Bioresource Technology 181 (2015) 330337

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Prospective technology on bioethanol production from

photofermentation
Rosangela Lucio Costa, Thamayne Valadares Oliveira, Juliana de Souza Ferreira, Vicelma Luiz Cardoso,
Fabiana Regina Xavier Batista
School of Chemical Engineering, Federal University of Uberlandia. Av. Joao Naves de Avila 2121, Santa Monica 38408-144, Uberlandia, MG, Brazil

h i g h l i g h t s
 C. reinhardtii and R. capsulatus were utilized to the ethanol production.
 Ethanol was produced from sulfur deprivation and mixotrophic carbon source.
 Hybrid system and co-cultivation increase the ethanol production by photofermentation.

a r t i c l e

i n f o

Article history:
Received 21 November 2014
Received in revised form 20 January 2015
Accepted 22 January 2015
Available online 30 January 2015
Keywords:
Ethanol
Chlamydomonas reinhardtii
Rhodobacter capsulatus
Hybrid system
Co-culture

a b s t r a c t
The most important global demand is the energy supply from alternative source. Ethanol may be considered an environmental friendly fuel that has been produced by feedstock. The production of ethanol by
microalgae represent a process with reduced environmental impact with efcient CO2 xation and
requiring less arable land. This work studied the production of ethanol from green alga Chlamydomonas
reinhardtii through the cellular metabolism in a light/dark cycle at 25 C in a TAP medium with sulfur
depletion. The parameters evaluated were inoculum concentration and the medium supplementation
with mixotrophic carbon sources. The combination of C. reinhardtii and Rhodobacter capsulatus through
a hybrid or co-culture systems was also investigated as well. C. reinhardtii maintained in TAP-S produced
19.25 4.16 g/L (ethanol). In addition, in a hybrid system, with medium initially supplemented with
milk whey permeated and the algal efuent used by R. capsulatus, the ethanol production achieved
19.94 2.67 g/L.
2015 Elsevier Ltd. All rights reserved.

1. Introduction
The use of ethanol to replace oil is the most viable way to
ensure a sustainable future. However, up to now more emphasis
has been given on the yeast performance in reactors for bioethanol
production (Andrietta et al., 2008). On the other hand, considering
the advantages of microalgae culture such as rapid growth rate and
productivity (Li et al., 2008) and their use to minimize contamination, since microalgae may to applied in wastewater treatment
from inorganic salts (NH+4, NO3 , PO34 ) using them as nutrient
materials (Mata et al., 2010), Chlamydomonas reinhardtii could be
preferentially selected as a prospective biological system for
bioethanol production instead of yeast.
Concerning on the application of microalgae, recent research
have shown that a diversity of strains may be cultivated to produce
Corresponding author. Tel.: +55 34 32309400.
E-mail address: frxbatista@feq.ufu.br (F.R.X. Batista).
http://dx.doi.org/10.1016/j.biortech.2015.01.090
0960-8524/ 2015 Elsevier Ltd. All rights reserved.

biodiesel, hydrogen, methane and ethanol. Specically to the

ethanol production from microalgae, it can be directly synthesized
by the cellular metabolism or from the fermentation of microalgae
biomass, mainly cellulose and starch that are readily converted to
fermentable products by enzymatic or acidic pretreatment
technology (Oncel, 2013; Chen et al., 2013). Microalgae based
ethanol may be considered as part of integrated process and a
promising environmentally friendly alternative, since they could
be present a potential for xing CO2, high growth at high yields
and low costs utilizing light as the energy source. Furthermore, they
do not require fertile land and portable water as feedstock based
biofuel (Chen et al., 2013; John et al., 2011; Hirano et al., 1997).
The unicellular green alga C. reinhardtii uses light to grow
photoautotrophically or mixotrophically in the presence of small
organic substrates (Goff et al., 2009). C. reinhardtii is widely studied
for hydrogen production by biophotolysis of water. This
method uses the same processes found in plants photosynthesis.
Photosynthesis involves the absorption of light by two distinct

