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RESUME TEKNOLOGI FERMENTASI

JURNAL EFFECT OF INOCULUM CONCENTRATION, PH, LIGHT INTENSITY AND


LIGHTING REGIME ON HYDROGEN PRODUCTION BY PHOTOTROPHIC
MICROBIAL CONSORTIUM OLEH CAROLINA ZAMPOL LAZARO, MARIA
BERNADETE AMANCIO VARESCHE, EDSON LUIZ SILVA

Disusun oleh
Deanda Putri Ekapaksi

H0912033

PROGRAM STUDI ILMU DAN TEKNOLOGI PANGAN


FAKULTAS PERTANIAN
UNIVERSITAS NEGERI SEBELAS MARET
SURAKARTA
2015

Pengaruh Konsentrasi Inokulum, pH, Intensitas Cahaya dan Pencahayaan


Rezim Produksi Hidrogen dengan Phototrophic Konsorsium Mikroba
Produksi hidrogen photobiological menggunakan konsorsium bakteri memiliki
keunggulan dibandingkan kultur murni dalam pengolahan air limbah. Dalam jurnal ini bertujuan
untuk menentukan kondisi kultur optimal untuk produksi hidrogen oleh konsorsium bakteri
phototrophic. Parameter yang diuji dalam jurnal ini yaitu konsentrasi inokulum, pH, intensitas
cahaya dan pencahayaan protocol (illumination protocol).
Kerusakan lingkungan yang disebabkan oleh pembakaran bahan bakar fosil, sedang
dicari alternatif untuk menanggulanginya dengan mengganti bahan bakar menggunakan H 2.
Karena H2 dianggap sebagai bahan bakar bebas polusi karena dapat diproduksi secara biologis
melalui fotosintesis dan fermentasi. Bakteri yang dapat menghasilkan oksigen melalui
metabolisme photoheterotrophic dengan mengkonsumsi asam organik (asetat, butirat dan
propionat) yaitu bakteri ungu non sulfur (PNS) (Rhodobacter, Rhodopseudomonas,
Rhodospirillum dan Rubrivivax). Limbah dark fermentation yang mengandung asam tersebut
dapat digunakan sebagai substrat bakteri PNS untuk memproduksi H 2 secara bersamaan untuk
penghapusan kebutuhan oksigen (COD).
A. Tahap-tahap penelitian
Inokulum PHPBC disusun oleh mikroorganisme dengan genera Rhodobacter,
Rhodospirillum, Rhodopseudomonas dan Sulfurospirillum. Sebelum penelitian produksi
hydrogen (HP), konsorsium bakteri diserahkan ke fase adaptasi, di mana sel-sel tumbuh pada
campuran sumber karbon. Medium kultur disiapkan menggunakan senyawa berikut (g L 1):
0,6 KH2PO4; 0,9 K2HPO4; 0,2 MgSO4; 0,4 NaCl; 0,05 CaCl2; Ekstrak ragi 0,2; 5 mL L 1 besi
solusi sitrat (0,1 g 100 mL- 1); 1 mL L 1 larutan logam jejak; 0,8 mL L 1 dari HCl (30%); 0,1
(mg L -1) vitamin B12. Sumber karbon adalah 1.93 g L 1 natrium asetat trihidrat dan 1,4 g L
1 natrium butirat. Percobaan dilakukan dalam botol kaca dengan total volume 0,6 L (0,2 L
volume kerja) yang disimpan di dalam inkubator dengan suhu terkontrol (30 0C) dan konstan
pencahayaan dengan lampu tungsten (60 W) atau berbeda.

