Journal of Biotechnology 150 (2010) 51–56

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Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec

Microalgae as substrates for fermentative biogas production in a combined

biorefinery concept
J.H. Mussgnug ∗ , V. Klassen, A. Schlüter, O. Kruse
Bielefeld University, Center for Biotechnology, Universitätsstrasse 27, 33615 Bielefeld, NRW, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Most organic matter can be used for bioenergy generation via anaerobic fermentation. Today, crop
Received 15 June 2010 plants like maize play the dominant role as substrates for renewable biogas production. In this work
Received in revised form 20 July 2010 we investigated the suitability of six dominant microalgae species (freshwater and saltwater algae and
Accepted 27 July 2010
cyanobacteria) as alternative substrates for biogas production. We could demonstrate that the biogas
potential is strongly dependent on the species and on the pretreatment. Fermentation of the green alga
Chlamydomonas reinhardtii was efficient with a production of 587 ml (±8.8 SE) biogas g volatile solids−1
Keywords:
(VS−1 ), whereas fermentation of Scenedesmus obliquus was inefficient with only 287 ml (±10.1 SE) bio-
Bioenergy
Biogas
gas g VS−1 being produced. Drying as a pretreatment decreased the amount of biogas production to ca.
Biorefinery 80%. The methane content of biogas from microalgae was 7–13% higher compared to biogas from maize
Fermentation silage. To evaluate integrative biorefinery concepts, hydrogen production in C. reinhardtii prior to anaer-
Methane obic fermentation of the algae biomass was measured and resulted in an increase of biogas generation
Microalga to 123% (±3.7 SE). We conclude that selected algae species can be good substrates for biogas production
and that anaerobic fermentation can seriously be considered as final step in future microalgae-based
biorefinery concepts.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction persed and strongly fluctuating nature in which solar irradiation

reaches the planet. These methods include photovoltaics, the col-
The global energy demand keeps rising at a dramatic speed since lection of solar heat and the biological production of plant biomass
the beginning of the industrial revolution in the late 18th century. In and subsequent conversion. Biomass can be converted into a num-
contrast, easy accessible fossil fuel reserves rapidly decrease which ber of different products, e.g. into bioethanol, biodiesel or biogas. In
leads to increasing energy prices. For these reasons, one of the major recent years it has increasingly become clear that “first generation”
challenges for industrialized countries today is it to ensure the biofuels such as ethanol production from plant sugars or biodiesel
energy supply for the future. Combustion of fossil energy carriers production from plant lipids have got comparably bad energy bal-
like petrol, natural gas or coal leads to the release of CO2 and there- ances and therefore most likely can never play a major role in global
fore to environmental problems which are projected to manifest in energy supply, whereas “second generation” biofuels, which con-
problematic climate changes (IPCC, 2007). In recent years, numer- vert the whole plant (e.g. biomass-to-liquid or biogas fermentation)
ous ideas have been considered to develop environmentally more offer far greater potentials (IEA, 2010).
friendly alternatives. The energy sources which are tapped include In general, the use of plant biomass for energy generation today
wind energy, geothermal temperature differences, kinetic energy is problematic because of the competition with food or feed produc-
stored in water (e.g. wave and tidal movements of the oceans or tion. This is because most of the plants used for energy generation
river dams) and the irradiation of the sun. From these, by far the today (crop plants, sugar cane, sugar beets, canola, etc.) have to be
biggest energy source is the solar irradiation. It has been calculated grown on arable land. Low demand alternatives like switchgrass
that the energy which reaches the earth’s surface equals to around are only beginning to emerge.
5600 times the global energy demand today (Schenk et al., 2008). Algae have got a number of potential advantages compared to
A variety of methods have been developed to harvest this huge higher plants because of faster growth rates and the possibility
energy source, which is technically challenging because of the dis- of cultivation on non-arable land areas or in lakes or the ocean,
therefore attenuating food and feed competition (Rittmann, 2008;
Stephens et al., 2010). A promising approach therefore seems to
Abbreviations: SE, standard error; VS, volatile solids.
be the use of fast-growing algae species for anaerobic fermen-
∗ Corresponding author. Tel.: +49 0521 106 12257; fax: +49 0521 106 12290. tation to produce biogas, which then can substitute natural gas
E-mail address: jan.mussgnug@uni-bielefeld.de (J.H. Mussgnug). resources. Research on anaerobic fermentation of algae biomass

