Thermophilic biohydrogen production using pre-treated algal biomass as substrate
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Thermophilic biohydrogen production usingpre-treated algal biomass as substrate
Shantonu Roy, Kanhaiya Kumar, Supratim Ghosh, Debabrata Das*
Department of Biotechnology, Indian Institute of Technology, Kharagpur 721302, India
a r t i c l e i n f o
Article history:
Received 19 August 2013
Received in revised form
5 December 2013
Accepted 8 December 2013
Available online 2 January 2014
Keywords:
Algal biomass
Pretreatment methods
Biohydrogen production
Thermophilic mixed culture
Gompertz equation
* Corresponding author. Tel.: þ91 3222 83758E-mail addresses: [email protected]
0961-9534/$ e see front matter ª 2013 Elsevhttp://dx.doi.org/10.1016/j.biombioe.2013.12.
a b s t r a c t
Algal biomass is rich in carbohydrates which can be utilized as a promising source of
substrate for dark fermentation. It becomes more significant when biomass is produced by
capturing atmospheric greenhouse gas, CO2. In the present study, clean energy was
generated in the form of biohydrogen utilizing algal biomass. Biohydrogen production was
carried out by thermophilic dark fermentation using mixed culture. The culture of Chlorella
sorokiniana was cultivated in helical airlift photobioreactor at 30 �C under continuous light
intensity of 120 mmol m�2 s�1 provided by white fluorescent lamps. Biomass reached to
stationary phase on 9th day giving maximum dry cell weight of 2.9 kg m�3. Maximum
carbohydrate and protein content observed was 145 g kg�1 and 140 g kg�1, respectively.
Maximum volumetric productivity of 334 g dm�3 d�1 was observed. Algal biomass was
subjected to various physical and chemical pre-treatments processes for the improvement
of hydrogen production. It was observed that the pretreatment with 200 dm3 m�3 HCl-heat
was most suitable pretreatment method producing cumulative hydrogen of 1.93 m3 m�3
and hydrogen yield of 958 dm3 kg�1 volatile suspended solid or 2.68 mol mol�1 of hexose.
Growth kinetics parameters such as mmax and Ks were estimated to be 0.44 h�1 and
120 g m�3, respectively. The relationship between biomass and hydrogen production was
simulated by the LuedekingePiret model showing that H2 production is growth associated.
The study thus showed the potential of algal biomass as substrate for biological hydrogen
production.
ª 2013 Elsevier Ltd. All rights reserved.
1. Introduction
The recent exponential increase in worldwide energy demand
caused depletion of energy reserves at greater pace. The
combustion of fossil fuels has serious negative effects on
environment because of CO2 emission. Algal biomass culti-
vation is gaining importance in recent times as they can
capture atmospheric CO2 and can produce carbohydrates rich
biomass which can be used for production of biofuels [1].
Unlike other crops such as corn or soybeans, algae can use
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various water sources ranging from wastewater to brackish
water and can be grown in small, intensive plots on denuded
land. Hydrogen from algae is possible by two biological pro-
cesses. The first is the biophotolysis involving light-driven
splitting of the water [1,2]. Hydrogen production by bio-
photolysis had been extensively studied on Chlamydomonas
reinhardtii or Anabaena variabilis [3]. Secondly, dark fermenta-
tion of biomass utilizing carbohydrates present in algal cells
using thermophilic and mesophilic hydrogen producing bac-
teria. Thermophilic dark fermentation shows favorable ther-
modynamics of reaction and with reduced risk of
n (D. Das)..
b i om a s s a n d b i o e n e r g y 6 1 ( 2 0 1 4 ) 1 5 7e1 6 6158
methanogenic contamination, higher rate of hydrolysis and
higher hydrogen yields [4]. Algal biomass seems to be a po-
tential feedstock source for biofuel production because they
have higher growth rate, rich carbohydrate content and sim-
ple to harvest. Different processes are available for the pro-
duction of fuels from algae [5] which includes anaerobic
digestion of microalgae biomass to produce methane [6].
Moreover, direct extraction of fuel oils in the form of biodiesel
that remains accumulated in certain microalgae [7]. Micro-
algae can also be used to produce hydrogen photosyntheti-
cally by direct or indirect photolysis of water [8]. At this point,
there is one report on biohydrogen production from thermo-
philic dark fermentation using biomass of Chlorella vulgaris [9].
In this study, different pretreatments improved hydrogen
production. Different physico-chemical pretreatments were
employed to increase the accessibility of different complex
sugars entrapped in algal biomass into simpler form. Carbo-
hydrates in algal biomass are found as intracellular complex
polymeric form bounded with rigid algal cell walls [10,11].
Therefore, it is necessary to break the algal cell wall alongwith
complex carbohydrate to facilitate the release of simple sugar.