B. Hasil Penelitian
Tabel 2. Hasil Variable Percobaan Konsentrasi Sel Awal

Efek konsentrasi inokulum yaitu jika kepadatan sel berlebihan menyebabkan


penurunan dalam rasio substrat mikroorganisme yang menyebabkan kekurangan substrat
untuk mendukung metabolisme mikroba. Dan kelebihan sel sel dapat mendukung
pembentukan agregat yang dapat menghambat penetrasi cahaya karena efek self shading atau
bahkan dapat membatasi difusi substrat menjadi flok bakteri.
Dari Tabel 2. diamati potensi produksi hidrogen (HPP) dan SCE sedikit meningkat
pada konsentrasi inokulum 0,03 VSS-g L-1 yaitu 25 mmol H2 L-1 kultur pada HPP dan 13,7%
pada SCE, pada konsentrasi inokulum 0,2 VSS-g L -1 yaitu 25,9 mmol H2 L-1 kultur pada HPP
dan 14,6% pada SCE. Pada laju produksi hydrogen untuk konsentrasi inokulum 0,03 VSS-g
L-1 sebesar 1,5 mmol H2 L-1 kultur-1 dan memiliki fase lag 25,0 jam. Sedangkan laju hydrogen
konsentrasi inokulum 0,2 sedikit meningkat sebesar 1,7 mmol H2 L-1 kultur-1 namun pada fase
lag mengalami penurunan sebesar 24,2 jam. Konsentrasi biomassa pada inokulum 0,2 VSS-g
L-1 mengalami peningkatan dari inokulum 0,03 VSS-g L-1.
Namun, peningkatan lebih lanjut konsentrasi biomassa (inokulum 0,4 dan 0,6 VSS-g
L-1) menyebabkan penurunan baik dalam HPP dan SCE yang mencapai nilai terendah yaitu
pada inokulum 0,4 VSS-g L-1 menghasilkan HPP 22,8 mmol H2 L-1 kultur dan SCE 13,2 %.
Untuk inokulum 0,6 VSS-g L-1 menghasilkan paling rendah HPP sebesar 12,7 mmol H2 L-1
kultur serta SCE 10,3 %. Laju produksi hydrogen inokulum 0,4 VSS-g L -1 juga mengalami
penurunan dibanding inokulum 0,2 VSS-g L-1 yaitu hanya sebesar 1,3 mmol H2 L-1 kultur-1.
Dan inokulum 0,6 VSS-g L-1 memiliki laju produksi hydrogen terendah yaitu 0,5 mmol H2 L-1
kultur-1. Fase lag pada inokulum 0,4 VSS-g L-1 lebih rendah dibanding inokulum 0,2 VSS-g
L-1 yaitu 13,5 jam tetapi konsentrasi inokulum 0,6 VSS-g L -1 memiliki fase lag lebih tinggi
yaitu sebesar 16,1 jam.

C. Kesimpulan
Membandingkan efek konsentrasi inokulum cukup sulit ketika memiliki rentang nilai
berbeda jauh dari penelitian atau bahkan ketika data dinyatakan dalam satuan yang berbeda.
Pada penelitian ini nilai optimum pada konsentrasi inokulum 0,2 VSS-g L-1 (OD660
0,555). Dipilih inokulum 0,2 VSS-g L-1 karena pada konsentrasi inokulum tersebut dapat
memberikan efek positif mempengaruhi produksi hydrogen (HP). Sebaliknya, konsentrasi
yang lebih tinggi menimbulkan efek negatif pada produksi hydrogen (HP). Sumber inokulum
(spesies dan kultur strain), umur kultur dan kondisi eksperimental, yang mencakup banyak
parameter, juga mempengaruhi produksi hydrogen (HP) langsung.

Renewable Energy 75 (2015) 1e7

Contents lists available at ScienceDirect

Renewable Energy
journal homepage: www.elsevier.com/locate/renene

Effect of inoculum concentration, pH, light intensity and lighting


regime on hydrogen production by phototrophic microbial consortium
^ncio Varesche b, Edson Luiz Silva a, *
Carolina Zampol Lazaro a, Maria Bernadete Ama
~o Carlos, Rod. Washington Luis, km 235, 13565-905 Sa
~o Carlos, Sa
~o Paulo, Brazil
Department of Chemical Engineering, Federal University of Sa
~o Carlos, University of Sa
~o Paulo, Av. Trabalhador Sa
~o-carlense, 400,
Department of Hydraulics and Sanitation, School of Engineering of Sa
~o Carlos, Sa
~o Paulo, Brazil
13566-590 Sa

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 22 October 2013
Accepted 18 September 2014
Available online 8 October 2014

Photobiological hydrogen production using a bacterial consortium has advantages over pure cultures
regarding application to wastewater treatment. Photo-H2 production from organic acids, which were
produced by dark fermentation, must be studied in detail to make a hydrogen production process in two
stages efcient. In this scenario, our study aimed to determine the optimized culture conditions for
hydrogen production by a phototrophic bacterial consortium. The inoculum concentration, pH, light
intensity and illumination protocols were the parameters that we evaluated. The optimal conditions for
hydrogen production were inoculum concentration of 0.2 g VSS L1, pH 7.0, light intensity of 5 klux, with
a constant illumination regime. The highest hydrogen production potential (P) and substrate conversion
efciency (SCE) were 41.5 mmol H2 L1 culture and 25.4%, respectively, with a COD removal of 95%.
2014 Elsevier Ltd. All rights reserved.