R.L. Costa et al. / Bioresource Technology 181 (2015) 330337

photosynthetic systems operating in series: a water splitting and

O2 evolving system (photosystem II or PSII) and a second photosystem (PSI), which generates the reductant used for CO2 reduction
(Das and Veziroglu, 2001). It is important to note that green algae
could produce hydrogen not only under light conditions, but under
dark anaerobic conditions (Gaffron and Rubin, 1942). Nevertheless,
if dark and anaerobic conditions are established on the microalgae
cultures, hydrogen yield is quite low corresponding to about onesixth of direct biophotolysis production (Kosourov et al., 2002).
Besides hydrogen, the oxidative reaction of starch become incomplete and depending on the type of the microalga, carbon dioxide,
ethanol, lactic acid, formic acid, acetic acid, malic acid, glycerol and
other compounds are produced in varying proportions (John et al.,
2011; Gfeller and Gibbs, 1984). In addition, the medium composition may be altered to induce anaerobic condition. Hemschemeier
and Happe (2004) reported that, under sulfur depletion,
C. reinhardtii stops growing and accumulates starch. The absence
of sulfur forces the algae to reorganize the whole metabolism.
Anaerobiosis is established and hydrogen and ethanol could be
produced. These authors discussed that the accumulation of ethanol already indicate the activity of pyruvate formate-lyase (PFL).
PFL cleaves pyruvate into acetyl-CoA and it can further be reduced
to acetaldehyde by acetaldehyde dehydrogenase. Furthermore,
ethanol can be formed from cleavage of pyruvate by pyruvate
decarboxylase (PDC) producing acetaldehyde as intermediate that
is converted to ethanol by alcohol dehydrogenase (ADH).
Previous work suggested it is possible to produce ethanol from
metabolism of C. reinhardtii maintained in a basal medium supplemented with the mixotrophic carbon source (Costa et al., 2014).
And, the purpose of this work was at rst to verify the possibility
of C. reinhardtii produce ethanol using a basal medium with sulfur
depletion added mixotrophic carbon source such as milk whey
permeate (rich in lactose) and sodium acetate. In the second step,
the algae association with the purple non sulfur bacterium, a
Rhodobacter capsulatus, to improve ethanol content into the
medium was also evaluated by hybrid system (two stages) and
co-culture. The hybrid systems and co-cultures approaches are
strategies used in order to improve the yield. The purpose is to
integrate microorganisms with distinct biological routes. Thus,
the metabolites produced by one type of microorganism may be
the substrate to the second type.
2. Methods
2.1. Algal biomass
C. reinhardtii CC-124 was purchased from the Canadian Culture
Collection, the Chlamydomonas Resource Center. The green alga
was maintained in the basal medium Tris Acetate Phosphate
(TAP) (Andersen, 2005) at initial pH of 7.0. In order to guarantee
enough amounts of cells for the fermentation assays, the algal
inoculum was subcultured with the addition of 250 mL of fresh
TAP medium in 250 mL of growing culture. The alga was kept in
Erlenmeyer (500 mL) at 25 C under light cycle (night/day) of
12 h at 30 lE m 2 s 1.
2.2. Photosynthetic bacterial biomass
R. capsulatus was purchased from DSMZ German Collection of
Microorganisms and Cell Culture. The strain was cultivated
anaerobically in Erlenmeyers (500 mL) maintained at 30 C using
RCV medium (Weaver et al., 1975), at initial pH of 6.8, under
photosynthetic conditions of 30 lE m 2 s 1 (light-grown cells).
Sodium glutamate was used as nitrogen source instead of
(NH4)2SO4.

331

2.3. Biological strategies to ethanol production by photofermentation

For ethanol production by fermentation, anaerobic condition
was used in all strategies: by C. reinhardtii cultures, hybrid system
and co-culture approach. Thus, 50 mL bioreactors were ushed
with Ar gas (99.999%) for 3 min. The inoculum was 10 days age
for green algae culture and ve days age for the purple non sulfur
bacteria culture. Five days of time fermentation was used in all
assays. The photoperiod of 12 h (12 h dark/12 h light) was used
in all experiments with algae. In the hybrid system, in the second
stage by R. capsulatus, the assays were carried out under light
continuously.
2.3.1. C. reinhardtii under sulfur depletion
In the rst step of the current work, assays were carried out to
investigate the ethanol production by C. reinhardtii in TAP-S (Tris
acetatephosphate-minus-sulfur) medium. The evaluated variables
were the initial cell concentration (0.05, 0.10 and 0.20 g/L) of green
alga and the type of carbon source in a mixotrophic pathway. In
these latter trials, besides acetic acid, as usually present in the
TAP medium, it was added more 0.1 or 1.0 g/L of sodium acetate
and milk whey permeate (MWP), individually or simultaneously.
As the manufacturer, Sooro Concentrado Industria de Produtos Lcteos Ltda from Brazil, milk whey permeate contain lactose (93%),
proteins (1.2%), ashes (4.6%), among others traces compounds.
2.3.2. Hybrid system
The hybrid system was evaluated as an alternative to increment
the biofuel accumulation. In the rst stage, C. reinhardtii was cultivated in TAP-S medium and the efuent, free of cell and rich in soluble metabolites, was used as substrate by R. capsulatus, since
these photosynthetic bacteria can convert the organic acids and
the non-consumed original carbon source for additional ethanol
production.
Initially the effects of supplementing R. capsulatuss medium
with sodium glutamate, malic acid, milk whey permeate and
micronutrient solution, according Table 1a, were evaluated.
Subsequently, similar assays were carried out to verify the inuence of mixotrophic carbon source on the ethanol production by
C. reinhardtii adding to the TAP-S medium different concentrations
of sodium acetate and milk whey permeate. In these assays, only
the crude efuent was used in the second stage of hybrid system
(Table 1b). For all assays, the initial cell concentration was 0.1 g/L
and they were performed in duplicate.
2.3.3. Co-culture of algae and bacteria
Co-cultures of C. reinhardtii and R. capsulatus, being both cultured in TAP-S medium and RCV medium, were evaluated. The initial cell densities were 0.1 g/L, mixed together at variant ratios
(from 0% to 100%). Co-culture grew in bioreactor of 50 mL (working
volume of 37.5 mL) at 25 C using 30 lE m 2 s 1 of light intensity.
2.4. Analytical methods
The growth of cells was measured via spectrophotometry
(UVmini-1240, Shimadzu) and biomass dry weight. One milliliter
of sample was appropriately diluted with deionizer water and
the absorbance of the sample was read at 665 nm (algae) and
660 nm (bacteria). The concentrations of metabolites were determined by HPLC (Shimadzu MODEL LC-20A Prominence, Supelcogel,
column C-610H), equipped by ultra-violet and refractive detectors.
The UVVIS was used to determine organic acid concentrations at a
wave length of 210 nm and the RID detector quantied the contents of lactose and ethanol. The column temperature was kept
at 32 C and an aqueous solution of 0.1% M H3PO4 was used for
elution at 0.5 mL/min. The sample volume injected into the HPLC

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R.L. Costa et al. / Bioresource Technology 181 (2015) 330337

Table 1
(a) Schedule of Hybrid System to ethanol production. (b) Evaluation of carbon source in the Hybrid System to ethanol production. A control was performed in TAP-S
medium without mixotrophic carbon source addition.
Experiment 1
Culture conditions

Experiment 2
Culture conditions

Experiment 3
Culture conditions

C. reinhardtii
TAP-S medium

R. capsulatus
(1) Crude Supernatant from C. reinhardtii;
(2) Supernatant from C. reinhardtii supplemented with
glutamate (2.54 g/L), malic acid (4.02 g/L), milk whey permeate
(6 g/L) and micronutrient solutiona.

