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 4 5 ( 2 0 2 0 ) 2 4 4 2 4 e2 4 4 5 2

Available online at www.sciencedirect.com

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

Review Article

A review of measurement methods of biological

hydrogen

Fatemeh Boshagh a,*, Khosrow Rostami b

a
Department of Chemical Technologies, Iranian Research Organization for Science and Technology (IROST), P.O. Box
3353-5111, Tehran, Iran
b
Department of Biotechnology, Iranian Research Organization for Science and Technology (IROST), P.O. Box 3353-
5111, Tehran, Iran

highlights

GC is accurate and needing small sample however is expensive and energy-intensive.

Gas meter is accurate and easy to use but is not suitable to measure low gas flow.

Water displacement is simple, easy to use, and inexpensive but is labor-intensive.

Using GC with water displacement or gas meter is accurate to measure hydrogen.

article info abstract

Article history: The current review purpose is to present a general overview of measurement methods of
Received 14 April 2020 hydrogen in dark fermentative hydrogen production. The gas measurement is performed
Received in revised form with manometric and volumetric devices by manual and automatic methods. The
8 June 2020 hydrogen gas measuring devices must be simple to construct and use, inexpensive, ac-
Accepted 9 June 2020 curate, durable, and economical. The methods of water displacement, plunger displace-
Available online 7 August 2020 ment, commercial gas meter, respirometer, and manometric were used to measure volume
and rate of biogas. For measurement of hydrogen fraction in the biogas, gas chromatog-
Keywords: raphy and hydrogen sensor were employed. The advantages and drawbacks of each
Biohydrogen production method are highlighted and some works of their uses in dark fermentation are presented.
Gas chromatography Literature shows that the water displacement method and gas meter were extensively used
Gas meter to measure biogas volume and flow and gas chromatography with thermal conductivity
Hydrogen sensor detector with a Molecular Sieve 5A column and nitrogen as carrier was widely employed to
Water displacement measure biohydrogen.
© 2020 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

* Corresponding author.
E-mail addresses: fatemehboshagh@gmail.com, boshagh@irost.ir (F. Boshagh).
https://doi.org/10.1016/j.ijhydene.2020.06.079
0360-3199/© 2020 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
 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 4 5 ( 2 0 2 0 ) 2 4 4 2 4 e2 4 4 5 2 24425

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24425
Methods of biogas collecting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24426
Methods of biohydrogen measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24426
Gas chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24426
Volume displacement devices (water displacement and plunger displacement method) . . . . . . . . . . . . . . . . . . . . . . . 24441
Commercial gas meter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24442
Hydrogen sensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24442
Respirometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24443
Manometric method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24443
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24443
Declaration of competing interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24444
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24444

Introduction
Nowadays, using fuels not compete with resources of water
and food and not emit CO2 is necessary to decrease green-
house gas emission and global warming [1,2]. In recent years,
hydrogen has received global consideration as future fuel and
a clean and efficient energy carrier which can replace fossil
fuels hydrogen can be directly employed as a transportation
fuel while solar and nuclear fuels do not have this feature [3].
The methods of production of hydrogen including physical/
chemical and biological [4]. Among hydrogen production
methods, biological methods as environmental friendly due to
reduction of CO2 production, less consumption of energy
(ambient pressure and temperature), and using organic
wastes and wastewaters as substrate are desirable. The bio-
logical hydrogen production methods are including dark and
photo fermentation, direct and indirect biophotolysis, and
water-gas shift reactions [5]. One of the hydrogen production
methods is dark fermentation that occurs under facultative or
strictly anaerobic conditions in the absence of light [6,7]. The
dark fermentation compared with photo fermentation pre-
sents advantages such as easier to conduct and control, no
need light, higher yield and production rate, stability and
feasibility for industrialization, using a wide variety of carbon
sources, and lower operating costs [8]. Also in this process
simultaneous produce bioethanol and biohydrogen from the
various substrate [9].
One of the important issues of biogas and biohydrogen
production is the detection and quantify measurement of
biogas composition. Biogas sampling and analysis is a major
challenge to control and adjust the cleaning process param-
eters [10]. The production of biogas in the laboratory scale is
low especially during the startup time. Therefore measuring
such a low flow rate is not an easy work [11]. The gas mea-
surement is carried out with manometric and volumetric
methods. In the volumetric method, the pressure was brought
to atmospheric and volume of gas was measured [12] while
the manometric method is based on measurement of pressure
increase in the system and the pressure difference was
converted into the volume of gas using the ideal gas law [13].
The biogas measurement can be done manual or automatic
[14]. Various biogas measurement devices were introduced in
the literature and explained that the ideal biogas meter must
be precise, need slight or no maintenance, durable, inexpen-
sive, and can measure a wide range gas flow at varying pres-
sure and temperature [15]. The approaches such as lubricated
syringes, pressure manometers or transducers, volume
displacement devices, manometer assisted syringes, or low
pressure switch meters were employed to determine volume
and rate of biogas. The biogas measurement at low headspace
pressure is an important issue to all manometric or volu-
metric methods of biogas measurement [14].
The method and process of gas measurement itself can
result in the inhibition of biogas and biohydrogen production.
Generally, the process of biogas or biohydrogen produce gases
such as methane, carbon dioxide, hydrogen, etc. Carbon di-
oxide is very polar and its solubility is high in water [16], where
methane and hydrogen have low solubility in water [17]. The
high amount of dissolved CO2 affects the pH of the medium
and consequently alters the microbial activity [18]. Two pro-
tocols to measure biohydrogen production in batch mode has
been recommended. A manual protocol with periodic mea-
surements of biogas, needing conventional laboratory mate-
rials and analytical equipment for biogas characterization and
an automatic protocol as a developed device for online mea-
surements of low biogas production [19,20]. Results indicated
that both protocols of manual and automatic are suitable to
get acceptable results. Typically manual methods are labour-
intensive and time-consuming, and unless data are recorded
at frequent intervals the sensitivity of measurement is
reduced and critical events may not be observed [21].
Various methods have been developed in literatures for
collecting, measurement, and calculation of biohydrogen gas
manually or automatically. In dark fermentation produced
biogas is a gas mixture which hydrogen and carbon dioxide
are the main components [22] and methane may also produce
[23]. Size of the bioreactor, and the sensitivity and capacity of
the gas-measuring apparatus affect selection of appropriate
method [24]. In dark fermentation, the volume of produced
24426 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 4 5 ( 2 0 2 0 ) 2 4 4 2 4 e2 4 4 5 2
biogas was measured by methods of water displacement [25],
plunger displacement [26], gas meter [27], respirometer [22],
manometer [24], pressure transducer [17], and pressure gauge
[23]. For measurement of hydrogen fraction in the biogas
hydrogen sensor [28], gas chromatography [29], and portable
hydrogen gas analyzer [30] were employed. Each method has
advantages and limitations that must be considered when
they are employed. Also, different types of the instrument of
displacement gas measurement depending upon the research
requirements were developed.
The present study covers the methods of hydrogen gas
measurement which are employed for collecting and mea-
surement of hydrogen gas in dark fermentative hydrogen
production. The methods of water and plunger displacement,
respirometer, manometric, gas meter, gas chromatography,
hydrogen sensor, and portable hydrogen gas analyzer were
employed to measure hydrogen gas in dark fermentation.
Each method briefly is introduced and the advantages and
disadvantages of its is discussed.
Methods of biogas collecting
In dark fermentation, biogas was collected in gasbag [31e39],
syringe [40], and helium balloon [41]. The biogas can be
collected in a large syringe [39,42] or several small syringes
[40]. Gas sampling was carried out with a syringe [43] and can
be collected in evacuated 6.8 mL exetainers [44] or syringe is
equipped with Mininert valves [45]. Gasbags are extensively
employed as a convenient portable system to collect biogas
[46]. The Tedlar bag was often used to collect biogas [47]. The
polyvinyl fluoride, polyethylene-terephthalate-nylon-
aluminum, and polytetrafluoroethylene were most widely
employed for making gasbag by Tedlar brand [46]. Other bags
such as Nalophan, Teflon, and Plastigas were also used in
some researches [47]. The bags or helium balloons are con-
nected to the bottle or bioreactor headspace for biogas col-
lecting [41]. Gasbags with various capacities of 30 L [35], 10 L
[33], 1 L [38], and 0.5 L [32] were used in dark fermentation.
Gasbags have advantages such as the sampling of simple and
fast, easy to use, no need a skilled user or additional equip-
ment [47] and disadvantages such as bags leakage may take
place especially near to the septum and connections [41].
Long-term storage of biogas in bags is not recommended [46].
To check leakage of bags or balloons after fill by biogas, they
are immersed in water and formation of bubbles are investi-
gated. The bags and ballons are limited in volume and if vol-
ume of produced gas is high those are not suitable. Compared
with the Tedlar bags, balloons of helium were more robust for
long term use [41]. Methods of biogas collecting are shown in
Fig. 1.
Methods of biohydrogen measurement
Gas chromatography
Gas chromatography is an important separation device in
which the components of a sample partition between two
phases of stationary and mobile [48] and is used to analyze
qualitative and quantitative compounds that can be vaporized
without decomposition [49]. Samples of solid, liquid, and gas
can be analyzed by gas chromatography [50]. Gas chroma-
tography as an analytical device can be used to analyze and
measure gases such as methane (CH4), carbon dioxide (CO2),
hydrogen (H2), and so on [10]. Gaseous samples are injected by
a gas-tight syringe or through a by-pass loop and valves. For
each gas, a calibration curve is drawn and according to it the
percentage of gas composition is determined. If moderate
accuracy is adequate, single-point linear calibration with pure
hydrogen is suitable. When high accuracy is required, multi-
point calibration preferably with bracketing approaches is
used [51]. The biogas was sampled from the headspace of the
bioreactors by using special gas-tight glass syringe (pressure-
lock syringe) and injected directly in the chromatographic
column. During sampling and injection of the gas sample,
proper attention is needed. Rapid injection of the samples into
the column of GC without delaying will give more precise re-
sults [16]. Selection of column (stationary phase and di-
mensions: column inner diameter, length, and film thickness),
selection of carrier gas (type and flow rate), determination of
temperature programing (initial temperature, initial hold,
ramp rate, final temperature, and final hold), selection of
injector and its temperature, and selection detector and its
temperature are several important issues in the separation
process and analyze by GC [52]. Advantage of GC compare to
other methods of gas measurement is its capability to mea-
sure both quantitative and qualitative produced biogas [16].
This method is very useful, easy to use, accurate and sensitive
to measure biohydrogen, but requires equipment and specific
installations [53]. More expensive and relatively larger size
than other methods of biogas measurement are disadvan-
tages of GC [54,55]. GC is relatively bulky and needs high power
with a typical peak power requirement of 2000e3000 v-
ampere. The challenge of new GC is low power, develop
compact, low cost, and field-portable GC instruments [56].
The column of gas chromatographic can be considered the
heart of the GC, where the separation of sample components
takes place [50]. The columns of GC are classified into two
categories packed and capillary columns (Fig. 2) [57]. In dark
fermentation the packed columns of Molecular Sieve 5A
[29,58,59], Molecular Sieve 6A [60], Molecular Sieve 13X [4,61],
Porapak Q [62,63], Porapak N [64,65], Porapak TDS-01 [66,67],
HayeSep D [68,69], HayeSep Q [70,71], Unibeads C [72], GDX-
102 [73,74], TDX-01 [75,76], Unibeads C [77,78], Carboxen 1000
[79,80], Chromosorb 102 [81], Carbon Shin [82e84], Carbosive
SII [85], and CTR1 [24] and capillary columns of Carboxen 1010
PLOT [86,87], GS-CarbonPLOT [88,89], CarboPLOT P7 [90,91],
HP-PLOT Q [92,93], HP-PLOT U [94], HP-PLOT Molecular Sieve
[9], and HP-PLOT Molecular Sieve 5A [94] were employed to
analysis hydrogen gas. Micro-GC with columns of CP-Cox
[95,96] and CP Molecular Sieve 5A [97,98], HP-PLOT Molecular
Sieve 5A [37,99], Porapak N [44], and HP-PLOT U [99] was also
used to measure hydrogen gas in dark fermentation. The
working principle of micro GC is similar to conventional GC,
except that the different components are miniaturized in
micro GC to reduce power consumption, increase portability
and analysis speed [56]. Selection of carrier gas as the mobile
phase is another important issue that must be determined.
The carrier gas is an inert gas (helium) or unreactive gas
 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 4 5 ( 2 0 2 0 ) 2 4 4 2 4 e2 4 4 5 2 24427

