Fermentative hydrogen and methane production from microalgal biomass (Chlorella vulgaris) in a two-stage combined process

Applied Energy 132 (2014) 108–117

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Fermentative hydrogen and methane production from microalgal biomass (Chlorella vulgaris) in a two-stage combined process Nils Wieczorek, Mehmet Ali Kucuker, Kerstin Kuchta ⇑ TUHH – Hamburg University of Technology, Institute of Environmental Technology and Energy Economics, Waste Resource Management, Harburger Schloßstr. 36, 21079 Hamburg, Germany

h i g h l i g h t s  Microalgae C. vulgaris was used for hydrogen production.  Hydrogen production increased significantly with the enzymatic pre-treatment.  The residues after H2 production was used for the conventional CH4 fermentations.  An innovative two-stage combined process was improved for H2 and CH4 production.

a r t i c l e

i n f o

Article history: Received 6 December 2013 Received in revised form 30 June 2014 Accepted 1 July 2014

Keywords: Microalgae (C. vulgaris) Biohydrogen Biomethane Dark fermentation Enzymatic pre-treatment

a b s t r a c t This study investigated the hydrogen fermentation in a two-stage combined fermentation process (combination of dark fermentation for hydrogen (H2) production from microalgae and methane (CH4) fermentation from residues following H2 production process). Microalgae Chlorella vulgaris culture was used as a substrate. The hydrogen production from C. vulgaris ranges 1.75 ± 1.50–19 ± 2.94 ml H2 g-VS1 at different substrate dosages without enzymatic pre-treatment. A seven-fold increase in H2 production yields (19 ± 2.94–135 ± 3.11 ml H2 g-VS1) was observed with enzymatic pre-treatment (Onozuka R-10 and Macerozyme R-10) of the microalgae. The results of the CH4 fermentation show that the methane yields could be increased from 245 ± 2.46 to 414 ± 2.45 ml CH4 g-VS1 by using enzymatic pretreatment. In addition, the yield of the CH4 fermentation (14.86 kJ/g-VS) has approximately the same order of magnitude in comparison to the two-stage combined fermentation process (14.46 kJ/g-VS) in terms of energy production. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Shortage of fossil fuel reserves, environmental pollution and climate change have increased the industrial and social awareness of the alternative fuel resources [1,2]. Hydrogen is one of the most important alternative energy resources with high energy content compared to hydrocarbon fuels [2–4]. Among various H2 production processes, biological methods have shown significant advantages over chemical methods, since it can be less energy intensive than operation under mild conditions [5]. Biological method mainly includes photosynthetic hydrogen production and fermentative hydrogen production. The efficiency of photosynthetic hydrogen production is low and it cannot be operated in the absence of light, while fermentative hydrogen production can produce hydrogen continuously without light using various kinds of ⇑ Corresponding author. Tel.: +49 (0) 40 42878 3054; fax: +49 (0) 40 42878 2375. E-mail address: [email protected] (K. Kuchta). http://dx.doi.org/10.1016/j.apenergy.2014.07.003 0306-2619/Ó 2014 Elsevier Ltd. All rights reserved.

substrates such as organic waste and biomass. Therefore, there has been an increasing research tendency towards terrestrial biomass, such as edible agricultural crops and lignocellulosic waste, for the last decade [6]. On the other hand, several serious issues have arisen such as an increase in food price, carbon debt, and demands for water [7]. Hence, microalgae feedstocks have been recently gained interest for energy scenario due to their fast growth potential, coupled with relatively high lipid, carbohydrate, nutrient and protein contents [8,9]. An example of a remarkable organism is the green algae Chlorella vulgaris which are microscopic photoautotrophic organisms with enormous growth rates. Thus they have been cultivated on an industrial scale in autonomous systems such as photobioreactors as a natural recycling for the pharmaceutical, cosmetic and food industries [10]. A large-scale application of algae as a renewable resource for energy production has great potential [9,11–13], but it has not been economically usable yet [3,14–16]. Currently, a combination of natural resource recovery and subsequent ener-