C. reinhardtii
(1) TAP-S medium supplemented with 0.1 g/L of
sodium acetate or/and milk whey permeate

R. capsulatus
Crude supernatant from C. reinhardtii

C. reinhardtii
(2) TAP-S medium supplemented with 1 g/L of
sodium acetate and milk whey permeate

R. capsulatus
Crude supernatant from C. reinhardtii

The micronutrient solution was composed according to RCV medium (Weaver et al., 1975).

was 20 lL. The produced gas was collected in graduated syringes,

and the oxygen concentration was determined by gas chromatography using the chromatograph Shimadzu model GC 17A,
equipped with a thermal conductivity detector (TCD) and a capillary column Carboxen 1010 (length 30 m, internal diameter
0.53 mm). The operating temperatures of the injection port, the
oven, and the detector were 230, 32, and 230 C, respectively.
Argon was used as carrier gas.

3. Results and discussion

Photobiological production of biofuel by green algae has great
potential to be used for generating renewable fuel from clean
resources. As reported by Oncel (2013), microalgae can also excrete
ethanol directly through the cell walls by means of intracellular
processes under dark. In fact, the degradation of intracellular
starch, which is the main endogenous carbon source stored during
aerobic phototrophic metabolism, into pyruvate is accomplished
by the EmbdenMeyerhofParnas and pentose phosphate pathways using pyruvate decarboxylase and alcohol dehydrogenase
enzymes.
The metabolism of C. reinhardtii is complex including a broad
variety of end by-products resulting from the fermentation under
different growth conditions in order to keep the cellular redox
and energy balance in the anaerobic medium. The fermentation
process occurs in dark and anaerobic environment or in the light
after sulfur depletion. These by-products are carbon dioxide,
hydrogen, acetate, formate, ethanol, lactate, glycerol, malate and
succinate (Philipps et al., 2011; Mus et al., 2007). According to
Gfeller and Gibbs (1984), acetate, formate and ethanol are the
major anaerobic compounds produced by sulfur-replete cultures
and, as reported by Kosourov et al. (2002), glycerol and lactate
are produced only at low pH.
Through glycolytic pathway, the starch degradation produces
pyruvate that is cleaved by pyruvate formate lyase (PFL1) into formate and acetyl-CoA. Acetyl-CoA can further be converted to acetate by the successive action of phosphotransacetylase (PTA) and
acetate kinase (ACK), (Philipps et al., 2011; Mus et al., 2007;
Gfeller and Gibbs, 1984). Another pathway leads to the conversion
of pyruvate into D-lactate catalyzed by D-lactate dehydrogenase (DLDH) (Philipps et al., 2011). Fig. 1b summarizes the metabolic route
clarifying the ethanol production.
The formation of glycerol, malate and succinate from starch by
C. reinhardtii follows an alternative pathway for pyruvate metabolism (Catalanotti et al., 2012; Philipps et al., 2011; Dubini et al.,
2009). In addition, Catalanotti et al. (2012) using a double mutant

lacking pyruvate formate lyase (PFL1) and alcohol dehydrogenase

(ADH1) obtained glycerol and lactate in a higher concentration
level. In respect to succinate formation that proceeds via malate
synthesis, Dubini et al. (2009) succeeded to achieve elevated content of this metabolite applying a mutant strain with no hydrogenase activity.
The following results present the ethanol formation by only
C. reinhardtii or in hybrid system and co-culture with R. capsulatus.
Besides the ethanol concentration, data of other metabolites are
shown, since the comprehension of the relationship between different metabolic pathways, might lead to achieve the conditions
that improve ethanol production by photobiological processes.

3.1. Ethanol production by C. reinhardtii

3.1.1. Effect of inoculum concentration
In respect to the inuence of initial cell concentration, the concentrations of 0.05, 0.10 and 0.20 g/L were applied to the assays of
the ethanol production by C. reinhardtii, and the results of O2 evolution, nal cell concentrations and metabolites concentrations are
presented in Fig. 1.
The analysis of Fig. 1 indicated that the ethanol production did
not show a direct relation to the variation of cell concentration. The
highest ethanol content 19.25 4.16 g/L (0.16 0.03 g/L h) was
achieved by using 0.05 g/L as inoculum concentration and, in this
case, it was observed an increase of 14% in cell concentration. At
second ethanol production, 13.78 3.90 g/L (0.12 0.03 g/L h)
was attained for inoculum concentration of 0.20 g/L, and in this
assay, a 22.5% reduction of biomass concentration was measured.
In the assay using 0.10 g/L of inoculum, the nal cell concentration
was 75% higher; however the production of ethanol was negligible.
The ethanol production by green algae in mixotrophic conditions is
not fully understood. The ndings indicated that under low cell
inoculum density (0.05 g/L) C. reinhardtii used available nutrients,
under specic operational culture conditions, to ethanol production instead of cell growth. Probably, when higher density was
used, growth factors could have been synthesized by cells and metabolic rotes favoring growth were preferential. In addition, Fig. 1a
and b also showed clearly that the condition that generated the
highest O2 evolution resulted in the lowest ethanol content.
Concerning on the production of by-products (Fig. 1b), two
organic acids were detected, that is, propionic and acetic acids.
The propionic acid was produced in trace amounts from
0.74 0.8  10 2 mg/L up to 6.67 0.2  10 2 mg/L. The analysis
of acetic acid concentration allows identify a trend between this
metabolite and ethanol. For the assays with the highest ethanol