Fig. 1 e Methods of biogas collecting (A: gasbags, B: helium balloon, C: syringes) [40,41].

Fig. 2 e Capillary column (A) and packed column (B) [119].

(nitrogen) [100]. The gases of nitrogen [101e104], argon
[60,105e108], and helium [109,110] were used as the carrier gas
in dark fermentation. The carrier gas flows through an
injector onto a column, which is in turn connected to a de-
tector [50]. The compounds are detected by the detector. Gas
chromatography is equipped with various detectors such as
thermal conductivity detector (TCD), flame ionization detector
(FID), helium ionization detector (HID), thermal ionization
detector (TID), photometric ionization detector (PID), catalytic
combustion detector (CCD), electron capture detector (ECD)
and so on. Each having its own advantages and limitations
[111]. The detectors of TCD [112] TCD and HID [113,114], and
TCD and FID [115,116] were used to detect hydrogen gas in
dark fermentation. The literatures show that TCD was widely
employed to detect hydrogen gas in dark fermentation. Most
analyses require the use of a temperature program. A tem-
perature program involves heating the oven at a controlled
rate during the run. Several temperature programs were used
in literatures to measure hydrogen gas in dark fermentative
hydrogen production. Ray et al. [82] measured biohydrogen
production by GC equipped with TCD with a column packed
with Carbon Shin. The nitrogen gas with 10 mL/min flow rate
was used as carrier gas. The operating temperatures of the
injector, detector, and oven were kept at 100, 200, and 200  C,
respectively. A gas chromatography equipped with a TCD and
1.5 m stainless-steel column packed with Molecular Sieve 5A
was used by Mu et al. [117]. Argon with 30 mL/min flow rate
was employed as carrier gas. The operating temperatures of
the injector, detector, and column were set at 100, 105, and
60  C, respectively. Sreela-or [77] employed a GC equipped
with a TCD and a 2 m stainless column packed with Unibeads
C to determine biogas. Argon was used as the carrier gas at a
flow rate of 25 mL/min. The operating temperatures of the
column, detector, and injector were 145, 150, and 150  C,
respectively. Biogas composition was measured by GC
equipped with a TCD with a stainless steel column packed
with Carbosive SII. The temperatures of the injector, detector,
and column were kept at 100, 105 and 60  C, respectively [85].
Zhao et al. [73] used a GC equipped with a TCD with a 2-m
stainless iron column packed with GDX-102 to determine
produced hydrogen. The temperatures of the injector, detec-
tor, and column were kept at 80, 100, and 50  C, respectively.
Nitrogen was employed as a carrier gas at a flow rate of 30 mL/
min. Biogas composition was analyzed using GC equipped
 24428
Table 1 e Measurement methods of biogas composition and volume in dark fermentative hydrogen production.
Microorganism Biogas composition Biogas composition Biogas volume and flow Substrate Ref.
measurement (CH4 and/or measurement (H2) measurement
CO2)
Seed sludge GCeTCD GC-TCD Water displacement Glucose [25]
Column: Porapak Q Column: Molecular Sieve 5A PLOT

Carrier: Helium Carrier: Argon

Clostridium sp. LS2 GCeTCD GCeTCD Water displacement POME [29]

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 4 5 ( 2 0 2 0 ) 2 4 4 2 4 e2 4 4 5 2
Column: Porapak T Column: Molecular Sieve

Carrier: Helium (30 mL/min) Carrier: Nitrogen (25 mL/min)

E.cloacae DM11 Remove CO2 by 30% (w/v) KOH GCeTCD Water displacement Glucose [62]
Column: Porapak Q

Carrier: Nitrogen (20 mL/min)

E. aerogenes Remove CO2 by NaOH GCeTCD Water displacement Xylose, arabinose, mannose, [63]
Column: Porapak Q galactose, and rhamnose

Carrier: Nitrogen (20 mL/min)

Mixed culture GCeTCD GCeTCD Water displacement Glucose [64]
Column: Porapak N Column: Porapak N
Mixed culture GCeTCD GCeTCD e Glucose [66]
Column: Porapak TDS-01 Column: Porapak TDS-01