Cost of pretreatment of biomass adds significantly to overall
hydrogen production process. Several methods of pre-
treatments such as physical (sonication, milling, grinding,
pyrolysis), chemical (acid, alkali, thermal, H2O2) and biological
methods (enzymatic, microbial) have been reported to break
algal cell wall, hydrolyze the complex carbohydrates and
release fermentable sugars. Each of the method has its own
merits and demerits [12]. Physical methods are based on
simpler technology but they are energy intensive processes
limiting their use for commercial application. Biological based
methods are costly and time consuming with low hydrolysis
rate. Preference of chemical method such as acid treatment
over others is because of higher conversion efficiency of
polymeric carbohydrates into simpler sugars in lesser time
[10,13]. Some reports are available on use of algal biomass as
substrate after pretreatment for hydrogen production using
mesophilic microorganisms [14]. In a study, algal biomasswas
used to produce H2 using Clostridium butyricum and subse-
quent use of produced organic acids for H2 production by
photo fermentation using Rhodobacter sphaeroides KD 131 [14].
Surface chemistry of biomass changes with different
pretreatments. X-ray diffraction (XRD) has been used to study
the changes in crystallinity with respect to different
pretreatment processes [15]. Effect of acid and enzymatic
pretreatment on surface chemistry of cornstalk for
improvement of biohydrogen productionwas studies by using
X-ray diffraction [16].
Similarly, fermentation of algal biomass by amixed culture
of lactic acid bacterium Lactobacillus amylovorus and photo-
synthetic bacterium Rhodobium marinum was used in the
single-step process to convert algal starch to H2 [11].
Monod model and Luedeking-Piret unstructured models
were used for the determination of the kinetic parameters.
These models allow us to relate microbial growth rate and
product formation in an aqueous environment to the con-
centration of a limiting nutrient. These kinetic parameters
helps us to understand the growth characteristics of the
microorganism, design the bioprocess based experiment and
scaling up the process.
Thus, the present study aimed to investigate the effect of
different pretreatment methods on saccharification of com-
plex carbohydrates present in algal biomass; Chlorella sor-
okiniana. Cell morphology was studied to investigate the
extent cell wall rupture using microscopic and X-ray diffrac-
tion (XRD) with respect to different pretreatment methods.
Optimized concentration of pretreated algal biomass was
further used as carbon source in H2 production medium for
thermophilic mixed culture. Attempts were also made to
determine the kinetics of hydrogen production using con-
ventional Monod, Luedeking-Piret and modified Gompertz
equations.
2. Materials and methods
2.1. Cultivation and harvesting of microalgae
The culture of C. sorokiniana was cultivated in helical airlift
photobioreactor at 30 �C under continuous light intensity of
120 mmol m�2 s�1 provided by white fluorescent lamps. The
helical photobioreactor was constructed of four parts: i) the
helical photostage; ii) the gas riser; iii) the degasser and iv) the
downstream tube. The construction material used was plex-
iglass (polymethacrylate). The helical photostage had tube
dimensions of 2.0 cm ID and 3.0 cm OD wound on a 15.0 cm
imaginary cylinder with a gap of 1.0 cm between the spirals. It
had 8 spirals in total with a height of 32.0 cm. The gas riser
consisted of a T-shaped joint which had a top diameter of
8.0 cm and a bottom diameter of 2.0 cm. The degasser was
cylindrical in shape with a height of 12.0 cm. The downflow
tube connected the bottom of the degasser with the end of the
helix via a T-piecewhich also served as a sampling port. Inside
the reactor, air sparging had a space velocity of 0.33 min�1 for
proper circulation andmixing of the culture. The C. sorokiniana
was grown in mTAP [-acetate] medium which was prepared
by substituting the nitrogen source NH4Cl by 1.5 kg m�3 of
NaNO3 [17]. Algal biomass was harvested at stationary phase
and centrifuged at 4053� g RCF for 5min. It was washed thrice
with distilled water and further air dried to get a powdered
algal biomass.
2.2. Microbial consortium
An enriched thermophilic mixed culture capable of producing
hydrogen was used in this study. Media used for this study
consist of Na2HPO4 (4.2 kg m�3), KH2PO4 (1.5 kg m�3), NH4Cl
(1.95 kg m�3), MgCl2 (0.18 kg m�3), yeast extract (2.0 kg m�3),
glucose (10 kg m�3), cysteine HCl (1 kg m�3), vitamins solution
(DSMZ medium No141, German Collection of Microorganisms
and Cell Cultures). Thismixed culture derived from a distillery
industry anaerobic digester [18] had predominantly Thermoa-
naerobacterium sp. genus and was mostly related to Thermoa-
naerobacterium thermosaccharolyticum which have hydrogen
production ability.
2.3. Algal biomass pretreatment
Dried algal biomass with initial concentration of 14 kg m�3 was
subjected to physical and chemical pre-treatments such as
b i om a s s a n d b i o e n e r g y 6 1 ( 2 0 1 4 ) 1 5 7e1 6 6 159
HCl�heat, H2O2, autoclaving, sonication etc prior to be used as
substrate for fermentation.Such typeofpretreatment facilitates
availability of simpler sugar frommorecomplexpolymeric form
of carbohydrates present in algal biomass [9]. In heat-HCl pre-
treatmentmethod, dried algal biomass wasmixedwith various
concentration of HCl from 50 dm3 m�3 to 250 dm3 m�3 and
autoclaved at 121 �C, 204.9 kPa for 20 min. Entire hydrolyzates
was neutralized with 0.4 g m�3 NaOH. In H2O2 treatment
method, dried algal biomass was mixed with 2% H2O2 for over-
night and then autoclaved at 121 �C, 204.9 kPa for 20 min. Son-
ication was performed at 50% of amplitude for 10 min (130 W,
SonicVibraCellUltrasonicProcessor). Theentire sonicatedalgal
biomass containing solubilizedcomplex carbohydratewasused
as substrate to prepare H2 production medium.