Keywords:
Phototrophic bacterial consortium
Culture conditions
Organic acids
COD removal efciency

1. Introduction
Considering the environmental damage caused by the combustion of fossil fuels, researchers have been looking for a new and
sustainable energy system [1]. H2 is considered a clean nonpolluting fuel that can be produced biologically through photosynthetic and fermentative pathways [2]. Purple non-sulfur bacteria
(PNS) (Rhodobacter, Rhodopseudomonas, Rhodospirillum and Rubrivivax) can produce hydrogen through photoheterotrophic metabolism by organic acid consumption (acetate, butyrate and
propionate). This process is an attractive alternative because dark
fermentation efuents containing such acids can be used as substrate for PNS bacteria to produce H2 simultaneously to chemical
oxygen demand removal (COD) [3].
The type and amount of organic acids produced during dark
fermentation depends mainly on the metabolic pathway carried
out by different bacterial species [4]. Acetate and butyrate has been
identied as the main fermentation products; lactate, propionate
and ethanol have been reported in fewer amounts [3]. Specialized
literature indicates that Rhodopseudomonas palustris WP3-5 was
able to produce hydrogen from acetate, butyrate and lactate individually [5] and also from a mixture of acetate and butyrate [6].
Other PNS bacteria, Rhodobacter sphaeoides O.U.001, grew and
produced hydrogen from a mixture of acetate, butyrate and
* Corresponding author. Tel.: 55 16 3351 8264; fax: 55 16 3351 8266.
E-mail address: edsilva@ufscar.br (E.L. Silva).
http://dx.doi.org/10.1016/j.renene.2014.09.034
0960-1481/ 2014 Elsevier Ltd. All rights reserved.

propionate [3]. Rubrivivax gelatinosus were able to grow on acetate


and butyrate; however, the hydrogen production occurred in the
presence of glucose and lactate as the sole carbon sources [7]. The
feasibility of hydrogen production was also assessed by phototrophic sludge from a mixture of acetate, butyrate and ethanol [8].
The main challenge of the process is to enhance the hydrogen
yield (HY) and hydrogen production rate (HPR) making it
economically viable. Thus, is necessary to study and evaluate the
effects of as many culture conditions as possible [9]. The concentration of the inoculum is an important parameter because an
excess of biomass can cause self-shading by reducing light penetration [10,11]. pH is another parameter that inuences the
hydrogen production (HP) by altering the enzymatic activity and
the biochemical reactions [12]. Furthermore, HP in large-scale
outdoor under natural sunlight is desirable. Thus, to carry out
experiments under light intensity and lighting regime as close as
possible to outdoor conditions is therefore recommended [9]. The
effect of various parameters has been studied, but there are discrepancies among the results obtained, even for different strains of
the same bacterial species, and further variation among species will
probably be encountered [10].
The previous studies primarily used pure cultures to investigate
the effects of those parameters on the performance of HP
[3,6,9,11,13e19], however for scale-up it is not practical. Although a
few studies evaluated HP using mixed cultures [20e23], the use of a
microbial consortium could be advantageous over pure cultures in
wastewater biotechnology processes [24]. The present study

C.Z. Lazaro et al. / Renewable Energy 75 (2015) 1e7

therefore aimed to optimize culture conditions (concentration of


inoculum, pH, light intensity and lighting regime) to enhance
photobiological HP by PHPBC using a mixture of acetate and
butyrate in batch reactors.

Table 1
Experimental conditions.
Runs

2. Material and methods


2.1. Inoculum, culture medium and culture conditions
The inoculum was PHPBC composed by microorganisms related
to the genera Rhodobacter, Rhodospirillum, Rhodopseudomonas and
Sulfurospirillum, that was provided by Lazaro et al. [21]. Prior to the
HP experiments the bacterial consortium was submitted to an
adaptation phase, in which the cells grew on a mixture of carbon
sources described hereafter. The culture medium was prepared
using the following compounds (g L1): 0.6 KH2PO4; 0.9 K2HPO4;
0.2 MgSO4; 0.4 NaCl; 0.05 CaCl2; 0.2 yeast extract; 5 mL L1 of iron
citrate solution (0.1 g 100 mL-1); 1 mL L1 of trace metal solution
described by Lee et al. [25]; 0.8 mL L1 of HCl (30%); 0.1 (mg L1)
vitamin B12. Carbon sources were 1.93 g L1 of sodium acetate trihydrate and 1.4 g L1 of sodium butyrate.
The experiments were carried out in glass bottles with a total
volume of 0.6 L (0.2 L working volume) that were kept inside an
incubator with a controlled temperature (30  C) and constant
illumination by tungsten lamps (60 W) or different (when
mentioned in the text). The light intensity was measure at the
surface of the bottles, see Table 1. N2 gas was ushed for 10 min to
create anaerobic conditions in the headspace prior to incubation.
The pH adjustment was made with NaOH solution (2 M) or HCl


SCE %

Run
Run
Run
Run
Run
Run
Run
Run
Run
Run
Run
Run
Run
Run

1
2
3
4
5
6
7
8
9
10
11
12
13
14

Parameters evaluated
Inoculum
concentration
(g VSS L1)

pH

Light
intensity
(klux)

Lighting regime

0.03
0.2
0.4
0.6
0.2

6.5

24 h light

24 h light

0.2

5.0
6.0
7.0
8.0
7.0

24 h light

0.2

7.0

3
5
8
5

(equation (2)). Thus, in case of a mixture using both substrates, the


theoretical yield will be 14 mol of H2.

