Algal Research 12 (2015) 295–299

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Algal Research

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

Fermentative hydrogen and polyhydroxybutyrate production from

pretreated cyanobacterial blooms
Jinling Cai a,c,⁎, Minglang Chen b, Guangce Wang d, Guanghua Pan a, Peng Yu b,c,⁎⁎
a
College of Chemistry Engineering and Material Science, Tianjin University of Science & Technology, Tianjin Key Laboratory of Marine Resources and Chemistry, Tianjin 300457, China
b
College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China
c
Tianjin International Joint Academy of Biomedicine, Tianjin 300457, China
d
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China

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

Article history: The cell wall and cytoplasmic membrane components of algal biomass (in cyanobacterial blooms) are resistant to
Received 8 April 2015 biodegradation during anaerobic digestion. Various pretreatment methods including thermal, alkaline and acid
Received in revised form 14 September 2015 pretreatments, were performed (each as a two-stage process) to increase the biodegradability of the algal bio-
Accepted 23 September 2015
mass in terms of hydrogen and polyhydroxybutyrate (PHB) production. Among the pretreatment methods, ther-
Available online xxxx
mal pretreatment achieved the highest hydrogen production (113 mL/g VS), followed by alkaline pretreatment
Keywords:
(94 mL/g VS). Following hydrogen production, phototrophic bacteria were inoculated into the fermentative
Hydrogen production broth, for PHB production. The group that had undergone alkaline pretreatment produced the highest amount
Polyhydroxybutyrate production of PHB (about 1.69 g/L). Our studies indicate that pretreatment is a feasible option for transforming algal biomass
Algal biomass into a bio-available material, for the purpose of microbial hydrogen production and PHB conversion.
Pretreatment © 2015 Elsevier B.V. All rights reserved.
Fermentation

1. Introduction from algal biomass is considered a low-cost, environment-friendly meth-

Due to the shortage of fossil fuels, as well as problems associated
with environmental pollution, there is an urgent need to identify
sources of clean, renewable, sustainable and carbon-neutral energy. Hy-
drogen is considered a favourable alternative energy source, because of
its high energy density and clean-combustion products. Among the var-
ious hydrogen production technologies, fermentative hydrogen produc-
tion, from biomass and organic wastes, is considered as one of the most
promising processes for developing sustainable energy [1,2].
Cyanobacterial blooms frequently occur in eutrophic lakes, reservoirs,
and polluted waters, worldwide. In recent years, cyanobacterial blooms
have increased in frequency and intensity in several lakes in China [3].
The excessive growth of cyanobacteria in lakes can detrimentally affect
ecosystem functioning in lakes. Moreover, many cyanobacterial strains
contain microcystins, which pose a threat to human and animal health
[4]. The treatment of algal blooms is a worldwide problem. Algal biomass
in cyanobacterial blooms, which contain high levels of starch, polysaccha-
rides, oligomers and hydrocarbons, are potentially suitable feedstocks for
anaerobic fermentation [4–8]. The fermentative production of hydrogen
* Correspondence to: Jinling Cai, College of Chemistry Engineering and Material Science,
Tianjin University of Science and Technology, Tianjin 300457, P. R. China.
** Correspondence to: Peng Yu, College of Biotechnology, Tianjin University of Science &
Technology, Tianjin 300457, China.
E-mail addresses: jinlingcai@tust.edu.cn (J. Cai), yupeng@tust.edu.cn (P. Yu).
od for the treatment of algal blooms, and for energy production.
Cell walls and cytoplasmic membranes are components of the algal
biomass that are resistant to biodegradation during anaerobic digestion.
This complicates chemical processing, which means that biodegrada-
tion of these components becomes the limiting factor in the digestion
of algal biomass [2,9]. Pretreatment destroys cell microstructure,
resulting in a reduction in particle sizes within microalgae particles, fa-
cilitating the release of chemical contents and improvement to diges-
tion efficiency [8,10,11]. Thermal, acid, and alkaline pretreatments are
standard methods used for algal digestion [9,12]. Acid and alkaline pre-
treatment result in the disruption of the microalgal cell wall, rendering
the inner complex organic matter more available to biodegradation
[10–13]. Treatment with 2% (v/v) HCl results in higher cumulative hy-
drogen production (of 1230 mL/L), compared with that of the control,
in which hydrogen is not produced [13]. Alkaline pretreatment at
pH 13 has the capacity to produce about 3.8 times more hydrogen
than that of the control [10]. Thermal pretreatment is effective in
terms of solubilizing complex sugars in water, thus improving hydrogen
production [13,14]. Thermal pretreatment (at 170 °C for 20 min) of
Laminaria japonica resulted in a 62% higher production of hydrogen,
compared with that of the control [14]. Comparisons of pretreatment
methods have indicated that the suitability of the pretreatment method
depends on the type of biomass and the nature of the biological conver-
sion process. Algal cells have a simpler structure than agro-biomass,
which allows them to be crushed under relatively mild conditions.