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R.L. Costa et al. / Bioresource Technology 181 (2015) 330337

(a)

(b)

(c)

hexose

CoA

PDC

Pyruvate
CO2

(PFR1)

AcetylCoA
NADPH

Acetaldehyde
NAD(P)H

Acetaldehyde

NADP+CoA
NAD(P)+

NADPH

NAD+
Ethanol

Ethanol
Fig. 1. (a) O2 productivity and biomass evolution. C. reinhardtii culture was kept in anaerobiose under sulfur depletion. (b) Monitoring of ethanol and organic acid
concentration. The fermentation time and inoculum age was 5 days and 10 days, respectively. (c) Metabolism pathways of Chlamydomonas during dark anaerobiosis to
ethanol production (Adapted from Philipps et al., 2011). PFR1: pyruvate ferredoxin oxidoreductase.

contents, 19.25 4.16 (0.16 0.03 g/L h) and 13.78 3.90 g/L
(0.11 0.03 g/L h), the production of acetic acid were the highest
as well, 3.22 0.28 g/L and 3.53 0.81 g/L, respectively. The assay
with negligible ethanol production presented only 1.81 0.098 g/
L of acetic acid, which is around 54% lower than the other assays.
Ethanol production from green algae has not been intensively
studied. Hirano et al. (1997) performed the screening for microalgae with high starch content and high ability for intracellular ethanol production. The green alga Chlorella vulgaris (IAM C-534)
showed the highest starch content (37%) and the ethanol-conversion rate was 65% compared to the theoretical rate from starch.
In the study of the intracellular production by Hirano et al.
(1997), a maximum ethanol concentration (1 w/w%) was obtained
from C. reinhardtii (UTEX2247) and Sak-l isolated from seawater.
Hirano et al. (1997) also evaluated ethanol production with
intracellular starch fermentation. In this work, pH medium, cell
concentration and different microalgae strains were investigated
under dark and anaerobic conditions. Ethanol was observed in
almost all of the tested strains and the highest ethanol productivity
was attained by C. reinhardtii (UTEX2247). In contrast of the current work, Hirano et al. (1997) observed that the formation of ethanol increased almost proportionally to the concentration of the
algal cells. The analysis of the organic metabolites in one of the
anaerobic fermented slurries of Chlamydomonas showed that ethanol was the main metabolite, followed by lactate and acetate.
The analysis of organic metabolites in hydrogen production by
C. reinhardtii CC-124 was performed by Philipps et al. (2011). These
authors determined the production of formate and ethanol

achieved 441.6 and 331.2 ng/lg chlorophyll, respectively and in

smaller production of D-lactate (102.68 ng/lg chlorophyll). In
respect to acetate, this compound was detected in amounts
1020 times lower than the concentration in TAP medium and, in
a medium without acetate; the synthesis of acetate by the alga
was 258.17 ng/lmol chlorophyll.
Mus et al. (2007) determined the nal composition of the
organic metabolites in their study of hydrogen production as well.
In dark and anoxic fermentation, C. reinhardtii produced malate,
formate, acetate and ethanol. The major components were formate,
acetate and ethanol in a molar ratio of 1:1:0.5. In the current work,
the ratio molar of ethanol and acetate varied from 0.4:0.05 to
0.3:0.06 for those testes that showed signicant amount of
ethanol.
Gfeller and Gibbs (1984) investigated the anaerobic degradation
of starch into end products by C. reinhardtii F-60 in the dark and
light. Their study resulted in the production of formate, acetate
and ethanol in the ratios of 2.07:1.07:0.91, with roughly 100% of
starch conversion. Specically to ethanol, the authors concluded
that ethanol synthesis was inhibited by the light in all conditions
tested and on the other hand, formate production was least
affected by light. Furthermore, the other organic compounds, glycerol and lactate, were detected in minor concentrations.
In the current work, besides ethanol, only propionate and acetate were detected in the trials carried out in the evaluation of
the effect of initial cell concentration. Lactate and formate were
not identied as the works aforementioned described. In this work,
formic acid was produced by C. reinhardtii only in medium