2.4. Hydrogen production in batch fermentation
Hydrogen production in batch with optimized pretreated algal
biomass was carried out in double jacketed bioreactor under
thermophilic condition. The volume of the bioreactor was
300 cm3 with working volume of 250 cm3. Temperature was
controlled at 60 �C by passing water in the outer jacket of the
bioreactor.
2.5. Analytical procedure
The amount of H2 was quantitatively measured by gas chro-
matography (GC, Agilent Technologies, USA) with a thermal
conductivity detector (TCD) with N2 as the carrier gas. The
column used for gas determination was a stainless column (1/
8 inch 15 ft) packed with 50/80 mesh Carboxen 1000 (Supelco)
[18].
Total sugar was estimated using Phenol sulfuric method as
described by Loewus [19]. The pH values were monitored
using a desktop pH meter (pH510, Cyberscan, Singapore). The
COD was measured according to APHA standard methods [20]
using a COD measurement instrument set (DRB200 & DR2800
Portable Spectrophotometer, HACH, USA).
The crystallinity index (CrI) of the algal biomass was
measured by XRD using an X-ray powder diffractometer
(Bruker Axs: D8 focus) with Co Ka radiation at 40 kV and
200mA. The CrI of the sampleswere calculated using thewide
angle X-ray diffraction counts at 2q angle close to 22� and 18�
according to the Segal empirical method [21,22].
CrI ¼ I22 � I18I22
� 100% (1)
where I22 is the peak intensity of the crystalline material
(2q ¼ 22�) and I18 is the peak intensity of the amorphous ma-
terial (2q ¼ 18�).
2.6. Determination of kinetic constants
The Monod model was used for determining the growth ki-
netics as follows [23]:
m ¼ mmaxSSþ Ks
(2)
where m (h�1) is the specific growth rate; mmax (h�1) is the
maximum specific growth rate respectively; Ks (g m�3) is the
substrate affinity coefficient; and S (g m�3) is the substrate,
concentration.
Linearization of Eq. (2) gives LineweavereBurk plot as
shown in Eq. (3):
1m¼ 1
mmax
þ Ks
mmax
1S
(3)
The relationship between biomass and the products for the
anaerobic hydrogen production by mixed anaerobic cultures
could be simulated by the LuedekingePiret model:
dPdt
¼ adxdt
þ bx (4)
The first term of Eq. (4), i.e., adX/dt, is referred as the
growth-associated product formation rate. This implies that
the growing cells produce the product in constant proportion
of their growth. Non-growth associated product formation
term (bX) shows that all the microorganisms produce the
product in a constant proportion of their concentration,
regardless of the growth phase. Eq. (4) could be changed into:
Qp ¼ dPxdt
¼ amþ b (5)
where dP/xdt is the specific product formation rate of product
(Qp) and m is the specific growth rate of the microorganisms. A
straight line could be obtained with an intercept of b and a
slope of a, if plotting Qp against m.
The modified Gompertz equation was used to fit cumula-
tive hydrogen production in the batch experiment performed
under optimized process parameters.
HðtÞ ¼ P exp
�� exp
�Rm
Pðl� tÞ þ 1
��(6)
where H (t) is the cumulative hydrogen production at time t
(h). The kinetic constants of modified Gompertz equation P
(mL H2), Rm (cm�3 h�1) and l (h) represent hydrogen produc-
tion potential, maximum hydrogen production rate and
hydrogen production lag time, respectively. Parameters (P, Rm
and l) were estimated using the solver function in Matlab
(Curve fitting tool box ver. 1.1.7.) [24].
3. Result and discussions
3.1. Production of C. sorokiniana biomass in helicalbioreactor
Growth curve, carbohydrate, protein and chlorophyll content
of C. sorokiniana was shown in Fig. 1.
The growth rate of C. sorokiniana was 0.28 d�1 which was
slower than the growth rate found in tubular airlift reactor
(0.48 d�1) [17]. Biomass reached to stationary phase on 9th day
giving maximum dry cell weight of 2.9 kg m�3. As the cells
reached stationary state, the carbohydrate and protein con-
tent also became constant. Maximum carbohydrate and pro-
tein content observed was 145 g kg�1 and 140 g kg�1
respectively. Volumetric productivity of 334 kg m�3 d�1 was
observedusing this helical reactorwhichwas comparablewith
the productivities that have been reported for different reactor
configurations. Volumetric productivity of 400e700 kgm�3 d�1
Fig. 1 e Profiles of growth, carbohydrate, protein and chlorophyll content for C. sorokiniana in helical airlift photobioreactor.
b i om a s s a n d b i o e n e r g y 6 1 ( 2 0 1 4 ) 1 5 7e1 6 6160
and 370 kgm�3 d�1was reported in shallowponds and outdoor
tubular reactors, respectively [25]. With Arthrospira platensis,
an indoor tubular coiled bioreactor reported productivity
values of 510 kg m�3 d�1 [26]. Where light could be one of the
limiting factors, comparing different bioreactor configurations
was difficult as each configuration have different surface areas
and illumination. The helical tubular design has certain ad-
vantages like it has a large surface-to-volume ratio, easy con-
trol on mass transfer property of gases and occupies a small
ground area thus considered as a convenient setup for indoor
pilot plant operation [27].