http://dx.doi.org/10.1016/j.algal.2015.09.014
2211-9264/© 2015 Elsevier B.V. All rights reserved.
296 J. Cai et al. / Algal Research 12 (2015) 295–299
Cyanobacterial biomass-based carbohydrates, mainly comprised of gly-
cogen, cellulose and exopolysaccharides (with the absence of lignin),
are thus more easily converted to monosaccharides than is the case
for lignocellulosic materials [15].
The production of valuable by-products during processing can great-
ly increase the value and applicability of various hydrogen production
methods. Polyhydroxybutyrate (PHB), a completely biodegradable and
biocompatible plastic is a common intracellular storage compound
found in many microorganisms, such as photosynthetic bacteria [16].
With increasing environmental pollution, the replacement of synthetic
plastic with biodegradable plastic becomes an important issue. Never-
theless, the use of PHB is currently limited by its high production cost.
Substrate costs represent a significant proportion of the total cost of
PHB production. The broth that forms after fermentative hydrogen pro-
duction is rich with organic acids, which maybe used as a substrate by
photosynthetic bacteria for PHB production. This would greatly increase
its commercial application potential.
Relatively little data is currently available for the purpose of an in-
tensive investigation into the maximum potential hydrogen and PHB
production from cyanobacterial blooms. In the present study, to demon-
strate the potential of hydrogen and PHB production from a
cyanobacterial bloom, we investigated the influence of pretreatment
methods on hydrogen production.
2. Materials and methods
2.1. Inoculum
Anaerobic sludge, for the purpose of hydrogen production, was ob-
tained from an anaerobic municipal wastewater treatment plant in
Tianjin, China. The inoculum was stored at 4 °C until the test. Prior its
use in the hydrogen production experiment, the sludge was thermally
pretreated (at 90 °C for 30 min) to inhibit non-hydrogen-producing
bacteria.
The phototrophic bacterial consort for use in of PHB production was
collected from the lake water of the Meiliang Bay, Taihu Lake (120°30′N,
31°27′E). The phototrophic bacterial consort was enriched by a culture
medium, according to the methods described in Cai et al. [17] with
some modifications. Butyrate (concentration: 20 mmol/L) and ammoni-
um chloride (concentration: 10 mmol/L) were used as carbon and nitro-
gen sources, respectively. A 20 mL aliquot of lake water was inoculated
into 80 mL of culture medium in a 130 mL glass vial at 35 °C and
pH 7.5 ± 0.2. Light intensity was maintained at 80 μmol/m2·s and
argon gas was purged into the vial, to create anaerobic conditions.
Under the experimental conditions, phototrophic bacterial growth
would result in a purple-colored broth, after which a 20 mL aliquot of
the purple culture was injected, as an inoculum, into 80 mL of culture
medium. This process was repeated five times, to harvest the purple
non-sulfur photosynthetic bacteria. The phototrophic bacterial consort
was used for PHB production.
2.2. Feedstocks and pretreatments
Mixtures of algae and lake water, collected from Meiliang Bay, Taihu
Lake were used as substrates in the experiment. Mixtures were stored at
4 °C until further use. The cyanobacterial biomass was rich in organic
Table 1
Characteristics of the cyanobacterial bloom.
Constituent Cyanobacterial bloom inoculum
Total solids (TS, %) 6.47 ± 0.26 6.53 ± 0.46
Volatile solids (% of TS) 74.29 ± 2.99 61.63 ± 2.61
Reducing sugar (% of TS) 5.57 ± 0.28 NA
NA—no analysis.
Data are the means of three measurements, and numbers in parentheses are the standard
deviations.
compounds (volatile solid: VS) (Table 1). Thermal, acid, and alkaline,