C2 H4 O2 2H2 O/4H2 2CO2

(1)

C4 H8 O2 6H2 O/10H2 4CO2

(2)

Besides the theoretical HY, it is possible to calculate the SCE


(equation (3)), to evaluate the microbial HP based on the stoichiometric conversion of each substrate.

moles of H2 that have actually been produced


100
moles of H2 expected through stoichiometric equation

(30%). Optical density (OD) equal to 1 (OD at 660 nm) correspond to


0.4 g VSS L1. Each set of experiments was performed in triplicate. A
summary of the experimental conditions is presented in Table 1.
2.2. Chemical and chromatographic analysis
The H2 content in the biogas was determined by gas chromatograph [26]. Acetic and butyric acid was measured by highperformance liquid chromatograph [26]. COD and VSS (volatile
suspended solids) were measured according to Standard Methods
[27]. The OD was measured in a spectrophotometer (Hach DR/2010)
at 660 nm. The light intensity was measured using a digital luximeter (Testo 545).
2.3. Experimental data tting
The experimental data were t to the mean values obtained
from the triplicates using Statistica software (version 8). The
maximum HPR was obtained by nonlinear sigmoidal adjustment of
the modied Gompertz function [28].
3. Results and discussion
HP varies according to the carbon source. Considering acetate
and butyrate, the sources applied in the present study, the theoretical hydrogen yield is 4 mol of H2 for each mol of acetate
(equations (1) and (10) moles of H2 for each mol of butyrate

12 h light e 12 h dark
18 h light e 6 h dark
24 h light

(3)

To produce H2, PNS bacteria are inuenced by several culture


conditions and the present study evaluated the effect of inoculum
concentration, initial pH, light intensity and lighting regime to
determine an optimal condition for biological HP by the PHPBC. The
inuence of each condition will be discussed in detail hereafter.
3.1. Effect of initial biomass concentration
Regarding to inoculum concentration effect, the rst known
point is that excessive cell density leads to a decrease in the substrate/microorganism (S/M) ratio that causes a shortage of substrate to support microbial metabolism. In addition, the excess of
cells can favor the formation of cell aggregates, which can hamper
the penetration of light due to the self-shading effect or can even
limit the diffusion of substrate into bacterial ocs [10].
It was observed that hydrogen production potential (HPP) and
SCE increased slightly from 25 mmol H2 L1 culture and 13.7 % to
25.9 mmol H2 L1 culture and 14.6% with an increase in the cell
concentration from 0.03 to 0.2 g VSS L1. However, a further increase in biomass concentration (0.4 and 0.6 g VSS L1) caused a
decrease either in the HPP and SCE that reached the lowest value
(12.7 mmol H2 L1 culture and 10.3%) at a biomass concentration of
0.6 g VSS L1 (Table 2).
According to Argun et al. [29] the highest HP (19.85 mmol H2 L1
culture) was obtained at 1.1 g L1 cell concentration. Ren et al. [30]
achieved the highest SCE (51.5%) at an inoculant volume of 10%
(v/v). Kim et al. [10] observed that HP was positively proportional to

C.Z. Lazaro et al. / Renewable Energy 75 (2015) 1e7

Table 2
Summary of results of the variable initial cell concentration experiments.
Inoculum
concentration (VSS e g L1)

Hydrogen production
potential (mmol H2 L1 culture)

0.03
0.2
0.4
0.6

25.0
25.9
22.8
12.7

1.5
2.6
1.2
0.6

Hydrogen production
rate (mmol H2 L1 culture h1)
1.5
1.7
1.3
0.5

0.2
0.1
0.1
0.04

Lag phase (hours)


25.0
24.2
13.5
16.1

1.1
0.8
0.2
5.6

SCE (%)
13.7
14.6
13.2
10.3

1.1
1.7
0.4
0.2

End biomass
concentration(VSS e g L1)
0.7
0.8
0.8
0.8

0.02
0.01
0.01
0.08

Table 3
Summary of results of the variable pH experiments.
pH

Hydrogen production potential


(mmol H2 L1 culture)

Hydrogen production rate


(mmol H2 L1 culture h1)

Lag phase (hours)

SCE (%)