Carrier: Nitrogen (70 mL/min) Carrier: Nitrogen (70 mL/min)

Enterobacter aerogenes ATCC 13048 GCeTCD GCeTCD e Glucose [32]
Column: Porapak Q Column: Molecular Sieve
Anaerobic sludge e H2 sensor e Glucose [28]
Enterobacter aerogenes MTCC 111 e H2 sensor Water displacement Glucose [144]
Anaerobic sludge CO2 sensor CH4 sensor H2 sensor e Glucose [175]
Mixed culture e H2 sensor e Chemical wastewater [176]
Mixed culture e H2 sensor e Chemical, domestic, and synthetic [170]
wastewater
Mixed culture e H2 sensor e Chemical wastewater [177]
Mixed culture H2 sensor e Wastewater [178]
Anaerobic sludge e H2 sensor Water displacement Glucose [167]
Anaerobic sludge CO2 sensor H2 sensor Water displacement Glucose [164]
CH4 sensor
Mixed culture e H2 sensor Water displacement Bean husk, corn stalk, and organic [166]
fraction of solid municipal waste
Mixed culture e H2 sensor e Chemical wastewater [168]
Mixed culture e H2 sensor e Dairy wastewater [179]
Mixed culture (soil and sludge) e H2 sensor e Glucose, untreated and pretreated [180]
soil and sludge
Clostridium Remove CO2 by KOH (30%) H2 sensor e Cheese whey [130]
saccharoperbutylacetonicum
Anaerobic sludge CO2 sensor H2 sensor and GCeTCD Gas flow meter and water Sewage biosolids [163]
CH4 sensor displacement
Anaerobic sludge e H2 sensor e Distillery wastewater [169]
Enterobacter MTCC 7104 e H2 sensor Water displacement Xylose [181]
E. aerogenes e H2 sensor Water displacement Rice mill wastewater hydrolysate [165]
Mixed culture GCeTCD GCeTCD Owen method Sucrose [4]
Column: HayeSep DB Column: Molecular Sieve13X
Carrier: Helium (140 mL/min)
Carrier: Nitrogen ( 75 mL/min)

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 4 5 ( 2 0 2 0 ) 2 4 4 2 4 e2 4 4 5 2
Mixed culture Remove CO2 by NaOH GCeTCD Owen and respirometric methods Glucose, molasses, sucrose, potato [22]
Column: Molesieve 5A starch, lactate, cellulose

Carrier: Nitrogen
Mixed culture GCeTCD GCeTCD Respirometer Glucose [127]
Column: Porapak Q Column: Molesieve 5A

Carrier: Hydrogen Carrier: Nitrogen

Mixed culture e GCeTCD Respirometer Domestic wastewater and food [173]
Column: Molesieve 5A processing wastewaters
Carrier: Nitrogen
Clostridium acetobutylicum ATCC 824 GCeTCD GCeTCD Respirometer Glucose [172]
Column: Porapak Q Column: Molesieve 5A

Carrier: Hydrogen Carrier: Nitrogen

Mixed culture GCeTCD GCeTCD e Cow manure [182]
Column: Porapak N Column: Porapak N
Carrier: Nitrogen Carrier: Nitrogen
E. aerogenes e GCeTCD Water displacement Glucose [6]
Column: Molesieve 5A
Carrier: Nitrogen
Mixed culture e GCeTCD Water displacement Acid hydrolyzed waste wheat [183]
Column: HayeSep D powder
Carrier: Nitrogen (30 mL/min)
Mixed culture GCeTCD GCeTCD Water displacement Pig slurry [184]
Column: Molesieve 5A and Porapak Column: Molesieve 5A and Porapak
Q Q
Carrier: Helium Carrier: Helium
E. aerogenes e GCeTCD Water displacement Glucose [7]
Column: Molesieve 5A
Carrier: Nitrogen
Anaerobic sludge e GCeTCD Water displacement Dairy wastewater [138]
Column: Molesieve 5A
Carrier: Nitrogen (25 mL/min)F
Mixed culture GCeTCD GCeTCD Water displacement Glucose [126]
Column: Porapak Q Column: Molesieve 5A PLOT
Carrier: Helium Carrier: Argon

24429
(continued on next page)
 24430
Table 1 e (continued )
Microorganism Biogas composition Biogas composition Biogas volume and flow Substrate Ref.
measurement (CH4 and/or measurement (H2) measurement
CO2)
Mixed culture e GCeTCD Water displacement Buffalo slurry, cheese whey, and [185]
crude glycerol
Sewage digester sludge GCeTCD GCeTCD Water displacement Glucose [70]
Column: HayeSep Q Column: HayeSep Q
Carrier: Argon (30 mL/min) Carrier: Argon (30 mL/min)

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 4 5 ( 2 0 2 0 ) 2 4 4 2 4 e2 4 4 5 2
Enterobacter aerogenes MTCC 2822 Remove CO2 by KOH (30%) GCeTCD and gas monitor Water displacement Cheese whey wastewater [129]
Vegetable waste e GCeTCD Water displacement Vegetable waste [186]
Column: HayeSep Q
Carrier: Nitrogen (35 mL/min)
Mixed culture GCeTCD GCeTCD Water displacement Glucose, lactose, cheese whey [118]
Column: HayeSep D Column: HayeSep D
Carrier: Nitrogen (12 mL/min) Carrier: Nitrogen (12 mL/min)
Anaerobic sludge e GCeTCD Water displacement Wheat powder [69]
Column: HayeSep D
Carrier: Nitrogen (30 mL/min)
Mixed culture e GCeTCD Water displacement Glucose [110]
Column: Molesieve 5A
Carrier: Helium (12 mL/min)
Mixed culture MicroGC -TCD MicroGCeTCD Water displacement Cattle manure, Slaughterhouse [44]
Column: Porapak N Column: Porapak N risk material
Carrier: Helium Carrier: Argon
Mixed culture e GCeTCD Water displacement Glucose [187]
Column: TDX-01
Carrier: Nitrogen (15.5 mL/min)
Mixed culture e GCeTCD Water displacement POME [38]
Column: HP- Molesieve
Carrier: Argon (45 mL/min)
Mixed culture and E. aerogenes GCeTCD GCeTCD Water displacement Glucose [188]
Column: Porapak Q Column: Porapak Q
Mixed culture e GCeTCD/HID Water displacement POME [114]
Column: Molecular Sieve 13X
Carrier: Nitrogen (30 mL/min)
Anaerobic sludge GCeTCD GCeTCD Water displacement Sewage sludge and fallen leaves [189]
Column: TDX-01 Column: TDX-01
Carrier: Argon Carrier: Argon
Anaerobic sludge GCeTCD GCeTCD Water displacement Sugar refinery wastewater [124]
Column: PoraPLOT Q Column: Molesieve 5A and
Carrier: Helium HayeSep Q
Carrier: Argon
Mixed culture e GCeTCD Water displacement Glucose [59]
Column: Molesieve 5A
Carrier: Helium (12 mL/min)
Clostridium sp. LS2
Thermoanaerobacterium aotearoense
SCUT27/Dldh
Enterobacter cloacae
Enterobacter aerogenes
GCeTCD GCeTCD Water displacement POME [121]
Column: Porapak T Column: Molesieve
Carrier: Helium (30 mL/min) Carrier: Nitrogen (25 mL/min)
GCeTCD/FID GCeTCD/ FID Water displacement Sugarcane bagasse [35]
Column: TDX-01 and AE electric Column: TDX-01 and AE electric
insulating oil analysis insulating oil analysis
e GCeTCD Water displacement Glucose and sucrose [190]
Column: Porapak Q
Carrier: Nitrogen (20 mL/min)
e Portable hydrogen gas analyzer Water displacement Glucose and glycerol [30]