Fig. 2 e Hydrogen production profile in different
pretreatment process of algal biomass. a) Autoclaved
biomass raw algal biomass. b) Sonicated biomass. c)
200 dm3 mL3 HCl treatment. d) 50 dm3 mL3 HCl treatment.
e) H2O2 treatment f) Raw algal biomass. Process conditions
are pH 6.5, algal biomass concentration 14 kg mL3 and
temperature of 60 �C. Fermentation time was of 24 h.
3.2. Effect of different pre-treatment methods onhydrogen production
Different pretreatment methods used in this study resulted in
improvedhydrogenproductionas compared tountreatedalgal
biomass. Acid and heat treatment converts complex carbo-
hydrates to simpler sugar. Under the condition of HCl-heat
pretreatment, algal starch was hydrolyzed to different soluble
sugars mainly consisting of dextrin, cellobiose and glucose at
various contents depending on the used HCl concentration.
Dextrin was only presented in the acid hydrolytic solution of
algal biomass treated by 50 dm3 m�3 HCl-heat [9]. With using
the 200 dm3 m�3 HCl concentration for algal biomass treat-
ment, the algal starch was almost converted to glucose with
the 100% of glucose conversion efficiency. Similarly
200 dm3 m�3 H2O2 treatment generates nascent oxygen that
breaks the glycosidic bondspresent in complex carbohydrates.
Fig. 2 shows effect of different pretreatments for improvement
of hydrogen production and also degree of saccharification.
The 200 dm3 m�3 HCl-heat treatment gave highest
hydrogen production of 1.33 m3 m�3 with reducing sugar
concentration of 9.6 kg m�3 among the other pretreatment
methods. The probable reason could be that this pretreatment
could have resulted inmaximumconversion of complex sugar
to hexose. Pre-treatment with physical methods also
improved hydrogen production. Pretreatment of algal biomass
by sonication gave better hydrogen production of 0.42 m3 m�3
than autoclaved biomass 0.100 m3 m�3. This could be due to
smaller-sized starch molecules generated by sonication was
easier and more suitable to convert to H2 than pure starch.
Moreover, reducing sugar concentration in sonicated algal
biomasswas 4.8 kgm�3 was higher as compared to autoclaved
biomass extract. Moreover, autoclaved biomass gave
improved hydrogen production than raw biomass. Probable
reason of this could be the solubilization of some of the nu-
trients present in algal biomass under autoclaving condition.
3.3. Study of effect of pretreatment on algal biomass
3.3.1. Microscopic study of microalgal biomass disruptionPre-treatment processes impart distortion of cellular
morphology and integrity. Microalgae consist of different
types of carbohydrate which are mostly stored in the cell wall
[28]. Therefore disruption of the microalgae cell walls is
necessary to release the entrapped carbohydrates for use as a
Fig. 3 e Microscopic observation (403) of effect of different pretreatment on hydrogen production. a) Raw algal biomass. b)
Pretreatment of algae by 50 dm3 mL3 HCl-heat c) Pretreatment of algae by 200 dm3 mL3 HCl-heat d) Pretreatment of algae by
H2O2 e) Pretreatment of algae by autoclave. f) Pretreatment of algae by sonication.
b i om a s s a n d b i o e n e r g y 6 1 ( 2 0 1 4 ) 1 5 7e1 6 6 161
carbon source during the fermentation process. In this study,
acid pre-treatment was used for microalgae cell wall disrup-
tion. Different concentrations of hydrochloric acid were
interacted with the microalgae cells and microscopic analysis
was performed to examine any morphological change after
different pre-treatment processes was shown in Fig. 3.
The image before pre-treatment shows that the cells were
intact, mostly spherical in shape and clumped together
(Fig. 3a). This suggests that the carbohydrates are intracellular
entrapped. On the other hand, the microscopic images of the
pre-treated microalgae with different concentration of HCl
shows ruptured cell wall evidenced by the broken cells. With
200 dm3 m�3 HCl treatment showed greater cell debris for-
mation than 50 dm3 m�3 HCl as shown in Fig. 3b and c. This
shows the greater efficacy of higher concentration hydro-
chloric acid for cell wall disruption. Similar observations was
Fig. 4 e XRD patterns of different pretreated algal biomass.
also made by Razhi et al., where algal biomass gave clumped
ruptured morphology when treated with HCl for ethanol pro-
duction [12]. The change from intact cells to broken cells after
the acid pre-treatment process confirms the disruption of the
microalgae cell wall to release entrapped carbohydrates.