pretreatments were performed, for the purpose of comparison of the
hydrogen yields. In the acid pretreatment experiment the algal biomass
samples were adjusted to pH 3 by addition of 6 mol/L HCl, for 30 min at
room temperature, after which neutral pH was maintained by the addi-
tion of NaOH. In the alkaline pretreatment experiment, the algal bio-
mass samples were adjusted to pH 13 by the addition of 6 mol/L
NaOH for 30 min at room temperature, after which the neutral pH
was maintained by the addition of HCl. In the thermal pretreatment ex-
periment, the algal biomass was heated to 170 °C and maintained at this
temperature for 20 min, after which it was cooled to room temperature.
2.3. Experimental procedure
Hydrogen production tests were carried out in 500 mL glass bottles,
with a working volume of 200 mL. Approximately 150 mL of pretreated
biomass solution and 50 mL of inoculum were added to each bottle.
Prior to purging with N2 gas, to create anaerobic conditions, the initial
pH was adjusted to 7.5 ± 0.2. Cultures were placed in a shaker-
incubator at 35 °C and 150 rpm. A control, containing raw cyanobacteria
and lake water, was also set up.
PHB production tests were carried out in 130 mL glass vials with a
100 mL working volume. After the cessation of hydrogen production, a
20 mL aliquot of precultured phototrophic bacteria was harvested and
inoculated into 100 mL of the fermentative broth. The pH was regulated
to 7.5 ± 0.2. Batch fermentation was carried out in dark aerobic condi-
tions in a shaker–incubator maintained at 35 °C and 150 rpm. The PHB
accumulation was analyzed after 3 d cultivation. All experiments were
carried out in triplicate and results were expressed as means.
2.4. Analytical methods
Total solid (TS) and volatile solid (VS) were measured according to
standard methods [18]. The pH value was measured with a digital pH
meter (delta-320, Mettler, USA). Total reducing sugars were evaluated
by the phenol–sulfuric acid method as described by Dubios et al. [19].
PHB content was measured using the FT-IR methods according to Cai
et al. [16]. The water replacement method was used to measure gas. A
gas chromatograph (Agilent, Model 6820, China) equipped with a ther-
mal conductivity detector (TCD) and a 2-m-long column packed with
5 Å molecular sieves (80/100 mesh), was used to determine hydrogen
content. Nitrogen was used as a carrier gas, at a flow rate of 30 mL/min.
The temperatures of the injector, detector, and column oven were
200 °C, 200 °C, and 40 °C, respectively.
The concentrations of organic acids, including acetic acid, propanoic
acid and butyric acid, were analyzed by gas chromatography (Agilent,
Model 6890 N, USA), using a flame ionization detector (FID) and a
30 m × 0.25 mm × 0.25 μm fused-silica capillary column (DB-FFAP).
The supernatants were centrifuged (12,000 rpm for 5 min), acidified
(by formic acid) and filtered (through a 0.2 μm membrane). The tem-
peratures of the injection port and the detector were 250 °C and
300 °C, respectively. The initial temperature of the oven was 70 °C, for
3 min, followed with a ramp of 20 °C/min for 5.5 min, to reach a final
temperature of 180 °C for 3 min. Nitrogen was used as the carrier gas
at a flow rate of 2.6 mL/min.
3. Results and discussion
3.1. Effect of pretreatment methods on algal biomass
During fermentative processes, an intact algal cell wall can protect
the cell from biodegradation [11,20]. Pretreatment can effectively dis-
rupt the cell microstructure and subsequently enhance hydrogen pro-
duction [8,11,13,20]. In this study, three pretreatment methods
(thermal, acid, and alkaline) were used to treat algal biomass.
 J. Cai et al. / Algal Research 12 (2015) 295–299 297