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 4 5 ( 2 0 2 0 ) 2 4 4 2 4 e2 4 4 5 2
Enterobacter aerogenes HU-101 GCeTCD GCeTCD Water displacement Glucose [139]
Anaerobic sludge e GCeTCD Water displacement Glucose [191]
Column: Molesieve 5A
Carrier: Helium (12 mL/min)
Escherichia coli DhycA DlacI (WDHL) e GCeTCD Water displacement Cheese whey [53]
Column: HayeSep D
Carrier: Nitrogen (12 mL/min)
Mixed culture e GCeTCD Water displacement Starch [192]
Column: Molesieve
Carrier: Helium (25 mL/min)
Mixed culture e GCeTCD/HID Water displacement POME [193]
Carrier: Helium (25 mL/min)
Mixed culture e GCeTCD Water displacement POME [194]
Column: Porapak Q
Carrier: Nitrogen (30 mL/min)
Clostridium butyricum EB6 e GCeTCD Water displacement Glucose [31]
Column: Porapak Q
Carrier: Nitrogen
Mixed culture GCeTCD GCeTCD Water displacement Rice, oil, fat, lettuce, lean meat, [195]
Column: Porapak Q Column: Porapak Q potato, and banyan leaves
Carrier: Argon (30 mL/min) Carrier: Argon (30 mL/min)
Mixed culture e GCeTCD Water displacement Glucose [196]
Column: Molesieve 5A
Carrier: Helium (12 mL/min)
Mixed culture e GCeTCD Water displacement Glucose [197]
Column: Molesieve 5A
Carrier: Argon (30 mL/min)
Anaerobic sludge e GCeTCD Water displacement Brewery wastewater [8]
Column: Molesieve 5A
Anaerobic sludge e GCeTCD Water displacement Ozonated POME [72]
Column: Unibeads C
Carrier: Helium
Mixed culture e GCeTCD Water displacement Waste wheat powder [68]
Column: HayeSep D
Carrier: Nitrogen (30 mL/min)
(continued on next page)

24431
 24432
Table 1 e (continued )
Microorganism Biogas composition Biogas composition Biogas volume and flow Substrate Ref.
measurement (CH4 and/or measurement (H2) measurement
CO2)
Enterobacter aerogenes HU-101 GCeTCD GCeTCD Water displacement Glucose, fructose, glycerol, [198]
mannitol, sorbitol, gluconate, and
galactose
Mixed culture GCeTCD GCeTCD Water displacement Cheese whey [199]
Column: Porapak Q Column: Porapak Q

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 4 5 ( 2 0 2 0 ) 2 4 4 2 4 e2 4 4 5 2
Carrier: Nitrogen Carrier: Nitrogen
Anaerobic mixed culture e GCeTCD Water displacement Sucrose [200]
Column: Molecular Sieve 5A
Carrier: Argon (30 mL/min)
Enterobacter cloacae IIT-BT 08 Remove CO2 by 40% (w/v) KOH GCeTCD Water displacement Glucose [131]
Column: Porapak Q
Carrier: Nitrogen (20 mL/min)
Mixed culture e GCeTCD Water displacement Glycerol [71]
Column: HayeSep Q
Carrier: Nitrogen (30e35 mL/min)
Enterobacter aerogenes Remove CO2 by 20 M NaOH GCeTCD Water displacement Rice straw [201]
Column: Porapakk Q
Carrier: Nitrogen (20 mL/min)
Mixed culture e GCeTCD Water displacement Hydrolyzed wheat powder [202]
Column: HayeSep D
Carrier: Nitrogen (30 mL/min)
Enterobacter cloacae IIT-BT 08 Remove CO2 by KOH (40%) GCeTCD Water displacement and gas flow Starchy wastewater, cane [203]
Carrier: Nitrogen (20 mL/min) meter molasses, and distillery effluent
Mixed culture e GCeTCD Water displacement Glucose [141]
Column: HayeSep D
Carrier: Nitrogen (30 mL/min)
Mixed culture e GCeTCD Water displacement Hydrolyzed wheat powder [140]
Column: HayeSep D
Carrier: Nitrogen (30 mL/min)
Anaerobic sludge GCeTCD GCeTCD Water displacement Food waste [204]
Column: Porapak Q Column: Molecular Sieve 5A
Carrier: Helium Carrier: Nitrogen
Mixed culture e GCeTCD Water displacement Sucrose [205]
Column: Molecular Sieve 5A
Carrier: Argon (30 mL/min)
Anaerobic sludge e Water displacement Sucrose [117]
Anaerobic sludge GCeTCD GCeTCD Water displacement Food waste, potato pulp, cattle [206]
Column: TDX-01 Column: TDX-01 manure, and pig manure
Carrier: Argon (40 mL/min) Carrier: Argon (40 mL/min)
Anaerobic sludge GCeTCD GCeTCD Water displacement Sucrose [207]
Column: PoraPLOT Q Column: Molecular Sieve 5A and
Carrier: Helium (20.7 mL/min) HayeSep Q
Carrier: Argon (20.7 mL/min)
Anaerobic sludge GCeTCD GCeTCD Plunger displacement Sucrose, Food waste and non-fat [26]
Column: Porapak T Column: Porapak Q dry milk
Carrier: Helium (35 mL/min) Carrier: Nitrogen (30 mL/min)
Mixed culture e GCeTCD Plunger displacement Sucrose [146]
Column: Porapak Q
Carrier: Argon (30 mL/min)
Mixed culture GCeTCD GCeTCD Plunger displacement Vermicelli processing wastewater [208]
Column: Molesieve 5A Column: Molesieve 5A
Carrier: Argon for CH4 and Helium Carrier: Argon
for CO2

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 4 5 ( 2 0 2 0 ) 2 4 4 2 4 e2 4 4 5 2
Anaerobic sludge e GCeTCD Plunger displacement Organic wastes [209]
Column: Molesieve 5A
Carrier: Argon
Mixed culture e GCeTCD Plunger displacement Molasses-based distillery [210]
Column: Molesieve wastewater
Carrier: Nitrogen (25 mL/min)
Mixed culture e GCeTCD Plunger displacement Glucose [73]
Column: GDX-102
Carrier: Nitrogen (30 mL/min)
Anaerobic sludge e GCeTCD Plunger displacement Sucrose and digestion sludge [102]
Column: Porapak Q
Carrier: Nitrogen (30 mL/min)
Anaerobic sludge e GCeTCD Plunger displacement Melon, papaya, pineapple, sewage [211]
Column: Molesieve sludge
Carrier: Nitrogen (20 mL/min)
Mixed culture GCeTCD GCeTCD Plunger displacement Starch and sucrose [61]
Column: Porapak T Column: Molesieve 13X
Carrier: Helium (35 mL/min) Carrier: Nitrogen (30 mL/min)
Anaerobic sludge e GCeTCD Plunger displacement Food and beverage processing [212]
Column: Molesieve 5A wastewater
Carrier: Argon
Mixed culture GCeTCD GCeTCD Plunger displacement Fruit and vegetable waste [213]
Column: HP-PLOT Q Column: HP-PLOT Q
Carrier: Helium Carrier: Helium
Anaerobic sludge GCeTCD GCeTCD Oil volume displacement Agro industrial wastewaters [214]
Column: HP-PLOT Q Column: HP-PLOT Q (Liquid cow manure, cheese whey,
Carrier: Nitrogen Carrier: Nitrogen olive mill wastewater)
Anaerobic sludge e GCeTCD Gas meter Glucose [107]
Anaerobic sludge e GCeTCD Gas meter Sucrose [215]
Column: Porapak Q
Carrier: Argon
Anaerobic sludge e GCeTCD Gas meter Sucrose [153]
Column: Porapak Q
Carrier: Argon
(continued on next page)

24433
 24434
Table 1 e (continued )
Microorganism Biogas composition Biogas composition Biogas volume and flow Substrate Ref.
measurement (CH4 and/or measurement (H2) measurement
CO2)
Anaerobic sludge e GCeTCD Gas meter Sucrose [154]
Column: Porapak Q
Carrier: Argon
Thermotoga neapolitana e GCeTCD Gas meter Xylose [216]
Column: Carboxen 1000