Treatment with 200 dm3 m�3 H2O2 gave lesser cell debris for-
mation (Fig. 3d). Nascent oxygen released from H2O2 oxidizes
the chlorophyll content of the cell along with other proteins
whichwere required formaintaining the integrity of cell. Thus
transparent cells were observed with H2O2 treatment. Sur-
prisingly, lesser cell debris formedwith physical pretreatment
viz. autoclaving and sonication (Fig. 3e and f). A large number
of small and transparent particles can be observed.
3.3.2. XRD (X-ray diffraction) analysisFermentation has been greatly influenced by the crystallinity
of the biomass as it governs the ease of hydrolysis. The
biomass composition significantly influences the CrI value
[29]. For algal biomass, the CrI measures the relative amount
of crystalline starch in the total solid.
The X-ray diffraction pattern of the algal biomass treated
under above mentioned pretreatments was shown in Fig. 4,
from which the CrI was summarized in Table 1.
Table 1 e Crystallinity index of pretreatedalgal biomass.
Sample CrI (%)
Autoclaved biomass 38.69158
Sonication 33.11965
200 dm3 m�3 HCl 3.80818
200 dm3 m�3 H2O2 24.26778
Table 2 e Kinetic parameters with various pretreatmentmethods.
Differentsubstrates
l
(h)Rm
(m3 m�3 h�1)P
(m3)H2 yield
on VS (dm3 kg�1)
Starch 1.17 0.1242 1.583 833
200 dm3 m�3
HCl-heat treated
algal biomass
1.05 0.3353 2.155 958
50 dm3 m�3
HCl treated algal
biomass
1.26 0.121 1.520 760
H2O2 treated algal
biomass
3.2 0.1120 0.180 63
Autoclaved algal
biomass
2.2 0.540 0.650 338
b i om a s s a n d b i o e n e r g y 6 1 ( 2 0 1 4 ) 1 5 7e1 6 6162
As shown in Table 1, the CrI value of 38.69 for autoclaved
algal biomass was the highest suggesting that the higher
crystallinity of starch as compared to other samples. The CrI
value of 10.38 was lowest for 200 dm3 m�3 HCl treated
biomass. The probable reason for such observation could be
due to complete hydrolysis of starchy component of algal
Fig. 5 e a) Hydrogen production profile in different initial biom
Process conditions are pH 6.5 and temperature of 60 �C. Fermen
fermented pretreated algal biomass.
biomass under acidic conditions. Another pretreatment that
showed decrease in CrI values is H2O2 treatment. Sonication
didn’t influenced CrI value much. The CrI value of 33.11 was
observed for sonicated biomasswas comparable to autoclaved
biomass. This could suggest that sonication didn’t change the
crystallinity of the starchy components of the algae. The re-
sults from the above seemed to suggest that there was a
profound effect of different pretreatment methods on the
crystalline structure of the biomass. Similar observation was
also made by Xing et al., in which corn stock was treated
enzymatically to improve hydrogen production [30]. They
showed similar decrement of crystallinity index of corn stock
under enzymatic pretreatment.
3.4. Improvement of hydrogen production with differentpretreatment methods
The three kinetic parameters were studied (Table 2) along
with H2 yield and maximum specific H2 production rate.
Result shows that lag time was least in the case of
200 dm3 m�3 HCl treated biomass than other pretreatments.
ass concentration pretreated with 200 dm3 mL3 HCl (v/v).
tation time was of 24 h b) Volatile fatty acid profile of
b i om a s s a n d b i o e n e r g y 6 1 ( 2 0 1 4 ) 1 5 7e1 6 6 163
Moreover highest hydrogen production potential of
2.15m3m�3 and rateofhydrogenproductionof 0.34m3m�3 h�1
was also observed in 200 dm3 m�3 HCl treated biomass. Prob-
able reason for higher hydrogenproduction could be due to the
greater availability of fermentable sugar in this pretreatment.
When compared to pure starch, the lag time (l) for fermenta-
tion of 200 dm3 m�3 HCl treated biomass wasmore. This could
be due to presence of some inhibitory components like furfu-
rals that were generated during pretreatment processes.
Pretreatment with H2O2 gave very less amount of hydrogen.
This could be due to excessive oxidation of the fermentable
sugars present algal biomass. Autoclaved biomass gave better
Fig. 6 e a) Batch fermentation using 50 dm3 mL3 HCl-heat treat
LineweavereBurk plot. c) Product kinetics study using Leudekin
hydrogenproduction thanH2O2 pretreatment. On autoclaving,
sugarsget solubilized thus improveshydrogenproduction.The
kinetic parameters were calculated from Gompertz model by
curve fitting with R2 value greater than 0.99.
3.5. Effect of pretreated initial biomass concentration onhydrogen production
After optimizing the pretreatment processes, 200 dm3 m�3
HCl-heat treatment was found to be most suitable amongst
the other pretreatment methods. The effect of microalgae
loading on the acid pre-treatment process was also studied.
ed algal biomass. b) Growth kinetics study by using
g Piret model.
Table 3 e Comparative study on hydrogen yield by the thermophilic mixed culture.