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R.L. Costa et al. / Bioresource Technology 181 (2015) 330337

supplemented with milk whey permeated and lactic acid was

produced by the green alga only in RCV medium as it will be
presented in the following sections.
3.1.2. Effect of carbon source
In order to evaluate the inuence of increasing the amount and
the type of the carbon source on the ethanol production by
C. reinhardtii, a set of trials were performed adding sodium acetate
and/or milk whey permeate (MWP) to the TAP-S medium (Table 2).
Heterotrophic cultivation is suitable for a restrict number of
microalgae. According to Perez-Garcia et al. (2011), sucrose, lactate, lactose and ethanol have been tested under heterotrophic
microalgae cultures with negative results in growth and metabolite production. Nevertheless, the results of Table 2 proved that C.
reinhardtii CC-124 cultivated in mixotrophic conditions has the
ability to produce ethanol under anaerobic fermentation with
day/light cycles of 12 h supplemented by sodium acetate and,
especially, with lactose from milk whey permeate.
Table 2 indicated that the TAP-S medium without supplementation of carbon source was not adequate to produce ethanol. This
result is in agreement with the aforementioned data. Nevertheless,
the additions of 0.1 g/L of sodium acetate or milk whey permeate
and the addition of both simultaneously promotes the production
of the biofuel. The concentration of ethanol attained was
9.64 1.84 g/L (0.08 0.016 g/L h), 13.11 2.57 g/L (0.11 0.02 g/
L h), 14.94 6.95 g/L (0.12 0.02 g/L h) in the presence of sodium
acetate, milk whey permeate and in the presence of both,
respectively. Therefore, the medium supplementation with milk
whey permeate resulted in increases of the target-product.
The data shown in Table 2 also indicate that increasing the supplementation to 1.0 g/L of the same mixotrophic carbon sources do
not improve the synthesis of ethanol, indicating that a substrate
inhibition could have occurred. Besides ethanol, further metabolites were detected such as acetic acid, propionic acid and formic
acid. Propionic acid and acetic acid are present in all compositions
of medium. The concentration of the propionic acid is in the order
of 1.4 0.08 mg/L to 8.8 1.75  10 3 mg/L. The content of acetic
acid was superior to 1.8 g/L and achieved 3.03.5 g/L at the highest
production of ethanol, similarly to the previous trials (Fig. 1b). Formic acid was synthesized only when milk whey permeate was
added to TAP-S medium. By taking into account the supplementation of the mixotrophic carbon source of 0.1 g/L, its concentration
range varied from 0.9 g/L for TAP-S medium with MWP to 1.54 g/
L for the medium with TAP-S medium with MWP and Na-acetate.
For the assay with addition of 1.0 g/L of MWP to TAP-S medium,
the nal concentration of formic acid was 1.11 0.45 g/L.
The number of developed studies concerning on the mixotrophic cultivation of algae are rare. The aim of these works was
to evaluate the effect of the different carbon source in growth rate

and bioproducts like biofuel and pigments (Perez-Garcia et al.,

2011; Sahu and Adhikary, 1981). Nevertheless, the inuence of
mixotrophic carbon source in the ethanol production by C. reinhardtii has not found in the literature.
The effect of lactose, fructose, mannose, xylose and sodium acetate on the growth rate and pigment composition by Anabaena sp.
under light and dark conditions was investigated by Sahu and
Adhikary (1981). They did not describe the ethanol formation,
but reported that lactose was the most appropriated carbon source
for growth of the alga. The present work indicated that industrial
wastes containing this disaccharide are promising resource that
can be mixed with water and be used as substrate to C. reinhardtii
in the production of ethanol.
Besides discussing the effect of carbon source as substrate to C.
reinhardtii, it is worth to emphasize that the ethanol amount
attained in the current work was considerable higher than certain
strains of yeast. For instance, Khattak et al. (2014) investigated the
production of ethanol by yeast varying the initial concentration of
glucose from 10 to 50 g/L. They found out that the substrate conversion was complete only at 10 g/L of glucose; and the conversion
decreased 33% at 50 g/L. The production of ethanol increased from
3.83 to 4.56 g/L corresponded to the initial glucose concentration
of 10 and 50 g/L, respectively. The ethanol concentration achieved
in Khattak et al. (2014) was approximately of 30% (4.56 g/L) of the
ethanol production in this paper (14.94 g/L). Nevertheless, low cost
feedstocks that are agroindustrial residues, such as whey cheese,
enables to combine the wastewater treatment efuent with the
production of value-added products and the addition of small
amounts of organic carbon sources leads to higher growth rate
without bacterial contamination (Perez-Garcia et al., 2011).
3.2. Ethanol from hybrid system: C. reinhardtii and R. capsulatus
An alternative biological route to obtain ethanol is to use the
efuent produced in the process by green algae as substrate to
photosynthetic bacteria, particularly, the purple non sulfur bacteria. The integration of these photobiological processes, named
hybrid system, may lead to higher degradation efciency, since
non purple bacteria may utilize the organic acids and non-converted carbon source present in the efuent by C. reinhardtii into
additional ethanol (Batista et al., 2014; Srikanth et al., 2009).
Hybrid systems have been widely studied for hydrogen formation, especially those based in the integration of dark fermentation
by anaerobic agents and photofermentation by sulfur non-purple
bacteria (Tawk et al., 2014; Urbaniec et al., 2014). Nevertheless,
they were not explored for ethanol production by microalgae and
photosynthetic bacteria as it was proposed in the present work.
In Experiment 1, TAP-S medium was used in the rst stage of
the hybrid system metabolized by C. reinhardtii. The potential of

Table 2
Effect of carbon source (0.1 and 1.0 g/L) on the production of ethanol.
Medium

Cacetic

Concentration of C source (0.10 g/L)

TAP-Sb
TAP-S + Na-acetate
TAP-S + MWPc
TAP-S + Na-acetate + MWP

1.79 0.07
2.21 0.64
3.04 0.75
3.49 0.83

1.48  10
5.1  10
4.4  10
8.8  10

0.002
4.3  10 3
3
0.001
3
1.75  10 3

NDa
NDa
0.90 0.37
1.54 0.07

NDa
9.64 1.84
13.11 2.57
14.94 6.95

Concentration of C source (1.0 g/L)

TAP-Sb
TAP-S + Na-acetate
TAP-S + MWPc

1.88 0.05
2.08 0.05
1.89 0.10

1.4  10
3.7  10
2.96  10

NDa
NDa
1.11 0.45

NDa
NDa
NDa

acid

(g/L)

The error estimates are derived from replicated (2) runs.

a
ND not detected.
b
Concentration of acetic acid in TAP-S medium: 1.04 g/L.
c
MWP, milk whey permeate.