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 4 5 ( 2 0 2 0 ) 2 4 4 2 4 e2 4 4 5 2
Carrier: Nitrogen (55 mL/min)
Anaerobic sludge MicroGC -TCD MicroGC -TCD Wet gas meter Glucose [217]
Column: Porapak Q Column: Molsieve 5A PLOT
Carrier: Helium Carrier: Argon
Mixed anaerobic culture e GCeTCD Gas meter Glucose [218]
Column: Porapak Q
Carrier: Argon
Sewage sludge e GCeTCD Gas meter Sucrose [219]
Column: Porapak Q
Carrier: Argon
Anaerobic sludge GCeTCD GCeTCD Wet gas meter Steam-exploded switchgrass [220]
Column: Carbon Shin Column: Carbon Shin liquor
Carrier: Nitrogen (10 mL/min) Carrier: Nitrogen (10 mL/min)
Anaerobic sludge GCeTCD GCeTCD Gas meter Sucrose [89]
Column: GS-CarbonPLOT Column: GS-CarbonPLOT
Carrier: Argon (3.67 mL/min) Carrier: Argon (3.67 mL/min)
Anaerobic sludge GCeTCD GCeTCD Gas meter Sucrose [88]
Column: GS-CarbonPLOT Column: GS-CarbonPLOT
Carrier: Argon (3.67 mL/min) Carrier: Argon (3.67 mL/min)
Anaerobic sludge GCeTCD GCeTCD Gas meter Glucose [125]
Column: Carboxen 1010 PLOT Column: Carboxen 1010 PLOT
Anaerobic sludge e GCeTCD Gas meter Glucose [221]
Column: Carboxen 1010 PLOT
Carrier: Argon
Anaerobic sludge GCeTCD GCeTCD Gas flow meter Chess whey [222]
Column: GS-Carbon Column: GS-CarbonPLOT
Carrier: Argon (3.67 mL/min) Carrier: Argon (3.67 mL/min)
Mixed culture e GCeTCD Gas flow meter Citric acid wastewater [76]
Column: TDX-01
Carrier: Argon (30 mL/min)
Clostridium butyricum GCeTCD GCeTCD Gas flow meter Glucose [91]
Column: Carboplot P7 Column: Carboplot P7
Carrier: Helium Carrier: Nitrogen
Anaerobic sludge e GCeTCD Wet gas meter Sucrose [223]
Column: HayeSep D
Carrier: Argon (20 mL/min)
Anaerobic sludge e GCeTCD Gas meter Sucrose [224]
Column: Molecular Sieve 5A
Carrier: Argon (30 mL/min)
Mixed culture e GCeTCD Gas meter Glucose [225]
Carrier: Argon
Mixed culture e GCeTCD Gas meter Starch [226]
Column: Column: Porapak Q
Carrier: Argon
Mixed culture GCeTCD GCeTCD Gas meter Corn stover [227]
Column: Carbon Shin Column: Carbon Shin

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 4 5 ( 2 0 2 0 ) 2 4 4 2 4 e2 4 4 5 2
Carrier: Nitrogen (25 mL/min) Carrier: Nitrogen (25 mL/min)
Mixed culture GCeTCD GCeTCD Gas meter Glucose [228]
Column: Porapak Q Column: Porapak Q
Mixed culture e GCeTCD Gas meter Cheese whey [87]
Column: Carboxen 1010 PLOT
Carrier: Argon
Anaerobic sludge GCeTCD GCeTCD Gas flow meter Glucose [123]
Column: TDX-01 Column: TDX-01
Clostridium butyricum GCeTCD GCeTCD Gas flow meter Glucose [103]
Carrier: Argon Carrier: Nitrogen
Clostridium butyricum GCeTCD GCeTCD Wet gas meter Glucose, xylose, fructose, [33]
Column: Molecular Sieve Column: Molecular Sieve 5A, galactose, and mannose
5A,Porapak Q, and Porapak N Porapak Q, andPorapak N
Carrier: Helium Carrier: Helium
Anaerobic sludge GCeTCD GCeTCD Gas meter Glucose [229]
Column: TDX-01 Column: TDX-01
Carrier: Nitrogen (50 mL/min) Carrier: Nitrogen (50 mL/min)
Seed sludge GCeTCD GCeTCD Gas meter Cassava starch [230]
Mixed culture GCeTCD GCeTCD Wet gas meter Galactose [231]
Carrier: Nitrogen Carrier: Nitrogen
Seed sludge Remove CO2 by NaOH (3e5%) GCeTCD Wet gas meter Molasses [128]
Column: Porapak Q
Carrier: Nitrogen (40 mL/min)
Mixed culture GCeTCD GCeTCD Wet gas meter Glucose [232]
Column: Porapak T Column: Porapak Q
Carrier: Helium (40 mL/min) Carrier: Nitrogen (40 mL/min)
Mixed culture e GCeTCD Wet gas meter Glucose and peptone [233]
Column: HayeSep Q
Carrier: Nitrogen (15 mL/min)
Seed sludge GCeTCD GCeTCD Wet gas meter Glucose [122]
Column: HayeSep Q Column: Molecular Sieve 5A
Carrier: Argon (30 mL/min) Carrier: Argon (30 mL/min)
Seed sludge GCeTCD GCeTCD Gas meter Sucrose [58]
Column: Molecular Sieve 5A Column: Molecular Sieve 5A
Carrier: Argon (30 mL/min) Carrier: Argon (30 mL/min)
Seed sludge GCeTCD GCeTCD Wet gas meter Molasses [27]
Column: Porapak Q Column: Porapak Q

24435
Carrier: Nitrogen (40 mL/min) Carrier: Nitrogen (40 mL/min)
(continued on next page)
 24436
Table 1 e (continued )
Microorganism Biogas composition Biogas composition Biogas volume and flow Substrate Ref.
measurement (CH4 and/or measurement (H2) measurement
CO2)
Anaerobic seed sludge e GCeTCD Gas flow meter Sucrose [234]
Column: Porapak Q
Carrier: Argon
Clostridium thermolacticum MicroGCeTCD MicroGCeTCD Gas flow meter Lactose [96]
Column: CP-Cox Column: CP-Cox

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 4 5 ( 2 0 2 0 ) 2 4 4 2 4 e2 4 4 5 2
Carrier: Argon Carrier: Argon
Anaerobic sludge e GCeTCD Wet gas flow meter Glucose [155]
Column: Porapak Q
Carrier: Nitrogen
Anaerobic sludge e GCeTCD Wet gas meter Condensed molasses [235]
Column: Porapak Q
Carrier: Argon
Anaerobic granular sludge GCeTCD GCeTCD Wet gas meter Galactose [236]
Mixed culture GCeTCD GCeTCD Wet gas meter Dairy manure [237]
Column: Porapak Q Column: Porapak Q
Carrier: Nitrogen (20 mL/min) Carrier: Nitrogen (20 mL/min)
Anaerobic sludge GCeTCD GCeTCD Wet gas meter Starch [43]
Column: Porapak Q Column: Porapak Q
Carrier: Helium (30 mL/min) Carrier: Nitrogen (30 mL/ min)
Mixed culture GCeTCD GCeTCD Gas meter Soft-drink wastewater [238]
Anaerobic sludge e GCeTCD Gas meter Sucrose [106]
Column: Molecular Sieve 5A
Carrier: Argon(30 mL/min)
E. coli (XL1-BLUE) GCeTCD GCeTCD Gas meter Formate [109]
Column: CarbonPLOT Column: Molecular Sieve 13X
Carrier: Nitrogen Carrier: Helium
Mixed culture GCeTCD/HID GCeTCD/HID Gas meter POME [239]
Carrier: Helium (25 mL/min) Carrier: Helium (25 mL/min)
Cow dung compost e GCeTCD/HID e Sucrose [101]
Column: Porapak Q
Carrier: Nitrogen (20 mL/min)
Mixed culture MicroGCeTCD MicroGCeTCD e Swine manure and fruit and [37]
For CO2; Column: Molecular Sieve 5A PLOT vegetable market waste
Column: HP- PLOT U Carrier: Nitrogen (30 mL/min)
Carrier: Helium (30 mL/min)
For CH4 ;
Column: Molecular Sieve 5A PLOT
Carrier: Nitrogen (30 mL/min)
Clostridium e GCeTCD e Glucose [79]
Pasteurianu Column: Carboxen- 1000
Carrier: Helium
Ethanoligenens harbinense B49 e GCeTCD e Glucose [67]
Column: Porapak TDS-01
Carrier: Nitrogen (70 mL/min)
Clostridium beijerinckii NCIMB8052 e GCeTCD e Glucose [60]
Column: Molecular Sieve 6A
Carrier: Argon (2 mL/min)
Mixed culture GCeTCD GCeTCD e Food and blood waste [105]
Column: HayeSep Q and Molecular Column: HayeSep Q and Molecular
Sieve Sieve
Carrier: Argon Carrier: Argon
Escherichia coli e GCeTCD e Glucose [108]
Anaerobic sludge GCeTCD GCeTCD e Glucose [240]
Column: Porapak Q Column: Porapak Q