Total reducing sugar concentration was enhanced after pretreat-

ment, compared with that in the control (Fig. 1). This result is consistent
with previous observations in which pretreatment significantly in-
creased the concentration of dissolved carbohydrates [11,20], suggest-
ing that pretreatment greatly increased the release of carbohydrates
from the cells and during the subsequent hydrogen production.
Among the three pretreatment methods, thermal pretreatment pro-
duced the highest level of reducing sugar (21.52 ± 1.27% of TS), indicat-
ing that thermal pretreatment resulted in the effective lysis of the rigid
algal cell wall. A similar observation was also made by Jun. et al. [14],
who observed a noticeable increase in reducing sugars, such as glucose,
xylose, and furfural, following thermal pretreatment. In the case of alka-
line pretreatment, the reducing sugar level increased to about 172.5%,
compared with that of the control. Under appropriate alkaline treat-
ment, it seems that the algal cell wall can easily disrupt the ester
bonds within lignin, hemicellulose, cellulose and the inner complex or-
ganic matter [10,12], resulting in a higher level of biodegradability. Fol-
lowing acid pretreatment, the concentration of the reducing sugar
Fig. 2. Effect of pretreatment methods on hydrogen production. (n = 3, error bars =
released from the algal biomass was only 6.48 ± 0.32% of TS. In this
s.e.m.).
case, however, the hydrogen production was greatly increased, com-
pared with that of the control. These results demonstrate that acid pre-
have been due to the formation of inhibitory compounds and toxicity ef-
treatment cannot efficiently hydrolyze the cyanobacterial biomass, but
fects [12,25]. On the other hand, equipment corrosion, high operational
can result in a breakdown of the cell wall structure, which facilitates hy-
and maintenance costs limited the utilization of acid pretreatment. The
drolysis. This is consistent with the results from the study by Liu et al.
hydrogen yields obtained in our experiments were comparable with
[21].
those reported in related studies (Table 2). It can therefore be concluded
that an efficient level of hydrogen production can be obtained from algal
3.2. Effect of pretreatment methods on hydrogen production
blooms. The hydrogen yield after thermal pretreatment was slightly
higher than that obtained after alkaline pretreatment. The thermal pre-
Batch tests were conducted on three pretreatment methods, as well
treatment method is an energy-intensive process, which limits its use in
as on the control, to investigate hydrogen productivity enhancement. A
commercial applications. It is not feasible to heat large quantities (for
low rate of hydrogen production (28 ± 2 mL/g VS) was observed in the
example, 100,000 tons) of refloated algal biomass on an annual basis.
control experiment, suggesting a high resistance of algal biomass to bio-
Results from a techno-economic analysis indicate that, among the
degradation and the need for biomass pretreatment (Fig. 2). Similar re-
three pretreatment methods mentioned in this study, alkaline pretreat-
sults were also noted in Chlorella vulgaris and Dunaliella tertiolecta [21,
ment may be the most efficient, and the most economically-feasible,
22]. Each pretreatment group exhibited different hydrogen yield abili-
method for large-scale applications.
ties. This may have been because of different reducing sugar concentra-
tions, as observed in the results (Fig. 1). After hydrogen production
process, more 95% of reducing sugar was removed in all the pretreat- 3.3. Effect of pretreatment methods on VFA production
ment groups and the control. Roy et al. [11], Ortigueira et al. [23] and
Ao et al. [24] also found that most reducing sugar was removed during Volatile fatty acid (VFA) production is generally associated with fer-
hydrogen production process. Among the three tested pretreatment mentative hydrogen production [1]. Our results indicate that, in the
methods, thermal pretreatment produced the maximum hydrogen three tested pretreatment methods that we assessed, acetic acid and bu-
yield (113 ± 5 mL H2/g VS). Jun. et al. also found that thermal pretreat- tyric acid were the major liquid by-products (about 70–80% of total
ment greatly increased hydrogen production [14]. A significant increase VFAs with the remainder being propionic acid Fig. 3). Approximately
in hydrogen production was also noted after alkaline pretreatment, the same amounts of the three components of VFAs were produced in
resulting in an overall increase in hydrogen yield (94 ± 4 mL H2/g a successful hydrogen production process, following thermal pretreat-
VS). Acid pretreatment showed the lowest hydrogen yield, which may ment of Chlorella sorokiniana [11], and acid pretreatment of Anabaena
sp. PCC 7120 [13] and Chlorella vulgaris ESP6 [21]. There are also some
reports indicating that butyric acid/acetic acid ratios are directly propor-
tional to hydrogen yields [13,28]. In our studies we also found that
the highest butyric acid/acetic acid ratio was observed in liquid by-
products of thermal pretreatment. This result is consistent with that
of Nayak et al. [13], who studied hydrogen production from a
cyanobacterial biomass (Anabaena sp. PCC 7120). The control group
generated the lowest butyric acid proportion of total VFAs.
A decline in pH is always associated with VFA generation. In the
present study, we found that final pH values ranged from 5.37–6.04,
and that the final pH values in thermal and alkaline pretreatment
groups were significantly lower than those in the acid and control
groups.