Final pH value

5
6
7
8

e
37.8 7.7
40.3 1.2
e

e
1.1 0.2
1.1 0.1
e

e
21.8 3.8
22.9 1.7
e

5.1
7.2
7.7
8.1

the initial biomass concentration up to 0.56 g dwt L1. In the study


of Han et al. [11] at initial inoculum quantity equal to OD660 0.931
it was observed the highest HY and SCE: 597 mmol H2 mol1 acetate and 14.9%, respectively.
In the present study, the optimum value was 0.2 g VSS L1
(OD660 0.555), while for Kim et al. [10] and Han et al. [11], the
highest HP occurred for higher initial cell concentrations of
0.56 g dwt L1 (OD660 1) and OD660 0.931, respectively. This fact
is probably related to the source of inoculum used in different
studies.
Compare the effects of inoculum concentration is quite difcult
when the range of values differs considerably from study to study
[10,29] or even when data are expressed in different units [11,30].
However, it seems to be an agreement that up to a certain value, an
increase in inoculum concentration positively affects the HP.
Conversely, higher concentrations pose a negative effect on HP.
Authors also speculate that sources of inoculum (species and culture strains), cultures age and experimental conditions, that includes many parameters, also inuences the HP directly.
3.2. Effect of initial pH
pH affects the bacterial activity and the metabolic pathway
because it determines the ionic form of the active enzymatic site
and its activity [11,31]. Photobiological HP has been reported over a
wide pH range (4e10); and it seems to vary among species.
In the variable pH experiments, pH 7 was found to be an ideal
value. At this pH, higher HPP (40.3 mmol H2 L1 culture) and SCE
(22.9%) and a lower lag phase (16.9 h) were observed (Table 3). At
pH 6, the HP was delayed for 52 h, and reached the level of
37.8 mmol H2 L1 culture and SCE of 21.8%, values close to the parameters obtained at pH 7 except for the lag phase (Fig. 1). At pH 5,
HP was not observed, nor was cell growth. At pH 8, slight cell
growth was observed. However, there was no H2 in the biogas
(Table 3). It is probably related to the source of the inoculum that
had no natural tolerance to acidic or alkaline conditions.
In the study of Cai et al. [20] the highest HP (42.4 mmol H2 L1
culture) observed by a marine mixed phototrophic bacterial consort
was obtained at an alkaline pH (8). For the isolated Rhodopseudomonas acidophilla KU001 studied by Merugu et al. [32] the HP
was observed at wide range of pH (4.5e7.5), however, the highest
value was obtained at pH 6. Interestingly is the results presented by
Tao et al. [14] that isolated a bacterial strain able to grow and
produce H2 under acidic and alkaline pH and that also had the
ability to adjust the pH (around 7) during the experiments, even
though the initial pH values ranged from 5.5 to 9.5. This

52 3.6
16.9 1.1
e

phenomenon had not been reported for other hydrogen-producing


purple non-sulfur bacteria [14] and this fact is of paramount
importance because there is no need of external pH control.
In general, pH 7 is the optimum value [10,11,14,30], however,
there is no consensus about what is the upper and lower pH
threshold within which HP can occur and in which it is maximized.
Thus, the ideal pH should be evaluated for each inoculum source.
3.3. Effect of light intensity
There is a minimum light intensity threshold at which the cell
growth of PNS bacteria starts. Cell growth and HP increase with an
increase in light intensity up to the saturation value that occurs
when the photosynthetic system of bacteria is supplied with an
excess of ATP and Fdred. In this case, nitrogenase does not have the
capacity to process the excess [33]. Light intensity is reported to
vary among species and even among strains of the same species
[24], which can be attributed to the photo-adaptation of each
microorganism and the self-shading effect, which can be aggravated by inefcient bioreactor design [34]. The effect of light intensity on HP should therefore be evaluated to maximize the
performance of the process.
According to our results, an increase in light intensity from 3 to 5
klux increased HPP and SCE from 32.7 mmol H2 L1 culture and 19 %
to 40 mmol H2 L1 culture and 31.6%, respectively. However, a

Fig. 1. Effect of pH on cumulative H2 production. A pH 6.0; - pH 7.0.