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 4 5 ( 2 0 2 0 ) 2 4 4 2 4 e2 4 4 5 2
Carrier: Nitrogen (30 mL/min) Carrier: Nitrogen (30 mL/min)
Anaerobic sludge GCeTCD GCeTCD e Glucose [241]
Column: Porapak Q Column: Porapak Q
Carrier: Nitrogen (30 mL/min) Carrier: Nitrogen (30 mL/min)
Anaerobic sludge e GCeTCD e Biodiesel waste and pure glycerol [94]
Column: HP-PLOT U
Carrier: Helium
and
Column: HP-PLOT Molecular Sieve
5A
Carrier: Nitrogen
Anaerobic sludge GCeTCD Wetted glass syringe Food waste [77]
Column: Unibeads C
Carrier: Argon (25 mL/min)
Mixed culture e GCeTCD Wetted glass syringe Waste glycerol and sludge [242]
Column: Unibeads C
Carrier: Argon (25 mL/min)
Mixed culture e GCeTCD Wetted glass syringe Pineapple waste extract [78]
Column: Unibeads C
Carrier: Argon (25 mL/min)
Anaerobic sludge GCeTCD GCeTCD Wetted glass syringe (a 500 mL Food waste [243]
Column: Porapak Q Column: Molecular Sieve 5A syringe)
Carrier: Nitrogen Carrier: Nitrogen
Anaerobic sludge e GCeTCD Wetted glass syringe Sweet sorghum syrup [244]
Column: Unibeads C
Carrier: Argon (25 mL/min)
Thermoanaerobacterium e GCeTCD Wetted glass syringe Sweet sorghum bagasse [83]
thermosaccharolyticum Column: Carbon Shin
KKU19
Thermoanaerobacterium e GCeTCD Wetted glass syringe (20e50 mL) Xylose and arabinose [245]
thermosaccharolyticum KKU-ED1 Column: Carbon Shin
Carrier: Helium (25 mL/min)
Mixed culture e GCeTCD Wetted glass syringe (10e100 mL) Glucose [74]
Column: GDX-102
Carrier: Nitrogen (30 mL/min)
Buffalo dung compost e GCeTCD Glass syringes (5-50 mL) Glucose and xylose [246]
Column: Porapak Q

24437
Carrier: Nitrogen (20 mL/min)
(continued on next page)

Table 1 e (continued )
Microorganism
Mixed culture
Mixed culture
Clostridium termitidis
Anaerobic mixed culture
Caldicellulosiruptor owensensis
Anaerobic sludge
Mixed culture
Clostridial species
24438
Biogas composition Biogas composition Biogas volume and flow Substrate Ref.
measurement (CH4 and/or measurement (H2) measurement
CO2)
e GCeTCD Glass syringes (5e100 mL) Thin stillage [247]
Column: Molecular Sieve 5A
Carrier: Argon (30 mL/min)
GCeTCD GCeTCD Glass syringes (5e50 mL) Microcrystalline cellulose [248]
Column: Porapak T Column: Porapak Q
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 4 5 ( 2 0 2 0 ) 2 4 4 2 4 e2 4 4 5 2
Carrier: Nitrogen (30 mL/min) Carrier: Nitrogen (30 mL/min)
e GCeTCD Glass syringes (5e100 mL) Glucose and cellobiose [249]
Column: Molecular Sieve 5A
Carrier: Argon (30 mL/min)
GCeTCD Glass syringes (5-50 mL) Cornstalk wastes [250]
Column: Porapak Q
Carrier: Nitrogen (20 mL/min)F
MicroGCeTCD MicroGCeTCD e Glucose and xylose [97]
Column: PoraPLOT Q Column: Molecular Sieve 5A
Carrier: Helium Carrier: Nitrogen
GCeTCD GCeTCD e Starch [251]
Column: Porapak Q Column: Porapak Q
Carrier: Helium (20 mL/min) Carrier: Helium (20 mL/min)
e GCeTCD/HID e Glucose and starch [113]
Carrier: Helium (25 mL/min)
GCeTCD GCeTCD Owen method Glucose [252]
Column: HayeSep Q Column: HayeSep Q
Carrier: Nitrogen (15 mL/min) Carrier: Nitrogen (15 mL/min)
 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 4 5 ( 2 0 2 0 ) 2 4 4 2 4 e2 4 4 5 2 24439

Table 2 e Manometric method to measure hydrogen in dark fermentation.

Microorganism Substrate Biogas composition Biogas volume Ref.
measurement (H2) measurement
E. coli (DJT 135) Format GC-TCD Manometer [253]
Column: Molecular Sieve
Carrier: Helium
Thermotoga maritima, Glucose GC-TCD Manometer [24]
Thermococcus kodakarensis Column:CTR1
KOD1 and Enterobacter Carrier: Argon (20 mL/min)
cloacae
Clostridium butyricum TM-9A Glucose GC-TCD Mechanical pressure gauge [93]
Column: HP-PLOT Q
Carrier: Nitrogen (2.8 mL/
min)
Anaerobic sludge Food waste GC-TCD Pressure gauge [23]
Column: HayeSep DB
Carrier: Argon (30 mL/min)
Anaerobic sludge Sewage sludge and sugar beet pulp GC-TCD Pressure transducer [254]
Column: Carboxen 1010
PLOT
Anaerobic sludge Sewage sludge and wine vinasse GC-TCD Pressure transducer [255]
Column: Carboxen 1010
PLOT
Anaerobic sludge Sucrose GC-TCD Pressure transducer [17]
Anaerobic sludge Lard GC-TCD Pressure transducer [13]
Column: HayeSep DB
Carrier: Argon (30 mL/min)
Activated sludge Food waste GC-TCD Pressure transducer [256]
Column: Molecular Sieve

with a TCD and a column packed by HayeSep D. Nitrogen at a
flow fate of 12 mL/min was used as carrier gas. The operating
temperatures of the injection port, detector, and oven were set
at 250, 250, and 60  C, respectively [118]. Mandal et al. [62]
employed gas chromatography equipped with a TCD and a
column packed with Porapak Q to analyze biogas. Nitrogen
was used as carrier gas at a flow fate of 20 mL/min. The
temperatures of the injector, detector, and oven were kept at
150, 200, and 80  C, respectively. The determination of pro-
duced hydrogen from glucose and starch by mixed culture
was performed using GC equipped with detectors of TCD and
HID. Helium gas at a flow fate of 25 mL/min was used as
carrier gas. The temperatures of the injector, detector, and
oven were held at 40, 220, and 40  C, respectively [113]. The
produced biohydrogen from sugarcane bagasse by Clostridum
thermocellum DSM1313 was measured with GC with detectors
of TCD and FID [115]. A gas chromatography equipped with
detector of TCD and capillary columns of HP-PLOT U and HP-
PLOT Molecular Sieve 5A was used to measure hydrogen gas.
The gases of helium and nitrogen were employed as carrier

Fig. 3 e Water displacement method [55]. Fig. 4 e Commercial gas meter [55].
24440 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 4 5 ( 2 0 2 0 ) 2 4 4 2 4 e2 4 4 5 2

Fig. 5 e Commercial hydrogen sensor [171].

gas for the columns of HP-PLOT U and HP-PLOT Molecular (30%) [129,130], and KOH (40%) [131] in a CO2 absorber and
Sieve 5A, respectively [94]. Table 1 indicates GC equipped with remaining gas was then measured with GC. The sodium hy-
thermal conductivity detector using nitrogen gas as carrier gas droxide (NaOH), potassium hydroxide (KOH), and calcium
and column of Molecular Sieve 5 A was extensively employed hydroxide (Ca(OH)2) were widely employed for CO2 scrubbing
for quantitative measurement of hydrogen gas. and their reactions with CO2 are shown in Eqs. (1)e(3). KOH is
GC is the best analytical device to analyze the different more expensive than NaOH, however, the K2CO3 formation
components of the biogas including methane, carbon dioxide, (Eq. (2)) has several industrial applications [132].
etc. [120]. In dark fermentative hydrogen production, the col-
umns of HayeSep DB [4], Porapak Q [62], Porapak N [64], Por- 2NaOH (aq) þ CO2 (g) / Na2CO3 (aq) þ H2O (l) (1)
apak T [121], Porapak TDS-01 [66], GS-CarbonPLOT [88],
CarboPLOT P7 [91], CP- Molecular Sieve 5A [98], HayeSep Q
[122], TDX-01 [123], and PoraPLOT Q [124] were employed to
analyze methane. The columns of HayeSep DB [4], HP-PLOT U
[37], HP-Molecular Sieve [38], Porapak N [64], GS-CarbonPLOT
[88], CarboPLOT P7 [91], CP-PoraPLOT Q [97], CP-PoraPLOT U
[98], Porapak T [121], HayeSep Q [122], TDX-01 [123], PoraPLOT
Q [124], Carboxen 1010 PLOT [125], and Porapak Q [126] were
used to measure carbon dioxide in dark fermentation. The
gases of nitrogen [27,109], argon [70,96], hydrogen [127], and
helium [4,124] were used as carrier gas. In some researches,
dioxide carbon gas was removed by NaOH [6,7,63,128], KOH

Fig. 6 e Commercial respirometer [174]. Fig. 7 e Manometric cap [253].