0168-1656/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbiotec.2010.07.030
52 J.H. Mussgnug et al. / Journal of Biotechnology 150 (2010) 51–56

goes back to more than 50 years ago (Golueke et al., 1957). Since were washed and re-suspended in sulfur-free medium. The culture
then quite a number of research projects have been carried out. was then sealed and incubated in the light (600 ␮mol m−2 s−1 ) at
The early research efforts peaked in the late 1970th and 1980th room temperature. Under these conditions, photosystem II is pro-
as a consequence of the first oil crises. Species under investigation gressively inhibited while mitochondrial respiration stays active,
included several macroalgae such as Macrocystis, Gracilaria, Hyp- leading to anaerobic culture conditions and subsequent hydrogen
nea, Ulva, Laminaria and Sargassum (Chynoweth, 2002). Recently, production approximately 24 h after cell transfer into sulfur-free
the identification of microalgal strains with promising character- medium (Melis et al., 2000). Cells were harvested when hydrogen
istics (Eroglu and Melis, 2010), progress in microalgae cultivation production had stopped (192 h after the transfer) and the biomass
(Posten, 2009) and harvesting techniques (Brennan and Owende, was used for fermentation tests.
2010) as well as the potential of some strains to produce valu-
able co-products (Spolaore et al., 2006) has raised the interest to
2.3. Anaerobic substrate fermentation and biogas analysis
use these organisms for bioenergy generation. In contrast to higher
plants and macroalgae, some microalgae like the green microalga
Substrate fermentation was conducted in 250 ml batch tests at
Chlamydomonas reinhardtii have the remarkable ability to produce
38 ◦ C according to the guideline VDI 4630 of the Verein Deutscher
hydrogen via hydrolysis of water during illumination (Kruse et al.,
Ingenieure (VDI, 2004). 250 ml biogas batch fermenters were filled
2005b; Melis et al., 2000), which represents an additional envi-
with 60 ml sludge from a local sewage plant, cellular material cor-
ronmentally friendly gaseous fuel. This potential has stimulated
responding to 0.5 g of dried biomass per test was loaded and the
the research interest in recent years (Doebbe et al., in press, 2007;
fermenter then sealed with a rubber septum. The amount of bio-
Hemschemeier et al., 2009; Nguyen et al., 2008; Ruhle et al., 2008;
gas produced was determined by measurements of the pressure
Timmins et al., 2009). Hydrogen generation is a two-phase pro-
(WAL-BMP-Test system 3150, WAL, Germany) building up in the
cess with an aerobic and an anaerobic stage, during which the cells
fermenter head space. Fermenters without addition of substrates
undergo major physiological changes. After hydrogen production,
were used as negative controls. Biogas composition was deter-
algal biomass remains as a waste product. In the context of bioen-
mined with an ATEX biogas monitor BM2000 (Ansyco, Germany).
ergy production with microalgae it has been suggested that residual
The individual biogas production curves were analyzed with the
algal biomass should be converted into biogas via anaerobic fer-
curve fitting software at Zunzun.com to derive the mathematical
mentation (Chisti, 2007; De Schamphelaire and Verstraete, 2009).
description of the curves and obtain specific values for each time
Although research in the field of microalgae as substrates for bio-
point. Cell degradation rates were determined by light microscopy
gas production is very limited (Golueke et al., 1957; Hernandez and
(Motic BA310, Motic, China) of fermenter samples and subsequent
Cordoba, 1993; Legros et al., 1983; Samson and LeDuy, 1986; Yen
cell counting.
and Brune, 2007), recent theoretical calculations (Sialve et al., 2009)
indicated their potential.