Cpropionic

acid

(g/L)

0.008
0.37  10 2
3
0.045  10 2
3

Cformic

acid

(g/L)

Cethanol (g/L)

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R.L. Costa et al. / Bioresource Technology 181 (2015) 330337

Table 3
Effect of the composition medium for R. capsulatus on the production of ethanol in a hybrid system by C. reinhardtii and R. capsulatus.
Triala

Composition of metabolites (g/L)c

C. reinhardtii

1
2

R. capsulatus

Cacet

Cprop

1.79 0.07
1.84 0.05

1.5  10
0.7  10

0.002
3
0.000

Ceth

Clact

Cform

NDb
NDb

0.16 0.11
5.95 4.02

0.01 0.04  10
0.21 0.025

Cacet

Cprop

Cbut

Ceth

2.58 0.41
4.38 0.97

0.80 0.56
5.52 3.89

NDb
2.38 1.60

12.61 2.01
18.22 1.89

The error estimates are derived from replicated (2) runs.

a
Trial 1: First stage, C. reinhardtii TAP-S medium, Second stage R. capsulatus crude efuent; Trial 2: First stage, C. reinhardtii TAP-S medium, Second stage R. capsulatus
efuent with supplements.
b
ND not detected.
c
Metabolites: Acet: acetic acid; prop: propionic acid; lact: lactic acid; form: formic acid; but: butyric acid; eth: ethanol.

Table 4
Effect of the enrichment with 0.1 g/L of extra heterotrophic carbon source in the medium for C. reinhardtii on the production of ethanol in a hybrid system by C. reinhardtii and R.
capsulatus.
Medium in the 1st stage of
hybrid systema

TAP-S
TAP-S + Na-acetate
TAP-S + MWPc
TAP-S + Na-acetate + MWP

Composition of metabolites (g/L)c

C. reinhardtii

R. capsulatus

Cacet

Cprop

1.79 0.07
2.21 0.64
3.04 0.75
3.50 0.83

1.48  10
5.1  10
4.4  10
8.8  10

0.002
3
4.3  10 3
3
0.001
3
1.75  10 3

Cform

Ceth

Cacet

Cprop

Cform

Ceth

NDb
NDb
0.90 0.37
1.54 0.07

NDb
9.64 1.84
13.11 2.57
14.94 6.95

2.32 0.41
3.20 0.22
4.08 0.24
2.90 0.28

0.36 0.056
0.01 0.003
0.05 0.56  10
0.03 0.02

NDb
NDb
0.05 0.01
NDb

10.26 2.01
16.13 2.69
19.94 2.67
17.17 2.63

The error estimates are derived from replicated (2) runs.

a
Second stage: R. capsulatus crude efuent;
b
ND not detected.
c
Metabolites: Acet: acetic acid; prop: propionic acid; lact: lactic acid; form: formic acid; but: butyric acid; eth: ethanol; MWP: milk whey permeate.

R. capsulatus of producing ethanol from the metabolites synthesized by the green alga was evaluated comparing the biofuel production using the crude fermentative broth and the efuent
supplemented with sodium glutamate, malic acid, milk whey permeate and micronutrients. In both cases, the efuent was free of C.
reinhardtii and fermentation time was 5 days in the two stages of
the hybrid system. The results are presented in Table 3.
The results shown in Table 3 proved that R. capsulatus synthesis
ethanol even though the macro and micronutrients, usually found
in RCV medium, were not present. Table 3 also indicated that ethanol production by R. capsulatus cultivated in efuent supplemented with malic acid, milk whey permeate and sodium
glutamate was 44.5% superior (18.22 g/L) than when bacteria were
cultivated in the crude efuent (12.61 g/L).
In respect to the metabolites, the amount of organic acids was
higher as well. In the crude efuent there were around
1.79 0.07 g/L to 1.84 0.55 g/L of acetic acid and at the end, the
efuent produced by R. capsulatus in the medium without
supplementation was composed by 0.16 0.11 g/L of lactic acid,
2.58 0.41 g/L of acetic acid, 0.01 0.04  10 4 g/L of formic acid
and 0.80 0.56 g/L of propionic acid. The composition of the
efuent by R. capsulatus in the supplemented medium was
5.95 4.02 g/L of lactic acid, 4.38 0.97 g/L of acetic acid,
5.52 3.89 g/L of propionic acid, 0.21 0.025 g/L of formic acid
and 2.38 1.60 g/L of butyric acid.
In the following Experiments 2 and 3, an efuent produced by C.
reinhardtii cultivated in a medium enriched with a carbon source
was used by R. capsulatus. In the Experiment 2, the medium used
by C. reinhardtii was supplemented with 0.1 g/L of sodium acetate
or milk whey permeate or both of the components and the composition of the efuent is shown in Tables 2 and 4. In the Experiment
3, the supplementation of the medium was made by the addition of
1.0 g/L the same components and the composition of the efuent is
shown in Tables 2 and 5.