Organism used Substrate Operationtemperature/pH
Volumetric hydrogenproduction on VS (dm3 kg�1)
References
Mixed culture Starchy wastewater 55 �C/7.0 365 [35]
Mixed culture Cassava starch 55 �C/7.0 249.3a [36]
Mixed culture Cellulosic wastewater 55 �C/5.5 102a [37]
Mixed culture Glycerol 60 �C/5.5 34.0 [38]
Mixed culture Wheat straw (untreated) 55 �C/8.0 24.5a [39]
Thermotoga neoplanita Algal biomass 60 �C/6.5 2.4b [7]
Mixed culture Algal biomass 60 �C/6.5 172 [40]
Mixed culture Algal biomass 60 �C/6.5 958
2.51bThis study
a dm3 kg�1 substrate.b mol mol�1 of glucose.
b i om a s s a n d b i o e n e r g y 6 1 ( 2 0 1 4 ) 1 5 7e1 6 6164
Thus with 200 dm3 m�3 HCl-heat treatment, different
initial biomass concentrations were used for thermophilic
biohydrogen production (Fig. 5a). Initial algal biomass was
varied from 8 kg m�3 to 16 kg m�3. At biomass concentration
of 14 kg m�3 biomass concentration gave highest cumulative
hydrogen production of 1.50 m3 m�3. Fermentation was car-
ried out for 24 h at 60 �C. A decrease in hydrogen production
beyond 14 kgm�3 was observed. Highermicroalgae loading up
to 16 kg m�3 resulted in lower hydrogen production. Although
higher concentrations of biomass was expected to release
higher amounts of fermentable sugars during the pre-
treatment process, it has been reported that higher biomass
concentrations reduces hydrogen production levels due to
substrate inhibition, metabolic burden and accumulation of
inhibitory substances like furfurals [31]. The volatile fatty
acids (VFAs) production is always associated with H2 pro-
duction [32]. In this experiment, the acetate and butyrate
accounted for 70e80% of total VFA concentration and rest was
ethanol (Fig. 5b). More or less same amounts of all three
components of VFAs were produced during H2 production
with acid pretreated cornstalk waste [33]. With increase in
initial biomass concentration butyrate and acetate concen-
tration increased. It has been reported that butyrate to acetate
(B to A) ratios are directly proportional to H2 yields [34].
Highest butyrate to acetate ratio was observed during
fermentation of 14 kg m�3 pretreated initial biomass.
3.6. Batch fermentation under optimized processparameters
Fermentation was performed with 200 dm3 m�3 HCl�heat
pretreated algal biomass having initial biomass concentration
of 14 kg m�3 for 24 h.
Cumulative hydrogen production, substrate consumption,
pH and dry cell weight profile are shown in Fig. 6a. Hydrogen
production ceased after 20th hour of fermentation. Total cu-
mulative hydrogen production and hydrogen yield were
1.93 m3 m�3 and 2.68 � 0.2 mol mol�1 of hexose, respectively.
Substrate conversion efficiency of 62% was observed.
Maximum dry cell weight and final pH at the end of the
fermentationwere found to be 1.9 kgm�3 and 4.2, respectively.
The pH of the system may be decreased due to production of
volatile fattyacidsas fermentationof reducible sugaroccurred.
Table 3 shows a comparative study on hydrogen produc-
tion with algal biomass.
Hydrogen yield obtained in this study was 958 dm3 kg�1 of
volatile suspended solid which was higher than the report of
365 dm3 kg�1 of volatile suspended solid with starchy waste-
water at thermophilic condition [41]. In another study,
hydrogen yield of 9 mol kg�1 COD reduced was observed with
pretreated algal biomass as substrate using Enterobacter cloacae
IIT-BT 08 under mesophilic conditions [10]. Thus algal digest
could be a promising substrate for hydrogen production.
3.7. Kinetic analysis
Plotting m�1 against S�1, a straight line was obtained with an
intercept of m�1max and a slope of Ks m
�1max. This plot was shown in
Fig. 6b, from which mmax and Ks were estimated was 0.44 h�1
and 120 g m�3, respectively. The regression line had a corre-
lation coefficient of 0.96, suggesting the applicability of Eq. (3).
The Ks value represents the substrate concentration required
to achieve 50% of the maximum specific growth rate.
The hydrogen production kinetics was shown in Fig. 6c for
hydrogen. The high correlation coefficients (R2 ¼ 0.98) indicate
that the LuedekingePiret model could properly describe the
relationship between biomass and product in the anaerobic
hydrogen production process. The estimated values of a and b
were 17 (mmol g�1 dry cell weight) and 0.726, respectively
indicating the dependence of product formation and biomass
concentration. These values could be used to predict the
productivity in the bioreactor design. Since b values were near
to zero the formation of hydrogen in the anaerobic hydrogen
production was growth associated [42].
4. Conclusion
Algal biomass of C. sorokiniana was used in dark fermentation
for H2 production using thermophilic mixed consortia.
Different physical and chemical pretreatments methods were
applied to rupture the algal cell walls, hydrolyze intracellular
complex carbohydrates and to facilitate the release of
fermentable sugars. HCl (200 dm3 m�3)-heat was found the
most suitable pretreatment method. Hydrogen production
was studied in batch in double jacketed bioreactor with opti-
mized concentration of pretreated algal biomass. Microscopic
and XRD analysis confirmed the rupture of cell walls neces-
sary for the release of fermentable sugars. Monod, Luedeking-
Piret equations were used to determine the hydrogen
b i om a s s a n d b i o e n e r g y 6 1 ( 2 0 1 4 ) 1 5 7e1 6 6 165
production kinetics. Hydrogen production was found to be
growth associated. Modified Gompertz equation was found to
reasonably fit with the experimental hydrogen production
data.