 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 4 5 ( 2 0 2 0 ) 2 4 4 2 4 e2 4 4 5 2 24441

More expensive, high power

Not suitable for high biogas

than other methods, errors

Labour-intensive, diffusion

Short lifetime, poisoned by

injection of the gas sample

evaporation, and corrosion

Not suitable for low biogas
2KOH (aq) þ CO2 (g) / K2CO3 (aq) þ H2O (l) (2)

and relatively large size

other gases or material

of biogas in the barrier
Disadvantage

during sampling and

Ca(OH)2 (aq) þ CO2 (g) / CaCO3 (aq) þ H2O (l) (3)

solutions, liquid

flow and rate

flow and rate

As indicated in Table 1, GCeTCD using a column of Porapak
Q and helium gas as the carrier gas was widely employed for
quantitative measurement of methane and dioxide carbon
gases. There is much research available in the literature on GC
to measure hydrogen gas in dark fermentation which some of
them are reported in Table 1.

Volume displacement devices (water displacement and

plunger displacement method)

The volume displacement devices such as water displacement

[25] and plunger displacement [26] were extensively used for
Simple, easy to use, smaller
Simple, easy to use, robust,

the measurement of biogas volume [133]. The water

Useful, accurate, sensitive,
and needing small sample

Simple, durable, accurate,

allow significant pressure

Quick and easy, accurate,
user-friendly precise, not
size than other methods

displacement devices are employed for laboratory-based vol-

ume measurement and are usually height or weight types [16].
Advantage

The general working principle of volume displacement in-

struments is the difference of pressure between the inlet and
inexpensive

easy to use

outlet of the device which causes the periodic filling and

build-up

emptying a defined volume of gas in the measurement vessel

[16]. These devices are robust, easy to set up and use, inex-
pensive, and capable of working for long periods without
maintenance [134]. Since water is almost incompressible, the
volume flow rate of water leaving the chamber is equal to the
gas volume flow rate entering it [135]. The produced biogas
inside the bioreactor moves into an external reservoir, con-
taining a barrier solution and displaces an equivalent volume
of liquid, that can be manually or automatically measured
[14]. These devices can be constructed with materials of sim-
ple and inexpensive such as plastic or glass jars or cylinders
[16]. The gases solubility in water and liquid evaporation are
limitations of this method [136]. Acidified saturated solutions,
Composition of biogas
Application

alkaline solutions, and saline solutions can be employed as

Volume of biogas

Volume of biogas

Volume of biogas

barrier solution in all types of liquid displacement devices to

Composition of

minimize solubility of the gases [134]. The manual water

biohydrogen

displacement method is labour-intensive. On the contrary,

the automatic water displacement device is less labour-
Table 3 e Methods of hydrogen gas measurement.

intensive but has problems such as corrosion, algae growth,

and complexity in managing CO2 solubility and control of
reservoir levels [137].
In dark fermentation the method of water displacement
with acidic water [29,30], NaOH solution [138], aqueous NaCl
[139], sulfuric acid (2%) and NaCl (10%) [68,140,141], and
NaHCO3 [33] was used to avoid CO2 absorption. As carbon di-
oxide is very polar and its solubility in water is high [16], the
absorption of carbon dioxide into water produces carbonic
acid according to Eq. (4) and reduces pH of water. The carbonic
acid may lose protons to form bicarbonate (HCO1 3 ) and car-
bonate (CO23 ) [142]. Therefore, the absorption of carbon di-
Gas chromatography

Water displacement

oxide into water causes an error in determining the volume of

Hydrogen sensor

the produced biogas.

Respirometer
Gas meter

CO2 (g) þ H2O (l) / H2CO3 (aq)