3.4. Effect of pretreatment methods on PHB production

To maximize low-cost practices, a phototrophic bacterial consort

Fig. 1. Effect of three pretreatments on the hydrolysis of cyanobacterial bloom. (n = 3, was operated as a second-stage fermentation system, for PHB produc-
error bars = s.e.m.). tion from hydrogen fermentative effluent. During the PHB production
298 J. Cai et al. / Algal Research 12 (2015) 295–299

Table 2
Hydrogen yields of different microalgal biomass.

Microalga Pretreatment Inocula Hydrogen yields Reference

Algal bloom Microwave + heat + acid + enzymatic (microwave + 140 °C 15 min + Anaerobic sludge 47.07 mL/g VS [8]
1%H2SO4 pH 1.4 + cellulase and glucoamylase)
Taihu blue algae Acid (pH 2 12 h) Anaerobic granular sludge 54 mL/g VSS [10]
(autoclaved 121 °C 15 min)
Taihu blue algae Alkaline (pH 13 12 h) Anaerobic granular sludge 105 mL/g VSS [10]
(autoclaved 121 °C 15 min)
Chlorella Heat-HCl (200 m3/m3 HCl + autoclave 121 °C 20 min) Mixed culture 958 m3/kg substrate, 2.51 [11]
sorokiniana mol/mol glucose
Chorella vulgaris HCl–heat (1.5% HCl + autoclaved at 121 °C 20 min) Clostridiuim butyricum CGS 5 81 mL/g algae [21]
ESP 6
Scenedesmus Autoclave 15 min Clostridium butyricum 113.1 mL/g VS [26]
obliquus
Scenedesmus Autoclave 15 min Enterobacter aerogenes 57.6 mL/g VS [26]
obliquus (wet)
Chlorella vulgaris Enzymatic Anaerobic municipal wastewater 135 mL/g VS [27]
treatment plant sludge
Taihu blue algae Thermal (170 °C 20 min) anaerobic sludge (90 °C 30 min) 113 mL/g VS Present
work
Taihu blue algae Alkaline (pH 13 30 min) Anaerobic sludge (90 °C 30 min) 94 mL/g VS Present
work