Acknowledgment
The authors gratefully acknowledge Council of Scientific and
Industrial Research (CSIR), Govt. of India for fellowship; IIT
Kharagpur and Ministry of New and Renewable Energy
(MNRE), Govt. of India for the financial support.
r e f e r e n c e s
[1] Nayak BK, Pandit S, Das D. Biohydrogen chapter 15. In:Kennes C, Veiga MC, editors. Air pollution prevention andcontrol: bioreactors and bioenergy. Chichester: John Wiley;2013. pp. 345e82.
[2] Melis A, Happe T. Hydrogen production. Green algae as asource of energy. Plant Physiol 2001;127(3):740e8.
[3] Levin DB, Pitt L, Love M. Biohydrogen production: prospectsand limitations to practical application. Int J Hydrogen Energ2004;29(2):173e85.
[4] Roy S, Vishnuvardhan M, Das D. Improvement of hydrogenproduction by newly isolated Thermoanaerobacteriumthermosaccharolyticum IIT BT-ST1. Int J Hydrogen Energ.http://dx.doi.org/10.1016/j.ijhydene.2013.06.128; 2013.
[5] Kosaric N, Velikonja J. Liquid and gaseous fuels frombiotechnology: challenge and opportunities. FEMS MicrobiolRev 1995;16(2):111e42.
[6] Markou G, Angelidaki I, Georgakakis D. Carbohydrate-enriched cyanobacterial biomass as feedstock for bio-methane production through anaerobic digestion. Fuel2013;111:872e9.
[7] Sawayama S, Inoue S, Dote Y, Yokoyama S-Y. CO2 fixationand oil production through microalga. Energy ConversManag 1995;36(6):729e31.
[8] Melis A, Zhang L, Forestier M, Ghirardi ML, Siebert M.Sustained photobiological hydrogen gas production uponreversible inactivation of oxygen evolution in the green algaChlamydomonas reinhardtii. Plant Physiol 2000;122(1):127e35.
[9] Nguyen TAD, Kim KR, Nguyen MT, Kim MS, Kim D, Sim SJ.Enhancement of fermentative hydrogen production fromgreen algal biomass of Thermotoga neapolitana by variouspretreatment methods. Int J Hydrogen Energy2010;35(23):13035e40.
[10] Kumar K, Roy S, Das D. Continuous mode of carbon dioxidesequestration by C. sorokiniana and subsequent use of itsbiomass for hydrogen production by E. cloacae IIT-BT 08.Bioresour Technol 2013;145:116e22.
[11] Kawaguchi H, Hashimoto K, Hirata K, Miyamoto K. H2production from algal biomass by a mixed culture ofRhodobium marinum A-501 and Lactobacillus amylovorus. JBiosci Bioeng 2001;91(3):277e82.
[12] Harun R, Danquah MK. Influence of acid pre-treatment onmicroalgal biomass for bioethanol production. ProcessBiochem 2011;46(1):304e9.
[13] Nguyen MT, Choi SP, Lee J, Lee JH, Sim SJ. Hydrothermal acidpretreatment of Chlamydomonas reinhardtii biomass forethanol production. J Microbiol Biotechnol 2009;19(2):161e6.
[14] Kim MS, Baek JS, Yun YS, Jun Sim S, Park S, Kim SC.Hydrogen production from Chlamydomonas reinhardtiibiomass using a two-step conversion process: anaerobic
conversion and photosynthetic fermentation. Int J HydrogenEnergy 2006;31(6):812e6.
[15] Zhao X, Chen J, Chen F, Wang X, Zhu Q, Ao Q. Surfacecharacterization of corn stalk superfine powder studied byFTIR and XRD. Colloids Surf B Biointerfaces2013;104:207e12.
[16] Pan CM, Ma HC, Fan YT, Hou HW. Bioaugmented cellulosichydrogen production from cornstalk by integrating diluteacid-enzyme hydrolysis and dark fermentation. Int JHydrogen Energy 2011;36(8):4852e62.
[17] Kumar K, Das D. Growth characteristics of Chlorellasorokiniana in airlift and bubble column photobioreactors.Bioresour Technol 2012;116(1):307e13.
[18] Roy S, Ghosh S, Das D. Improvement of hydrogen productionwith thermophilic mixed culture from rice spent wash ofdistillery industry. Int J Hydrogen Energ2012;37(21):15867e74.
[19] Loewus FA. Improvement in anthrone method fordetermination of carbohydrates. Anal Chem1952;24(1):219e22.
[20] APHA/AWWA/WEF. In: Eaton AD, Clesceri LS, Rice EW,Greenberg AE, editors. Standard methods for theexamination of water and wastewater. 21st ed. Washington,DC: American Public Health Association/American WaterWorks Association/Water Environment Federation; 2005.