Method

(4)
24442 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 4 5 ( 2 0 2 0 ) 2 4 4 2 4 e2 4 4 5 2
Plunger displacement method or wetted glass syringe
method or Owen method was introduced by Owen et al. [143].
This type of device is employed for laboratory-based volume
measurement and was also used for dark fermentation. There is
a large number of research work available in the literature on
volume displacement devices for dark fermentation that a few
are reported in Table 1. A schematic of the water displacement
method is shown in Fig. 3. Generally, this method follows by gas
chromatography or hydrogen sensor to measure the amount of
generated hydrogen gas. For example, hydrogen production
from glucose by Enterobacter aerogenes was investigated in
250 mL conical flask by Karthic et al. [144] and the water
displacement method was used to measure the volume of
hydrogen gas. Water was adjusted to pH 3 or less in order to
prevent dissolution of the biogas. Amount of hydrogen gas was
then estimated using an electrochemical sensor. The hydrogen
production from corn stalk by Xing et al. [145] was investigated.
The method displacement of saturated brine was employed to
measure the volume of biogas at regular interval and GC-TCD
was used to measure the concentration of hydrogen in biogas.
Dark fermentative hydrogen production from artificial waste-
water was investigated by Zhang et al. [146] in 1 L completed
stirred tank reactor (CSTR). Plunger displacement method fol-
lowed by GC was used to estimate volume and composition of
hydrogen gas.
Commercial gas meter
The diffusion of biogas in the barrier solutions is a disadvan-
tage of the water displacement method which the designed
gas meter overcomes this problem. Gas meter as an instru-
ment for the measurement of gases is simple, easy to use,
accurate, repeatable, and durable [16] and for measurement of
hydrogen gas flow was employed. Gas meters can be used for
large-scale commercial applications [134]. The ideal gas meter
must be affordable, accurate, be able to measure a wide flow
range, capable of operation at low gauge pressures, data-
logging, need little maintenance, and produce insignificant
pressure variations [147]. The gas meter classified into two
types of dry and wet and two uses of commercial and exper-
imental [148]. The dry and wet gas meter are similar in oper-
ation, but in a dry gas meter, the water seal is replaced by a
sliding valve seal [149]. Wet gas meters are heavy. Under
suitable conditions, wet gas meters are very accurate, partic-
ularly at low flow rates. Dry gas meters are more convenient
but have lower accurate than wet gas meters [150]. In exper-
iments of laboratory scale, the biogas production is usually
low, often below 5 mL/min [151], and commercial gas flow
meters are usually not suitable to measure small amounts of
biogas with low flow rates [11]. To solve this problem gas flow
meters were designed and devised. The gas meters with an
adjustable resolution meter, minimal back-pressure and wide
flow rate capability were developed [133,147,152].
Both wet- and dry-type commercial gas meters were
employed for biohydrogen measurement. However, the dry-
type gas meter was used more than the wet-type gas meter.
Beckers et al. [91] used two digital flow gas meters to monitor
the continuous biohydrogen where flow meters were placed in
series for the validation of the results. The experiments of
hydrogen production were performed by Clostridium butyricum
CWBI1009 and glucose monohydrate as the substrate in a 2.5 L
AnSBR. The percentage of hydrogen in biogas was then
measured using a GC equipped with TCD and CarboPLOT P7
column and nitrogen as the carrier gas. Amount of generated
hydrogen through dark fermentation of glucose in an anaerobic
fluidized bed bioreactor with expanded clay as support was
investigated by Amorim et al. [107]. They used a gas meter
follow by GC-TCD to measure rate and fraction of produced
hydrogen gas. A wet gas meter was employed to record daily
produced biohydrogen by anaerobic digested sludge. The gas
characterization was carried out by a GC equipped with TCD
and columns packed with Porapak Q and Porapak T [43]. There
is a lot of literature available (Table 1) on commercial gas me-
ters for dark fermentative hydrogen production. The commer-
cial gas meter brands of Ritter Inc. (Germany) [107,153,154],
Changchun Filter Co., Ltd. (China) [27], Far Asia Co. Ltd. (China)
[128], Mettler Toledo (Switzerland) [123], Shanghai A.K. In-
struments Co., Ltd. (China) [33], Toshniwal Instruments Pvt.
Ltd, (India) [155], and Shinagawa Co. (Japan) [122] were used in
dark fermentation. A schematic of the commercial gas meter is
shown in Fig. 4.
Hydrogen sensor
A sensor is an electronic instrument designed to measure and
monitor changes in the environment, convert those changes
to electrical signals and communicate that information to
other electronic instruments to compute and analyze [156].
Hydrogen sensor is transducer devices that detect hydrogen
gas and has advantages such as lower cost, smaller size, and
fast response when compared with conventional methods of
hydrogen detection. There are different types of hydrogen
sensors include catalytic, mechanical, optical, electro-
chemical, metal oxide, thermal conductivity, etc. [54]. Each
type of hydrogen sensor has advantages and disadvantages in
terms of performance. The performance parameters of sensor
types are measuring range, sensitivity, selectivity, stability,
accuracy, and response time [157]. The poor selectivity, high
power consumption, poisoned by other gases or material,
short lifetime, and slow response time can limit the perfor-
mance of sensors. A suitable sensor must be low cost, low
maintenance, fast response time, easy to install and use, and
accurate. Also choice a sensor is according to ambient work-
ing conditions and detection requirements and sensor per-
formance capabilities [158,159]. The measurement sensor
should not inhibit the biogas production [160]. Many types of
hydrogen sensors based on various mechanisms to detect
hydrogen are available in the market. The sensors of hydrogen
are employed in applications of hydrogen production, storage,
transportation, and use [161]. As hydrogen is colorless, odor-
less, tasteless, and explosive, industry often used hydrogen
sensors to detect leakage of hydrogen. Hydrogen sensors are
applicable in many industries such as petroleum, petro-
chemical, medical diagnostics, fuel cells, nuclear power
plants, etc. The challenge of new hydrogen sensors is the
improvement of performance and reduction of costs [162].
Biological hydrogen production from primary sewage bio-
solids was investigated in 5 and 10 L bioreactors by Massanet-
Nicolau et al. [163]. Both bioreactors were equipment by H2
sensor, CH4 sensor, and CO2 sensor to measure biogas
 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 4 5 ( 2 0 2 0 ) 2 4 4 2 4 e2 4 4 5 2
continuously. A custom monitoring program written by the
LabView programming package was used for logging of sensor
data. In the mentioned research, when sparging was carried
out, sensors and online gas flow were not used to measure
gases and water displacement method and gas chromatog-
raphy were employed instead of those. Because during
sparging, sensors confine the gas flow out of bioreactor and
cause a build-up of pressure in the bioreactor. Faloye et al.
[164] investigated optimum conditions for biohydrogen pro-
duction from glucose by mixed culture. The composition of
biogas was determined by H2 sensor, CH4 sensor, and CO2
sensor with measuring ranges of (0e100 vol%), (0e100 vol%),
(0e50 vol%), respectively. Sensors were interfaced to the Flab-
Biogas software. The volume of produced hydrogen gas was
measured with the water displacement method. The
hydrogen production from rice mill wastewater by Enterobacter
aerogenes was monitored using a hydrogen sensor. The vol-
ume of produced hydrogen gas was measured with the
method of water displacement [165]. More researches about
using hydrogen sensors in dark fermentation were reported in
Table 1. The commercial hydrogen sensor is shown in Fig. 5.
The commercial sensor brands of Bluesens (Germany)
[28,166,167], ATMI GmBH Inc. (Germany) [168e170], and H2
Scan (USA) [165] were used in dark fermentation.
Respirometer
The respirometric method is purpose-designed to automatically
present accurate instantaneous measurements of the production
of gas on a continuous basis in increments as small as. The
method is precise and also has advantageous as it does not allow
significant pressure build-up. Anaerobic respirometer can be
overcome limits such as labour-intensive and time-consuming
and save considerable test time [21,24]. The volume of head-
space can be reduced to as little as 10 mL, thereby minimizing
changes in gas composition due to dilution of a large volume of
headspace gas. This feature allows the attachment of carbon di-
oxide adsorption devices in the gas line. Anaerobic respirometers
indicate a significant improvement to measure the production of
gas accurately for small bench-scale bioreactors [21].
Oh et al. [127] used respirometric method to measure the vol-
ume of produced biohydrogen from glucose. Then, the composi-
tion of hydrogen in biogas was determined by GC equipped with
TCD and a column packed with Molesieve 5A and nitrogen gas as
the carrier gas. Logan et al. [22] studied the production of bio-
hydrogen from glucose either by Owen method or respirometric
method. The results showed that the respirometric method pro-
duces 43% hydrogen gas more than the Owen method. The bio-
hydrogen production from glucose by Clostridium acetobutylicum
ATCC 824 was investigated by Oh et al. [172]. A Respirometer was
used to measure the volume of produced biohydrogen and a GC-
TCD equipped with a column of Molesieve 5A and nitrogen as
the carrier was employed to determine the amount of bio-
hydrogen. A commercial respirometer is shown in Fig. 6. The res-
pirometers in dark fermentation were not extensively used and
application is limited by performed works by Logan et al.
[22,127,172,173]. The commercial respirometer brand of Challenge
Technology Inc. (USA) [22] was used in dark fermentation.
24443
Manometric method
The manometric method as an alternative to the liquid
displacement method commonly was employed when dealing
with low volumes of gas production [134]. Pressure measure-
ment devices such as manometer [24,253], pressure gauge
[93,254], and pressure transducers [13,17,255,256] were used in
dark fermentation (Table 2). Bakonyi et al. [253] employed a
manometer to investigate dark fermentation hydrogen pro-
duction by E. coli. The bioreactor was sealed by a special mano-
metric cap (Fig. 7) which was able to register the increasing
pressure of the biogas. The composition of biogas was investi-
gated by GC equipped with TCD and helium gas as the carrier
gas. Gannoun et al. [24] investigated hydrogen production by
Thermotoga maritima, Thermococcus kodakarensis KOD1, and
Enterobacter cloacae. A gas pressure manometer for measurement
total pressure in the headspace of 116 mL serum bottles was
used. The main compounds in the headspace of serum bottles
were carbon dioxide, hydrogen, and water vapor. The compo-
sition of biogas was determined by GC-TCD, CTR1 column, and
argon gas at flow rate of 20 mL/min as carrier gas. A mechanical
pressure gauge was used by Junghare et al. [93] for measurement
of biogas pressure. The composition of produced biogas from
glucose by Clostridium butyricum TM-9A was measured with GC-
TCD equipped with column of HP-PLOT Q and nitrogen at flow
rate of 2.8 mL/min as carrier. Montanes et al. [255] and Tena et al.
[256] used a pressure transducer to monitor the pressure and a
GC-TCD equipped with Carboxen 1010 PLOT column to deter-
mine composition of biogas. The pressure of biogas in the
headspace of bioreactor was measured using a pressure trans-
ducer. Then biogas in headspace was released and pressure of
biogas in the headspace was measured again. The difference of
pressure was converted into volume of biogas with the ideal gas
law under the standard conditions [13,254].
Conclusion
The biogas measurement is carried out with manometric and
volumetric devices by manual and automatic methods. The
volumetric methods are most commonly used in dark
fermentation to determine the volume and flow of hydrogen.
Both manual and automatic methods are used to measure
biological hydrogen production. The manual methods are
labour-intensive, however, they are simple, inexpensive, and
easy to use. The generated gas during the fermentation pro-
cess can be collected into a gasbag or helium balloon on-line
and its volume can be measured using a gas meter, water or
plunger displacement method, respirometer and manometric
method. For measurement of hydrogen fraction in the biogas
gas chromatography and hydrogen sensor were employed.
Each method has advantages and limitations (Table 3). The
water displacement method and gas meter were extensively
used to measure volume and flow of biogas and gas chroma-
tography was widely employed to measure the composition of
produced biohydrogen in laboratory scale. Using these
methods together is accurate and sensitive to determine
hydrogen gas.
24444 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 4 5 ( 2 0 2 0 ) 2 4 4 2 4 e2 4 4 5 2
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
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