N. Wieczorek et al. / Applied Energy 132 (2014) 108–117

getic source seem more promising. Therefore, the combined processes such as combined H2/CH4 fermentation and conventional CH4 fermentation have to be investigated in order to exploit the energy from microalgae. Hydrogen emerges as an intermediary during the anaerobic substrate oxidation of organic matter to CH4 and carbon dioxide (CO2). The challenge of a combined hydrogen and methane fermentation lies in the decoupling of the syntrophic co-cultural from anaerobic fermentation. Dark fermentative H2 production of biomass in anaerobic digestion is also more amenable for practical application [17,18]. The dark fermentative hydrogen production process is basic and energy-saving compared with traditional hydrogen production processes. Previous studies have also reported hydrogen production via dark fermentation from various biomass, such as wheat straw [19], cornstalk [20], food waste [21–24] and microalgae [8,25–27]. In addition, there are a lot of studies about CH4 fermentation from algae; however, there is limited information for a twostage combined process described in the present work which is a combination of dark fermentation for H2 production and CH4 fermentation from residues following H2 production process. In the first stage glucose in microalgae is fermented to acetate, CO2 and H2 in an anaerobic dark fermentation at thermophilic conditions. This is followed by a successive second stage where residues which have resulted from H2 production are converted to CH4 and CO2 at mesophilic conditions. The purpose of this study is to examine if the chemical energy stored in algae can be put into effective use by two-stage combined fermentation processes. The focus is on the energy production during hydrogen and methane fermentation.

2. Material and methods 2.1. Feedstock and inoculum Two main types of large-scale algae cultivation systems are open ponds and closed photobioreactors (PBRs) [28–30]; open ponds have a low productivity compared to photobioreactors [31]. In this study, the microalgal species C. vulgaris (Göttingen Culture Collection of Algae, Germany, strain SAG 211-12) which was cultivated using the outdoor flat panel PBRs was obtained from the microalgal cultivation plant in Hamburg-Reitbrook in Northern Germany. Flat panel PBRs were employed, since these kind of PBRs consume less energy than tubular reactors with a high cultivation efficiency [13]. The harvested microalgae were concentrated by means of a concentrative separator (Westphalia GEH). Total crude protein was determined according to AOAC International Method 2001.11 [32], where the TKN was multiplied by a conversion factor of 6.25. The pH of C. vulgaris biomass was adjusted to 7.0 ± 0.2 and the biomass slurries were stored at 18 °C until used in the dark fermentation. On the other hand, the algal biomass was pre-treated with enzymes such as Onozuka R-10 (SERVA Electrophoresis, Cat. Nr. 16419), Macerozyme R-10 (SERVA Electrophoresis, Cat. Nr. 28302) and mixed with them in order to increase in H2 production. Anaerobic inocula were obtained from anaerobic municipal wastewater treatment plant sludge in Hamburg, Germany. The inoculum was stored at 35 °C until the test over a period (7 days) without any addition of nutrients. In order to check the activity in the experimental procedures, trial references were prepared with glucose (Merck, Germany). Before beginning the experimental procedures, the inoculum was thermally pre-treated to inhibit methanogenic organisms and to prevent a reduction of the hydrogen production. The thermal treatment was carried out at 85 °C and over a period of 35 min. The amount of inoculum was generally 25% of the reaction volume.