In this study we determined the potential of six dominant 3. Results and discussion
microalgal species as a substrate for biogas production. In addition,
we tested the influence of drying as a pre-treatment. The appli- 3.1. Microalgal biogas production is strongly dependent on the
cation of microalgae in a two-step biorefinery process (1st step selected strain
hydrogen production, 2nd step fermentative biogas production)
was investigated with the green microalga C. reinhardtii. The microalgal species selected for this approach are all com-
mon in moderate climate zones and show fast growth rates in
the nature and under standard growth conditions in the labo-
2. Materials and methods
ratory, therefore they represent a selection of dominant strains.
Five eukaryotic microalgal species were selected; four green algae
2.1. Growth and culture conditions
(C. reinhardtii, Dunaliella salina and Scenedesmus obliquus from
the class Chlorophyceae and Chlorella kessleri from the class Tre-
C. reinhardtii strain cc124 was obtained from the Chlamy-
bouxiophyceae) and one euglenoid species (Euglena gracilis from
domonas Center (Duke University, Durham NC, USA). All other
the class Euglenoidea) as well as the prokaryotic cyanobacterium
microalgal strains used in this study were obtained from the SAG
Arthrospira platensis (class Cyanophyceae). D. salina and A. platen-
algae collection (Goettingen University, Germany). Liquid cultures
sis are halophilic species; all other species tested are fresh water
were grown in continuous white light (40 ␮mol m−2 s−1 ), TAP
microalgae.
medium (Harris, 2009) was used for C. reinhardtii, C. kessleri and E.
The suitability of fresh microalgal biomass as substrate for the
gracilis (in the latter case, Thiamin (0.1 mg/l), Biotin (0.5 ␮g/l) and
production of biogas was assessed in anaerobic fermentation batch
vitamin B12 (0.5 ␮g/l) were added), Spirulina medium (Aiba and
tests over a period of 32 days (Fig. 1). Equal amounts of biomass (on
Ogawa, 1977) was used for A. platensis, ProF medium (Provasoli et
the basis of dry biomass) were loaded.
al., 1957) was used for S. obliquus and 2 M NaCl medium (Pick et al.,
As a first important result, the experiments revealed that
1986) was used for D. salina. Algae cells were harvested by centrifu-
the biogas quantity produced in the fermenters was strongly
gation (6 min at 3.100 × g) and the content of organic dry biomass
dependent on the species. The green freshwater alga C. rein-
of the pellets was determined by drying at 105 ◦ C for 24 h. For com-
hardtii was identified as the most efficient biogas substrate
parative fermentation tests, fresh or dried cells corresponding to
(587 ml ± 8.8 SE g VS−1 ), followed by the halophilic green alga D.
equal organic dry biomass were applied as substrates.
salina (505 ml ± 24.8 SE g VS−1 ). Compared to the standard sub-
strate control Z. mays silage (653 ml ± 37.7 SE g VS−1 ), these two
2.2. Hydrogen production in C. reinhardtii algae produced 90% (C. reinhardtii) and 77% (D. salina) of the bio-
gas amount (Fig. 1), respectively. Application of biomass from the
Hydrogen production in C. reinhardtii was induced via the sulfur prokaryotic cyanobacterium A. platensis or the euglenoid alga E.
deprivation method established by Melis et al. (2000) as described gracilis as substrates also resulted in comparably high biogas pro-
in detail elsewhere (Doebbe et al., 2007). Briefly, cells were grown duction (both 74% of the control) with 481 ml ± 13.8 SE g VS−1 for
in sulfur-containing medium until they reached the early stationary A. platensis and 485 ml ± 3 SE g VS−1 for E. gracilis, respectively. Bio-
growth phase and then harvested by centrifugation. The cell pellets gas production from C. kessleri was significantly lower (335 ml ± 7.8
 J.H. Mussgnug et al. / Journal of Biotechnology 150 (2010) 51–56 53