C.Z. Lazaro et al. / Renewable Energy 75 (2015) 1e7

Table 4
Summary of results of the variable light intensity experiments.
Light intensity (klux)

Hydrogen production potential


(mmol H2 L1 culture)

Hydrogen production rate


(mmol H2 L1 culture h1)

Lag phase (hours)

SCE (%)

3.0
5.0
8.0

32.7 2.1
40.0 5.5
34.0 3.3

1.1 0.1
1.2 0.3
1.4 0.1

35.6 0.1
31.6 1.2
31.8 1.0

19.0 0.1
23.5 2.0
19.3 2.4

further increase from 5 to 8 klux led to a decrease in HPP and SCE


(34 mmol H2 L1 culture and 19.3%). In this study, the saturation
value was thus found to be 8 klux. However, no photoinhibition was
observed and the maximum HPR had a positive relationship with
light intensity (Table 4).
Similar trend was observed in the literature [19,25,34]. Tao et al.
[14] afrm that by increasing the light intensity from 1.5 klux to 5
klux, there is an increase in the total H2 evolved (from 47.7 mmol H2
L1 to 127.1 mmol H2 L1) and the SCE (from 26.9 to 71.3 %).
However, HP and SCE slightly decrease at 7 klux (123.5 mmol H2
L1 and 69.2%). For Han et al. [11] the maximum value of HP and HY
occurred at 6 klux (21.6 mL H2 and 0.395 mol H2 mol1 acetate,
respectively). Cai et al. [20] observed the highest HP at 4 klux
(42.11 mmol H2 L1 culture) than at 2 klux (32.78 mmol H2 L1
culture) and 6 klux (36.01 mmol H2 L1 culture) (Table 4). Uyar et al.
[16] reported similar results and suggested that light intensity
should be at least 4 klux in the darkest area of the reactor to obtain
a high H2 production rate. However, Akroum-Amrouche et al. [35]
obtained the highest HP (298.7 mmol H2 L1 culture) at higher
light intensity (8.5 Klux).
Different ideal light intensities for HP and SCE were observed by
Li et al. [36] under standing (4 klux, 3.6 L H2 L1 culture and 89.7%)
and shaking (8 klux; 3.6 L H2 L1 culture and 88.3%) culture conditions. This difference is probably associated with the light
attenuation in the shaking culture due to scattering of light by
suspended cells.
3.4. Effect of illumination protocol (lighting regime)
In addition to other parameters, the illumination pattern also
affects HP and should be investigated when it is desirable to achieve a high rate and high HP under large scale outdoor conditions
[16].
According to our results, the increase in the light period over
dark in lightedark cycles promoted an increase in HPP, maximum
HPR and SCE (Table 5). The highest values were 41.5 mmol H2 L1
culture, 1.1 mmol H2 L1 culture h1 and 25.4% achieved with
continuous illumination. By increasing the hours of light exposure,
the lag phase decreased from 55.4 h (12 h light-12 h dark) to 37
(24 h light) (Fig. 2).
Our results are similar to the ndings of Uyar et al. [16], Argun
and Kargi [37] and Li et al. [38]. For Uyar et al. [16] the highest HP,
maximum HPR and SCE (54.74 mmol H2 L1 culture, 0.6 mmol H2
L1 culture h1 and 68%) were achieved under constant illumination. Argun and Kargi [37] observed an increase in the HP increased
from 21.5 mmol H2 L1 culture to 42.5 mmol H2 L1 culture for
0.5/0.5 (h light/h dark) reactor cycle and continuous illumination,

respectively. Li et al. [38] also observed higher levels of HP in reactors under constant illumination (147 mmol H2 L1 culture) than
for those reactors exposed to lightedark cycles (139 mmol H2 L1
culture). The authors afrm that sufcient illumination is essential
for HP and the reduction in the maximum HPR and SCE is due to
restriction on illumination. Furthermore, they add that a decrease
in SCE could be due to the cell metabolism adaptation in the
absence of light or even due to the cell activity in this period.
Furthermore, Uyar et al. [16], who evaluated the indoor HP, and
Adessi [39], who applied a 50-L reactor to outdoor HP, an articial
light supply during dark periods for reactors exposed to natural
dayenight cycles is recommended to keep the overall HPR and total
amount of H2 high. Distinct result was reported by Eroglu et al. [40]
that did not observe any difference in the cumulative HP (5 mmol
H2 L1 culture) in photobioreactors exposed to continuous illumination and photobioreactors under 12-12 lightedark cycles. However, the lag phase was at least four times higher for
photobioreactors under diurnal cycles (26 h) compared to constant
illumination (6 h) because cell growth and HP are light-dependent
and the metabolism was down-regulated in periods of the absence
of light in the reactors under 12-12 lightedark cycles.
The contrary results were reported by Koku et al. [9] that obtained more H2 (29 mmol H2 L1 culture) when the reactor was
exposed to lightedark cycles (14 h light-10 h dark) than for constant illumination (23.6 mmol H2 L1 culture). The authors noted
that although little or no hydrogen was produced in absence of
light, the overall amount of gas produced in the cycle reactor was
appreciably more than in the continuously illuminated reactor,
possibly due to the achievement of high cell densities on the cycle
reactor or due to any benecial effect of illumination cycles on
nitrogenase.
In terms of cost of production, it is desirable to save as much
energy as possible. The ultimate goal of photo-H2 evolution is its
occurrence in outdoor reactors under natural sunlight. Thus, the
study of light/dark cycles is crucial to provide a basis for further
experiments in hydrogen production in outdoor reactors under
sunlight diurnal cycles [9]. Most of the cited studies achieved
higher HP under a constant illumination pattern and just one of
them [9] reported the contrary result. Thus, because there is no
agreement regarding this topic, further studies are needed.
3.5. Comparison of the hydrogen production with the literature
Studies have been performed to determine optimum culture
conditions, varying pH, temperature, inoculum quantity, light intensity, and lighting regime, among other parameters, to enhance
HP using diverse microorganisms in pure culture [3,9e11,16,20,30]

