control strategies will upgrade the H2 productivity of forth-
all SSI systems, which adversely affects the H2 productivity of coming SSI systems towards the achievement of theoretical
these systems. Arrangements of proper pH regulation in SSI maximum.
systems can, therefore, significantly enhance the H2 genera- It is notable that, till date, the feedstock preference of SSI
tion performance of both microbes and the system as a whole. systems has mostly been different pure simple sugars or
The pH control of SSI systems may be achieved by using a complex starch, as evident from the data presented in Table 2.
buffer or base dosing during the prevalence of the acidogenic Both type of the carbohydrate sources belong to edible food
phase. Zagrodnik and Laniecki, 2015, experimentally observed sector, hence, occurrence of a ‘food Vs fuel’ conflict is highly
that the co-culture produced H2 with a high yield of 7.02 mol possible in SSI systems, which may hinder its further
progress. This classifies the H2 produced in SSI systems to be

of first generation (1G), which is not sustainable in a long run. Concluding remarks
In this context, utilization of non-food lignocellulosic feed-
stocks can provide a feasible way of tackling this issue. It is Over the last decade, biohydrogen production through the
well documented that the dark fermentative microbes, viz, systematic integration of dark and photofermentative pro-
the different Clostridia and the anaerobic consortia are capable cesses has grabbed attention of researchers worldwide.
of producing H2 from various pretreated lignocellulosic feed- Although research has been more extensively conducted on
stocks, including abundant agricultural wastes [119e124]. TSI systems, recently, the SSI systems are gaining increased
These microbes can efficiently utilize the simple sugars interest due to their cost-effectiveness and lesser operation
derived from the pretreated and hydrolysed lignocellulosic time. This article particularly reviews the state-of-the-art of
biomass for production of H2 and VFAs. Hence, it is justified to the SSI bio-H2 systems simultaneously utilizing the meta-
claim that lignocellulosic wastes can become dependable bolic capabilities of the dark and photofermentative micro-
feedstocks for SSI bio-H2 systems, upon addition of a pre- organisms for production of higher cumulative amount of H2
treatment and hydrolysis step up-stream it. The promising over the individual fermentative processes. Survey of litera-
scheme is represented in Fig. 4. Although, the use of chemical/ tures reporting SSI systems indicated that significant incre-
physical pretreatments are expensive, increasing the total ment of H2 yields have been attained from the SSI systems,
operational cost, use of inexpensive and eco-friendly which dissolves the issue of lower H2 yields associated with
biochemical pretreatment can make the overall process the individual dark and photofermentative processes. In
cost-effective [125]. Upon the facilitation of lignocellulosic most of the SSI systems studied till date, simple sugar and
feedstocks the H2 produced in SSI systems will belong to the starch have been used as the carbon sources for the dark
class of second generation (2G) biofuels, safeguarding its long microbe. No study is available reporting utilization of ligno-
term sustainable production. In some studies, organic cellulose based feedstocks in SSI systems. The ratio of the
wastewater has been successfully used as the feedstock for dark and photo fermentative microbes in the SSI systems is
hydrogen production in SSI systems, Table 2 [98e100]. Sig- another vital factor which influences the overall hydrogen
nificant scope of research and development is awaited in this outcome of the process. Due to the slower rate of growth of
field of integrated biohydrogen production, which will make it the PNSBs compared to the dark fermentative heterotrophs,
a dependable biohydrogen technology of future. Therefore, the studies using higher concentration of the former mi-
single stage integrated processes should be given adequate crobes obtained better cumulative H2 production. From the
research focus to overcome existing barriers and to facilitate profound discussions provided in the article a few scopes for
amelioration of the existing process outcomes. improvement of the performance of SSI systems have been
Fig. 4 e The operational scheme of SSI bio-H2 systems using lignocellulosic biomass resources as feedstocks. (* Double
column fitting image).
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i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 7 ) 1 e1 7

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journal homepage: www.elsevier.com/locate/he

A review on single stage integrated dark-photo

Shiladitya Ghosh a, Ranjana Chowdhury a,*, Pinaki Bhattacharya b

article info abstract

Feedstock usage pattern in SSI biohydrogen systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

with regard to - savings in energy expenditure, reutilization

Biochemical basis of integration of dark and

research studies investigating single stage integrated dark-

progress. This classifies the H2 produced in SSI systems to be

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