Table 1
Summary of the microalgal strains used and the fermentative biogas production characteristics. The biogas yield is calculated relative to the control substrate maize silage.

(P)ro- or (E)ukaryotic species Fresh (F) or salt (S) water Biogas production (ml g VS−1 ) CH4 content Methane yield (% control)

Arthrospira platensis (P) S 481 ± 13.8 61% 83%

Chlamydomonas reinhardtii (E) F 587 ± 8.8 66% 111%
Chlorella kessleri (E) F 335 ± 7.8 65% 62%
Dunaliella salina (E) S 505 ± 24.8 64% 93%
Euglena gracilis (E) F 485 ± 3 67% 93%
Scenedesmus obliquus (E) F 287 ± 10.1 62% 51%
Zea mays (E) F 653 ± 37.7 54% 100%

SE g VS−1 , 51% of the control), but still superior compared to S. tigated the cellular disintegration of the algal substrate by light
obliquus (287 ml ± 10.1 SE g VS−1 , 44% of the control), which repre- microscopy. Fresh algal substrate was centrifuged and added to
sented the worst strain in terms of anaerobic degradability (Fig. 1). batch fermenters and the kinetics of cell disintegration determined
These results clearly showed that the suitability of microalgae for by cell counting. Interestingly, the salt water species disintegrated
anaerobic fermentation and biogas production cannot be predicted very fast after addition to the fermenter sludge (A. platensis and D.
from the classification of the organism and indicates that the biogas salina; Fig. 2).
potential is strain-specific and always needs to be tested individu- Here, very few (Fig. 3B, arrow) or no (Fig. 3C) indigestible
ally. residues of the cells were detected via light microscopy. In contrast,
The main components of biogas are methane and carbon diox- all fresh water microalgae generally showed slower decomposition
ide. The variable, relative amount of methane determines the biogas rates (Fig. 2) with some indigestible residues remaining (Fig. 3A,
quality and depends on the substrate and the fermentation con- D–F).
ditions (Sialve et al., 2009). All microalgae tested showed higher In general, the decrease of the cell degradation correlated well
specific methane contents (ranging from 61% to 67%) compared to with the amount of biogas produced. The species with a high degree
the standard substrate maize silage (54%; Table 1). This result is in of decomposition and low amount of indigestible residues (C. rein-
good agreement with theoretical considerations and previous stud- hardtii, D. salina, A. platensis and E. gracilis) showed higher amounts
ies (Sialve et al., 2009) and indicates the potential of algal substrates of biogas production compared to the species with a lower degree
for superior biogas quality compared to traditionally used higher of decomposition and higher amount of indigestible residues (C.
plants. Taking this higher specific methane content into account, kessleri and S. obliquus) (Figs. 1 and 2; Table 1). Consequently, our
fresh biomass from C. reinhardtii produced 11% more pure methane results indicate that without a pretreatment, the accessibility to
when compared to fresh biomass derived from Z. mays (Table 1). cell disintegration is most likely a major factor for the efficiency of
Hydrogen sulfide (H2 S) is commonly found in biogas produced from fermentative biogas production.
organic substrates in small amounts. Because of its toxic and corro- It should be noted that all easy degradable species investigated
sive nature, low amounts of H2 S are desirable. Although we did not in this study have got no cell wall (D. salina (Sheffer et al., 1986))
determine H2 S levels within the biogas from microalgal substrates, or a protein-based cell wall containing no cellulose or hemicellu-
it has been suggested that the H2 S levels should be low because of lose (C. reinhardtii (Miller et al., 1972), A. platensis (van Eykelenburg
the comparably low amount of sulfurated amino acids in microal- et al., 1980), E. gracilis (Nakano et al., 1987)). In contrast, C. kessleri
gae (Sialve et al., 2009). However, future studies on the combustion and S. obliquus are characterized by having carbohydrate-based cell
and purification characteristics of biogas from microalgae will be walls containing hemicellulose (Takeda, 1991, 1996). The cell wall
necessary to exclude unknown and potentially detrimental aspects of S. obliquus has been described as particular rigid because it con-
before large scale application can be considered. tains a sporopollenin-like biopolymer (Burczyk and Dworzanski,
1988) which explains why no cell degradation of this strain could
3.2. The biogas potential correlates with the level of cellular be detected (Figs. 2 and 3F). It is worth noting that we were able
disintegration to detect intact Scenedesmus cells (as assessed from microscopic
images) more than six months after the transfer into the fermenter
The degree of cell degradation is crucial for the conversion (data not shown). During this time, the fermenter was kept in dark-
efficiency from algae biomass to biogas. Consequently, we inves- ness, therefore preventing photosynthetic reactions. It has been

Fig. 1. Net biogas production of six microalgal strains. Fresh algal biomass was sub-
jected to fermentation on the basis of equal dry biomass content. Maize silage was Fig. 2. Kinetics of microalgal cell disintegration in the fermenter. Fresh microalgal
used as a positive control. The gas amount produced by fermenters without substrate biomass was added to the fermenter sludge and the cell number was monitored by
addition (negative control) was subtracted. Error bars represent standard errors. light microscopy. Error bars represent standard errors.
54 J.H. Mussgnug et al. / Journal of Biotechnology 150 (2010) 51–56

Fig. 3. Light microscopic images of microalgal cells before (−) and after (+) incubation in the fermenter sludge for 28 days in darkness at mesophilic temperatures (38◦ C).
(A) C. reinhardtii; (B) D. salina; (C) A. platensis; (D) E. gracilis; (E) C. kessleri; (F) S. obliquus. Scale bars represent 10 ␮m.