As it was discussed aforementioned, the medium enriched with

0.1 g/L of milk whey permeate increased the ethanol production by
C. reinhardtii. The efuent produced from this medium led to a
higher nal production of the biofuel, 19.94 2.67 g/L
(0.17 0.02 g/L h) by R. capsulatus, as Table 4 demonstrated.
Although the efuent utilized by the bacteria was not supplemented with malic acid and MWP, the acetic acid and formic acid
may have provided carbon source enough to R. capsulatus
metabolism.
Concerning on the organic acids, except to TAP-S medium with
sodium acetate and milk whey permeate, acetic acid and propionic
acid were synthesized in both stages of the hybrid system. In the
trials with TAP-S medium containing MWP, the formic acid produced in the rst stage was consumed by the photosynthetic bacteria. Acetic acid produced by C. reinhardtii was consumed by R.
capsulatus in the assay with TAP-S medium supplemented with
sodium acetate and MWP.
The analysis of Tables 2 and 5 showed that no production of
ethanol was veried at higher concentration of carbon source
(1.0 g/L), independently whether sodium acetate or milk whey permeate were added. Besides the ethanol, acetic acid was synthesized by R. capsulatus only in the trial of TAP-S medium without
supplementation. In further trials, the addition of 1.0 g/L of sodium
acetate or milk whey permeate led to a metabolic pathway in
which consume of acetic acid and no production of ethanol by
the bacterium were observed.
Complementarily, to produce biofuels as hydrogen and ethanol,
purple non-sulfur bacteria commonly synthesize polyhydroxyalkanoates. In the current work, the consumption of the organic acids
without production of ethanol by R. capsulatus suggested that further metabolites could be synthesized, as it was demonstrated by
Sawayama (2001) and Maeda et al. (1998).
Hybrid systems incorporating two stages of algae and photosynthetic bacteria are hardly to nd published, since the focus of

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R.L. Costa et al. / Bioresource Technology 181 (2015) 330337

Table 5
Effect of the enrichment with 1.0 g/L of extra heterotrophic carbon source in the medium for C. reinhardtii on the production of ethanol in a hybrid system by C. reinhardtii and R.
capsulatus.
Medium in the 1st stage of hybrid systema

Composition of metabolites (g/L)c

Chlamydomonas reinhardtii

TAP-S
TAP-S + Na-acetate
TAP-S + MWPc

Cacet

Cprop

1.88 0.16
2.08 0.30
1.89 0.35

1.4  10
3.7  10
2.96  10

Rhodobacter capsulatus

0.17
3
0.001
3
0.001

Ceth

Cacet

Cprop

Ceth

NDb
NDb
NDb

4.09 0.15
1.08 0.37  10
0.95 0.28

NDb
0.03 0.003
0.02 0.002

12.57 0.01
NDb
NDb

The error estimates are derived from replicated (2) runs.

a
Second stage: R. capsulatus crude efuent;
b
ND not detected.
c
Metabolites: Acet: acetic acid; prop: propionic acid; lact: lactic acid; form: formic acid; but: butyric acid; eth: ethanol. MWP: milk whey permeate.

Table 6
Composition of metabolites produced by co-culture system based on different C reinhardtii (A) to R. capsulatus (B) ratios.
Composition of metabolites (g/L)b
Strain ratio

Clact

Basal medium

RCV

TAP-S

RCV

TAP-S

RCV

TAP-S

RCV

TAP-S

A
A
A
A
A
A
A

0.57 0.18
0.15 0.07
0.07 0.02
0.31 0.22
0.41 0.28
0.78 0.18
0.27 0.07

NDa
0.01 0.65  10
NDa
0.27 0.19
NDa
0.26 0.02
NDa

0.58 0.33
1.47 0.09
0.46 0.10
0.32 0.067
0.37 0.069
0.93 0.15
2.58 1.00

3.45 0.29
3.42 0.33
4.94 1.60
3.73 1.82
3.64 0.73
2.37 2.20
3.09 0.15

0.54 0.15
0.20 0.14
NDa
NDa
NDa
0.33 0.20
0.91 0.08

NDa
1.1  10 2 0.83  10
4.4  10 2 0.03  10
0.91 0.64
2.4  10 2 0.11  10
6.7  10 3 0.53  10
1.5  10 3 0.02

9.00 6.36
NDa
0.79 0.06
NDa
NDa
NDa
12.61 8.91

NDa
0.08 0.06
0.25 0.12
0.22 0.16
NDa
0.12 0.02
0.09 0.02

(0%): B (100%)
(10%): B (90%)
(30%): B (70%)
(50%): B (50%)
(70%): B (30%)
(90%): B (10%)
(100%): B (0%)

Cacet

Cprop

Ceth

2
2

2
2

The error estimates are derived from replicated (2) runs.

a
ND not detected.
b
Metabolites: Acet: acetic acid; prop: propionic acid; lact: lactic acid; but: butyric acid; eth: ethanol.