[21] Segal L, Creely JJ, Martin Jr AE, Conard CM. An empiricalmethod for estimating the degree of crystallinity of nativecellulose using the X-ray diffraction. Textile Res J1959;29:786e94.
[22] Tserki V, Matzinos P, Kokkou S, Panayiotou C. Novelbiodegradable composites based on treated lignocellulosicwaste flour as filler. Part I. surface chemical modification andcharacterization of waste flour. Compos Part A Appl Sci2005;36(7):965e74.
[23] Shuler ML, Kargi F. Bioprocess engineering: basic concepts.2nd ed. Prentice Hall; 2001.
[24] Khanna N, Kotay SM, Gilbert JJ, Das D. Improvement ofbiohydrogen production by Enterobacter cloacae IIT-BT 08under regulated pH. J Biotechnol 2011;152(1):9e15.
[25] Richmond A. Large scale microalgal culture and applications.Prog Phycol Res 1990;7:1e62.
[26] Watanabe Y, de la Noue J, Hall DO. Photosyntheticperformance of a helical tubular photobioreactorincorporating the cyanobacterium Spirulina platensis.Biotechnol Bioeng 1995;47(2):261e9.
[27] Scragg AH, Illman AM, Carden A, Shales SW. Growth ofmicroalgae with increased calorific values in a tubularbioreactor. Biomass Bioenergy 2002;23(1):67e73.
[28] Hu Q, Kurano N, Kawachi M, Iwasaki I, Miyachi S. Ultrahigh-cell-density culture of a marine green alga Chlorococcumlittorale in a flat-plate photobioreactor. Appl MicrobiolBiotechnol 1998;49(6):655e62.
[29] Chu Y, Wei Y, Yuan X, Shi X. Bioconversion of wheat stalk tohydrogen by dark fermentation: effect of different mixedmicroflora on hydrogen yield and cellulose solubilisation.Bioresour Technol 2011;102(4):3805e9.
[30] Xing Y, Fan SQ, Zhang JN, Fan YT, Hou HW. Enhanced bio-hydrogen production from corn stalk by anaerobicfermentation using response surface methodology. Int JHydrogen Energy 2011;36(20):12770e9.
[31] Cao GL, Ren NQ, Wang AJ, Guo WQ, Xu JF, Liu BF. Effect oflignocellulose-derived inhibitors on growth and hydrogenproduction by Thermoanaerobacterium thermosaccharolyticumW16. Int J Hydrogen Energy 2010;35(24):13475e80.
[32] Prakasham RS, Sathish T, Brahmaiah P, Subba Rao C,Sreenivas Rao R, Hobbs PJ. Biohydrogen production fromrenewable agri-waste blend: optimization using mixerdesign. Int J Hydrogen Energy 2009;34(15):6143e8.
b i om a s s a n d b i o e n e r g y 6 1 ( 2 0 1 4 ) 1 5 7e1 6 6166
[33] Zhang ML, Fan YT, Xing Y, Pan CM, Zhang GS, Lay JJ.Enhanced biohydrogen production from cornstalk wasteswith acidification pretreatment by mixed anaerobic cultures.Biomass Bioenergy 2007;31(4):250e4.
[34] Kim MS, Oh YK, Yun YS, Lee DY. Fermentative hydrogenproduction from anaerobic bacteria using a membranebioreactor. In: Proc. 16th world hydrog. energy conf. Lyon Fr,vol. 50; 2006.
[35] Lay JJ. Modeling and optimization of anaerobic digestedsludge converting starch to hydrogen. Biotechnol Bioeng2000;68(3):269e78.
[36] O Thong S, Hniman A, Prasertsan P, Imai T. Biohydrogenproduction from cassava starch processing wastewater bythermophilic mixed cultures. Int J Hydrogen Energy2011;36(5):3409e16.
[37] Liu H, Zhang T, Fang HHP. Thermophilic H2 production froma cellulose-containing wastewater. Biotechnol Lett2003;25(4):365e9.
[38] Ito T, Nakashimada Y, Senba K, Matsui T, Nishio N. Hydrogenand ethanol production from glycerol-containing wastes
discharged after biodiesel manufacturing process. J BiosciBioeng 2005;100(3):260e5.
[39] Kongjan P, Angelidaki I. Extreme thermophilic biohydrogenproduction from wheat straw hydrolysate using mixedculture fermentation: effect of reactor configuration.Bioresour Technol 2010;101(20):7789e96.
[40] Carver SM, Hulatt CJ, Thomas DN, Tuovinen OH.Thermophilic, anaerobic co-digestion of microalgal biomassand cellulose for H2 production. Biodegradation2011;22(4):805e14.
[41] Yang Y, Tsukahara K, Sawayama S. Biodegradation andmethane production from glycerol-containing syntheticwastes with fixed-bed bioreactor under mesophilic andthermophilic anaerobic conditions. Process Biochem2008;43(4):362e7.
[42] Luedeking R, Piret EL. A kinetic study of the lactic acidfermentation. Batch process at controlled pH. Reprintedfrom Journal of Biochemical and Microbiological TechnologyEngineering Vol. I, No. 4. Pages 393-412 (1959). BiotechnolBioeng 2000;67(6):636e44.