Table 5
Summary of results of the variable lighting regime experiments.
Lighting regime
(hours light-hours dark)

Hydrogen production potential


(mmol H2 L1 culture)

Hydrogen production rate


(mmol H2 L1 culture h1)

Lag phase (hours)

SCE (%)

12-12
18-6
24-0

32.6 5.9
34.5 6.5
41.5 5.0

0.4 0.2
0.8 0.1
1.1 04

55.4 3.0
43.8 2.4
37 0.2

20.7 3.4
21.8 3.7
25.4 0.1

C.Z. Lazaro et al. / Renewable Energy 75 (2015) 1e7

Fig. 2. Effect of lighting regime on cumulative H2 production. : 12-12 L-D; - 18-6 LD; A 24-0 L-D.

and, even less frequently reported, using mixed cultures (Cai et al.
[20] and the present study).
As seen from literature data, HP varied over a wide range
(5.4e255.4 mmol H2 L1 culture) according to the microorganism
and substrate used. Lower HP was reported for the pure culture of
Rhodopseudomonas palustris W004 (7.4 mmol H2 L1 culture) [19],
mixed photoheterotrophic culture (5.4 and 5.8 mmol H2 L1 culture) [23] and PHPBC (7.8 and 9.0 mmol H2 L1 culture) [21]
(Table 6).
In the present study, under the best culture conditions, HPP of
41.5 mmol H2 l1 culture was observed, which is similar to the
values reported for pure and mixed culture: Rhodopseudomonas sp.
(35.2 mmol H2 L1 culture) [19], Rhodopseudomonas palustris W004
(43.4 mmol H2 L1 culture) [19] and marine mixed phototrophic

bacterial consort (42.11 mmol H2 L1 culture) [20], Rubrivivax


gelatinosus L31 (42.14 mmol H2 L1 culture) [18], phototrophic
sludge hydrogen-producer (69 mmol H2 L1 culture) [22]. Higher
hydrogen production was observed by Uyar et al. [3] using Rhodobacter sphaeroides O.U.001 (88 mmol H2 L1 culture), Wu et al. [19]
using Rhodopseudomonas sp. (87.6 mmol H2 L1 culture) and Li &
Fang [18] using Rubrivivax gelatinosus L31 (90.56 mmol H2 L1
culture) (Table 6).
COD removal is not always reported. However, when the
reduction of organic matter is one of the goals of photo-H2 evolution, reduction of organic matter should be highlighted. In the
present study, COD removal was at least 94% for all the experiments, except for the experiments at pH 5 and 8 and for the higher
initial biomass concentration (0.6 g VSS L1) in which the COD
removal was only 18%, 18% and 77%, respectively (data not shown).
In terms of COD removal, our experiments were very satisfactory
and similar to the literature ndings (Table 6). Although COD
removal achieved signicant values SCE were not high as expected.
The reason could be the capability of phototrophic bacteria to
produce alternative metabolites under aerobic and anaerobic conditions. Poly-b-hydroxybutyrate (PHB) is a biodegradable polymer
synthesized during unfavorable growth conditions by diverse
bacteria. Although PHB has many industrial uses it is known that
PHB production competes with phototrophic hydrogen production
[41]. The nature of the substrate and pH seems to play an important
role to determine the metabolic routes that leads to the PHB synthesis or hydrogen production. Acetate and butyrate seems to be a
very efcient substrate for PHB, while lactate, malate and succinate
favor H2 production [9]. In this sense, considering that acetate and
butyrate are the major byproducts of fermentation, strategies to
enable H2 production from these acids should be explored in a way
to achieve increased H2 yields. The use of mutant strains is a
promise option to match this goal [42].
By comparing the cited studies, we can conclude that pure
cultures did not offer always such an advantage over the bacterial
consortium for HP. Upon comparing the present result with the

Table 6
Summary of results from the literature.
Inoculum

Substrate

Hydrogen production
(mmol H2 L1 culture)