the works developed by Maeda et al. (1998), Miura et al. (1997)

and Miura et al. (1992) was the hydrogen production.
In order to investigate hydrogen production, Maeda et al. (1998)
proposed an integrated process where the efuent produced by
Chlamydomonas sp. MGA161, cultivated under dark and anaerobic
conditions, was used by Rhodovulum suldophilum W-1S. In the
rst stage, acetic acid, ethanol and glycerol were the main products
excreted into the medium during Chlamydomonas sp. MGA161. In
the second stage, there was consumption by R. suldophilum W1S of all these organic acids immediately after hydrogen evolution
started. The amounts of the metabolites were not discussed by the
authors.
Miura et al. (1997) scaled up photobiological hydrogen production system to a pilot plant scale in the photosynthetic starch
accumulation, followed by the fermentative production of metabolites from starch by Chlamydomonas MGA 161 and the hydrogen
photoproduction by R. sulfidophilum W-1S from fermentative
broth produced by Chlamydomonas MGA 161 in the natural day/
night cycle. The yield of acetic acid, ethanol and glycerol from
starch of Chlamydomonas MGA 161 was 80100% of the theoretical yield.
Maeda et al. (1998) and Miura et al. (1997) also described that
the decrease in the conversion of substrate into hydrogen production was due to the accumulation of polyhydroxybutyrate (PHB).
Similar conclusion was withdrawn by Sawayama (2001) that studied the consumption of acetate by photosynthetic bacteria. The
author reported the production of polyhydroxybutyrate (PHB) by
phototrophic bacteria using as substrate an efuent from lighted
upow anaerobic sludge blanket (LUASB) reactor with sodium
acetate under anaerobic light condition. The concentration of
PHB enhanced in a secondary incubation of this efuent with
Na-acetate.

3.3. Ethanol from co-culture: C. reinhardtii and R. capsulatus

In this current work, strategies of co-cultivation of C. reinhardtii
(green alga) and R. capsulatus (PNS Bacteria) to ethanol production
were also attempted. In this coupled system, R. capsulatus could
use the organic acids excreted by C. reinhardtii cells during anaerobic photofermentation process. Furthermore, the integration of
these two biological systems may enhance the ethanol production
with a reduction in energy cost, due to better solar irradiance utilization (visible and infrared) and integrated use of the nutrients
(Melis and Melnicki, 2006). Table 6 summarizes the results of cocultivation. Two fermentation media were tested in the co-cultivation, that is, RCV and TAP-S.
In the RCV medium used in the co-cultivation, lactic and acetic
acids were produced in all algae to bacteria ratios. The production
of propionic acid and ethanol did not present a trend by increasing
the alga to bacteria ratio. Surprisingly, isolated green alga [Algae
(100%): Bacteria (0%)] cultured in RCV medium (a basal medium
to PNS bacteria) was fully capable to synthesize ethanol resulting
in 12.6 8.91 g/L (0.11 0.07 g/L h).
Similar procedure was also performed to co-culture maintained
in TAP-S medium. However, R. capsulatus has not presented a good
performance in relation to the ethanol production in this medium
formulation, probably due the lack of essential nutrients.
In TAP-S medium, our ndings showed that C. reinhardtii and R.
capsulatus contributed equally to acetic acid production, since at
isolated bacterial culture [Algae (0%): Bacteria (100%)] resulted in
3.45 0.29 g/L and isolated algal culture [Algae (100%): Bacteria
(0%)] produced up to 3.09 0.15 g/L. In addition, propionic acid
was produced when the variant ration of 1:1 of cells was used in
the co-cultivation. On the other hand, the nal ethanol concentration [Algae (30%): Bacteria (70%)] was at least 3.2-fold lower than

R.L. Costa et al. / Bioresource Technology 181 (2015) 330337

the minimum ethanol content observed in the co-culture maintained in RCV medium (0.79 0.06 g/L) at the same variant ratio.
The co-culture approach of microalgae and photosynthetic bacteria has not been explored and the inuence of lots of variables is
still unknown. However the results of ethanol content obtained
through the co-cultivation strategy were lower than those from
mixotrophic photofermentation by C. reinhardtii isolated or in
hybrid system with R. capsulatus. It is necessary to enlarge the conditions of this technique by evaluating the composition of the medium, including the supplementation of organic carbon source,
nutrients and light intensity as Melis and Melnicki (2006) did.
These authors investigated the hydrogen production by coupling
Rhodospirillum rubrum and C. reinhardtii, in a bioreactor containing
all the nutrients of both Ormerod and TAP that are media used to
the maintenance of bacteria and algae cultures, respectively.
Moreover, other parameter evaluated was the light intensity
(30150 lmol photons m 2 s 1), since the level of irradiance could
regulate the relative growth and production of metabolites of the
two types of cells. Melis and Melnicki (2006) did not present data
related to the composition of organic acids and ethanol.
In general, for each system approached in this work, it is important to emphasis that a full study must be performed in order to
enhance the comprehension of the effect of the medium composition and operational conditions on the ethanol production by
C. reinhardtii and R. capsulatus. For instance, the light intensity,
residual sulfur content, photoperiod and bioreactor congurations
may inuence the biofuel synthesis. The comprehension of
metabolism of different organic compounds by C. reinhardtii and
R. capsulatus requires more detailed studies. Further researches
may lead to signicant progress in improving the biomass accumulation, starch production and co-production of ethanol. The results
of this work may be used in future design of ethanol production in
integrated systems weather by hybrid or co-culture systems.
4. Conclusion
In this study the effect of inoculum concentration and carbon
source to C. reinhardtii, as well as the inuence of hybrid system
and co-culture (C. reinhardtii and R. capsulatus) on the photofermentative ethanol production were investigated. Maximum ethanol content 19.94 2.67 g/L (0.17 0.02 g/L h) was achieved by
hybrid system in which the efuent of C. reinhardtii containing
organic acids was used as substrate to R. capsulatus. The results
from this work are benecial to comprehend the potentiality of
green alga and PNS photosynthetic bacteria to synthesize ethanol
concerning several strategies such as media composition and different culture systems (hybrid and co-cultivation).
Acknowledgements
The authors wish to thank Brazilian agencies CNPq, CAPES and
FAPEMIG (Grant No. APQ-01020-12) for nancial support.
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