shown that Scenedesmus can utilize a wide variety of sugars (e.g. This was true for the control, Z. mays (−21 ± 2.4%) and also
glucose, fructose or galactose) and organic acids (e.g. acetate or for the two algal cell lines tested, C. kessleri (−23 ± 2.8%) and C.
pyruvate) for heterotrophic growth (Dvorakov, 1966). Therefore reinhardtii (−20 ± 2.7%). The most likely reasons for the decreased
our results indicate that the cells, protected from bacterial disin- biogas production are the loss of volatile organic compounds of
tegration, were indeed able to survive by uptake of fixed carbon high fermentation potential and/or a decreased accessibility of the
compounds from the fermenter sludge. However, we did not see dried organic compounds for the bacterial biocenosis within the
any evidence for algal cell growth or division within the fermenter. fermenter sludge. In any case, our results demonstrate that drying
Interestingly, a comparably low, but significant biogas production is detrimental in terms of biogas production and should be avoided.
was measured with S. obliquus substrate despite the fact that the Since drying of the biomass would require energy of some sort
cell number remained constant. A possible explanation for the bio- it can be concluded that the most energy efficient way of using
gas production could be that to a certain extent, dead/broken cells algal biomass for fermentation is to use fresh biomass and avoid
originating from the cell cultivation were transferred to the fer- transportation if possible. This could be achieved by building and
menter and, in contrast to the living cells, served as substrate for operating the algal production facility in close proximity to the
biogas production. Another explanation could be that the surviving biogas fermentation plant.
Scenedesmus cells actively promoted degradation of organic com-
pounds present in the fermenter sludge, which were not accessible
3.4. Hydrogen production in C. reinhardtii leads to higher
to the bacterial community.
subsequent biogas production levels
In conclusion, our data indicate that the presence and com-
position of the cell wall is the main reason for the differences
Industrial large scale growth of microalgae still is in its infancy
observed in the cell disintegration characteristics and subsequent
and the algae biomass therefore rather expensive. The general con-
biogas production. In terms of biogas production efficiency, strains
sensus today seems to be that biorefinery concepts have to be
with no cell wall or a protein-based cell wall should be pre-
adopted to achieve economical feasibility, where algae are used
ferred because disruptive, energy consuming pretreatments can
to produce a valuable substance prior to being subjected to fer-
be avoided. However, we cannot exclude the possibility that even
mentation (Chisti, 2007; Schenk et al., 2008; Spolaore et al., 2006;
microalgae without a rigid cell wall could be bad substrates for
Stephens et al., 2010). The green microalga C. reinhardtii has the
fermentative biogas production. This is because it is likely that
ability to produce biosolar hydrogen (H2 ) under anaerobic con-
some microalgae will produce compounds which exert detrimental
ditions (Doebbe et al., 2007; Hemschemeier et al., 2009; Kruse
effects on the bacterial biocenosis of the fermenter (Klocke et al.,
2007; Schlüter et al., 2008), e.g. by inhibition of the methanogenic
archaea. This could explain why D. salina and A. platensis substrates,
although rapidly and completely degraded, resulted in less biogas
production than the C. reinhardtii substrate (Figs. 1 and 2).

3.3. Drying as a pretreatment decreases the fermentative

potential of the substrates

Microalgae are grown in liquid medium for mass cultivation and

the dry matter content usually is below 15 g/l culture, although
up to 84 g/l have been reported (Hu et al., 1998). Efficient biogas
production will therefore require a concentration step, e.g. by fil-
tration or centrifugation. Depending on the concentration method,
the fresh algal biomass still contains a high degree of water, e.g. in
our case a typical Chlamydomonas pellet after centrifugation con- Fig. 4. The influence of drying and hydrogen production as pretreatments on the
tained ca. 6% VS and 94% water. For transportation and storage it biogas production potential of the substrate. Fresh biomass (F) was directly used
could be desirable to use dry algal biomass instead of algal biomass for fermentation or dried (D) at 105 ◦ C for 24 h prior to fermentation. In addition,
concentrate. We therefore tested the effect of drying of the sub- C. reinhardtii cells were subjected to hydrogen production and fresh biomass sub-
sequently used for fermentation (F/H2 ). Equal amounts on the basis of dry biomass
strate on biogas fermentation. As can be seen in Fig. 4, drying of the
were loaded. The gas amount produced by fermenters without substrate addition
biomass resulted in a general decrease of around 20% of the biogas (negative control) was subtracted. Z.m., Zea mays; C.k., Chlorella kessleri; C.r., Chlamy-
production potential. domonas reinhardtii. Error bars represent standard errors.
 J.H. Mussgnug et al. / Journal of Biotechnology 150 (2010) 51–56 55

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