process the reducing sugar and VFAs were almost completely con-
sumed, which indicated that the fermentative effluent is a favorable
feedstock for PHB production. The VFAs could be successfully used as
substrates by photosynthetic bacteria, for the production of PHB [16,
29–31]. Results illustrated in Fig. 4 indicate that alkaline pretreatment
resulted in the highest production of PHB (1.69 ± 0.17 g/L), followed
by thermal pretreatment (1.13 ± 0.10 g/L) and acid pretreatment
(0.62 ± 0.04 g/L). The lowest PHB production occurred in the control.
Numerous factors could affect the phototrophic bacteria culture perfor-
mance including bacterial strain, pH, carbon source and concentration,
etc. There was a positive correlation between PHB production and
total VFA concentration. This is consistent with results from the study
by Sangkharak and Prasertsan [32], in their research when acetate con-
centration was below 4 g/L (about 66.7 mmol/L), the PHB production
was increased with VFA concentration increasing. Fradinho et al. [33]
found that acetic acid and butyric acid mainly resulted in PHB produc-
tion, while propionic acid led little PHB production by phototrophic bac-
teria. However, in this test total concentration of acetic acid and butyric
acid in thermal pretreatment was similar to that of alkaline pretreat-
ment. But, the PHB production of alkaline pretreatment was much
more than thermal pretreatment. This difference may be result by the
different bacterial strains used. There have been reports that different
bacterial strains prefer different carbons for PHB production [16,34].
Based on the experimental results of hydrogen and PHB productions,
and considering the energy input in thermal pretreatment, the alkaline
pretreatment was selected as the optimal pretreatment methods for hy-
drogen and PHB production.
A few previous studies have reported on PHB production. Bacillus ce-
reus produced 2.26 g/L PHA at flow rate of 120 L/h until the 42nd hour
fermentation [10]. PHB produced by a mixed photosynthetic consor-
tium of bacteria and algae was 4.4 C mmol/L (about 0.09 g/L) [31]. A ma-
rine phototrophic bacterium Rhodovulum sulfidophilum P5 could
accumulate 6.07 g/L PHB under optimum culture condition [16]. Co-
culture of Enterobacter aerogenes and Rhodobacter sphaeroides produced
bout 6.25 g/L PHB under alternate dark-photo fermentative periods
[29]. In this test, even under non-optimal conditions, the PHB yields
were promising. In the future, we should further optimized culture con-
dition to increase PHB yield. Cyanobacterial blooms frequently occur all
over the world. It is becoming increasingly expensive to dispose of. Con-
version of waste materials to PHB would not only generate waste treat-
ment credits, but would also to some extent improve the economics of
the PHB production process.
4. Conclusion
In this study, we demonstrated the feasibility of hydrogen and PHB
production from cyanobacterial blooms, and the influence of pretreat-
ment methods. We also found that pretreatment significantly improved

Fig. 3. Influence of pretreatment methods on liquid fermentation products and pH value. (n = 3, error bars = s.e.m.).
 J. Cai et al. / Algal Research 12 (2015) 295–299
Fig. 4. Effect of pretreatment methods on PHB production. (n = 3, error bars = s.e.m.).
hydrogen production from cyanobacterial blooms. Thermal pretreatment
produced the highest hydrogen yield (113 mL H2/g VS), followed by alka-
line pretreatment (94 mL H2/g VS). Subsequent to the hydrogen produc-
tion process, the fermentative broth was inoculated with phototrophic
bacteria for the purpose of PHB production. The highest production of
PHB occurred after alkaline pretreatment (1.69 g/L). Considering the
huge energy input in thermal pretreatment, alkaline pretreatment can
be a regarded as an optional pretreatment method for destroying the
algal biomass, for the purpose of hydrogen and PHB production. These re-
sults demonstrate that algal biomass in cyanobacterial blooms could be a
potentially-valuable environmental-friendly feedstock for hydrogen and
PHB production.
Acknowledgments
This work was support by the Key Natural Science Foundation of
Tianjin of China (14JCZDJC40200) and the Science Foundation of Tianjin
University of Science & Technology (20130114).
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