H2 yield
(mmol H2 g1 COD)

COD
removal (%)

SCE (%)

Reference

Rhodobacter sphaeroides O.U.001

Acetate, Butyrate
and Propionate
Acetate
Butyrate
Malate
Lactate
Malate
Acetate
Butyrate
Acetate, Butyrate,
Propionate and Ethanol
Acetate
Butyrate
Acetate, Butyrate,
Propionate, Ethanol
Butyrate
Acetate
Butyrate
Acetate and Butyrate
Acetate
Butyrate
Acetate and Butyrate

100e

47

[3]

0.55a
0.61a
1.39a
90.72e
42.14e
35.2e
87.6e
255.4e

165b
242b
540b
e
e
12.75e
18.7e
8.6e

e
e
e
e
e
72.8
85.3
96.6

9**
50.5
24.6
e
e
56.5

7.4e
43.4e
233.2e

2.7e
9.3e
7.7e

40.6
35.9
93.7

e
e
51.6

0.21d
0.40d
13.2

e
e
e
43.4
e
e
95

21.03
14.5
13.8
28.1e
5.33
4.03
25.4

Rhodobacter sphaeroides RV

Rubrivivax gelatinosus L31


Rhodopseudomonas sp.

Rhodopseudomonas palustris W004

Marine mixed phototrophic bacterial consort


PHPBC
Phototrophic sludge hydrogen-producer
Mixed Photoheterotrophic Culture
PHPBC
a

42.11e
7.8
9.0
69e
5.8e
5.4e
41.5

e
e
e
e

4.1c
2.4c

[17]

[18]
[19]

[20]
[21]
[22]
[23]
Present study

Expressed as mmol H2.


b
Expressed as mmol H2 mol1 substrate.
c
Converted from original data expressed as mmol H2 mol1 substrate to substrate conversion efciency, considering the theoretical H2 production for 4 mol H2 mol1
acetate and 10 mol H2 mol1 butyrate.
d
Expressed as mol H2 mol1 substrate.
e
Converted from literature data.

C.Z. Lazaro et al. / Renewable Energy 75 (2015) 1e7

previously reported results [21], an improvement in the SCE and


maximum HPR was observed. SCE increased from 14.5% (acetate)
and 13.8% (butyrate) to 25.4% (mix of acetate and butyrate).
Regarding a maximum HPR, Lazaro et al. [21] observed values of 0.4
and 0.9 mmol H2 L1 culture d1 (0.02 and 0.04 mmol H2 L1 culture h1) for acetate and butyrate, respectively. In the present
study, 1.1 mmol H2 L1 culture h1, which is almost 30 times higher,
was observed. Therefore, the improvements achieved in the present study warrant further studies on HP by a bacterial consortium
using organic acid-rich wastewaters. The use of bacterial consortium in reactor in continuous mode fed with real wastewater
will require a close monitoring of the microbial structure once the
substrate can act as inoculum. In this case the operational conditions such as pH, light intensity, temperature, composition of
substrate, hydraulic retention time will contribute or not to the
establishment of new species inside the reactor. This fact could be
an issue if non hydrogen-producers or hydrogen-consumers predominate over hydrogen-producers purple bacteria or even
compete for substrates.
4. Conclusions
From the presented data (literature and present study) it is
possible to conclude that there is an agreement regarding optimum
culture conditions such as pH (7), light intensity (5e8 klux) and
lighting regime (constant illumination). However, there are also
peculiarities reported in the literature that are out of the cited
range: pH values (6.0 and 8.0), light intensity (8.5 klux) and lighting
protocol (lightedark cycles), which could be due to the source of
inoculum and substrates used in the experiments. The use of
phototrophic microorganisms to produce hydrogen through
organic acids present in wastewaters is desirable once energy can
be produced while removing COD from efuents. The use of a
bacterial consortium is advantageous over pure culture for HP.
Thus, the PHPBC has the potential to use liquid residues containing
organic acids as the inoculum for HP. The results achieved in the
present work encourage additional studies aiming to further
improve H2 yield by PHPBC when an increase in HP, maximum HPR
and SCE were observed in comparison to the results previously
reported.
There are many possibilities that should be taken into account to
get better results on the HP. The reactor conguration seems to play
an important role in the performance of the systems because the
phototrophic hydrogen production is a light dependent metabolism. Thus, studies on reactor design and illumination systems,
that avoid light conversion efciency limitation, are desired.
Moreover, the future of hydrogen economy depends also on the
advances in genetic engineering that involves the manipulation of
key enzymes (hydrogenases, nitrogenases and PHB synthases).
Acknowledgments
The authors would like to thank FAPESP (Process number 2009/
15984-0 and 2012/03979-5) for the nancial support.
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