109

2.2. Experimental design and procedure Dark fermentation is a biological process performed in anaerobic conditions with bacteria grown in the absence of light sources under optimum conditions to produce H2 and CH4 from biomass [4,17,18,33,34]. There has been an increasing tendency towards dark fermentative H2 production from microalgal biomass over the last few years [33]. Therefore, H2 and CH4 production from microalgal biomass has been investigated using dark fermentation process in this study. A total of 23-batch test sets were carried out in series using 500 ml glass bottles (Schott-Duran). The dark fermentation system is shown in Fig. 1. The working volume was 250 ml and the bottles were seeded with heat-treated anaerobic sludge up to 25% of the working volume, and filled with a specified amount of microalgal biomass and water. Before purging with N2 gas to provide an anaerobic condition, the initial pH was adjusted at 7.5 ± 0.2 by adding either 3 N NaOH or 3 N HCl solutions. All batch tests for H2 fermentation were conducted in batch incubators (Memmert U30) with a magnetic stirrer (Aqualytik AL-353) at 60 °C, this system were also used for CH4 fermentation at 37 °C and 130 rpm (20 min/day), which were carried out in triplicate. Fermentative temperature can affect hydrogen production by influencing the microorganism composition and activity of enzymes such as hydrogenases [35]. According to Luo et al. [36], the hydrogen production obtained was strongly temperature dependent, i.e. maximum hydrogen yield (53.8 ml H2 g-VS1) was obtained under thermophilic condition (60 °C), 53.5% and 198% higher than the values under mesophilic (37 °C) and extreme thermophilic (70 °C) conditions respectively. The volume of gas was measured by means of milligas counter (Ritter, MGC-1) and all data from milligas counter were logged online. The reactor and experimental procedure employed for the confirmation test were the same as those used for the batch test. There are 3 different experimental data sets in this study. The first experimental set (Set 1) was conducted for the determination of optimum substrate dosage which ranged between 5 and 30 g-VS l1. The enzymatic pre-treated algal biomasses were used in order to produce H2 in the second experimental set (Set 2) in which the investigation of hydrogen production was carried out with 10 g-VS l1 microalgae treated by Onozuka R-10 enzyme (B1), Macerozyme R-10 enzyme (B2) and a mixture of them (B3) in 500 ml batch glass bottles. The conventional CH4 fermentations from residues resulting from the H2 production process and enzymatic pre-treated microalgae were investigated in the third set (Set 3). The summary of the experimental sets is given in Table 1 and additionally the experimental diagram is illustrated in Fig. 2. 2.3. Theoretical and cumulative approach for H2 and CH4 production The theoretical approach for the reaction formation of H2 and CH4 are known, starting from glucose. For the calculation of the maximum theoretical volume (Vmax), the following equation can be used.

V max ¼

Vm  n M

ð1Þ

where Vm is the molar volume (22414 ml/mol), n is the number of moles of formed gas and M is the molecular weight of glucose (181.16 g/mol).

C6 H12 O6 ! CH3 ðCH2 Þ2 COOH þ 2CO2 þ 2H2

ð2Þ

C6 H12 O6 þ 2H2 O ! 2CH3 COOH þ 4H2 þ 2CO2

ð3Þ

4C6 H12 O6 ! 2CH3 COOH þ 3CH3 ðCH2 Þ2 COOH þ 8CO2 þ 10H2

ð4Þ

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Fig. 1. Representation of the dark fermentation system.

Table 1 Experimental procedure of the test series.

a b c d

Substrate concentration

Inoculum (ml)

Water (ml)

Total volume (ml)

Enzymatic pre-treatment

Fermentation

Temperature (°C)

Set 1 A1 A2 A3 A4 A Blank A Reference

5 (g-VS l1) Microalgae 10 (g-VS l1) Microalgae 20 (g-VS l1) Microalgae 30 (g-VS l1) Microalgae No substrate 10 (g-VS l1) Glucose

62.5 62.5 62.5 62.5 62.5 62.5

187.5 187.5 187.5 187.5 187.5 187.5

250 250 250 250 250 250

No No No No No No

H2 H2 H2 H2 H2 H2

60 60 60 60 60 60

Set 2 B1 B2 B3 B Blank B Reference

10 (g-VS l1) 10 (g-VS l1) 10 (g-VS l1) No substrate 10 (g-VS l1)

62.5 62.5 62.5 62.5 62.5

187.5 187.5 187.5 187.5 187.5

250 250 250 250 250

Yesa Yesb Yesc No No

H2 H2 H2 H2 H2

60 60 60 60 60

Set 3 C1 C2 C3 C4 C Blank C Reference

Digestate B3 (250 ml) Digestate B Blank (250 ml) 10 (g-VS l1) Microalgae 10 (g-VS l1) Microalgae No substrate 10 (g-VS l1) Cellulose

130 130 130 130 130 130

No No No No No No

380 380 130 130 130 130

Yesd No Yesc No No No

CH4 CH4 CH4 CH4 CH4 CH4

37 37 37 37 37 37

Microalgae Microalgae Microalgae Glucose

Onozuka R-10. Macerozyme R-10. Mixture of Onozuka R-10 and Macerozyme R-10. Substrate was pre-treated in Set 2/B3.

C6 H12 O6 ! 3CH4 þ 3CO2

ð5Þ

In the reaction of glucose to butyric acid (2) at most 2 mol of gas also 249 l of hydrogen are formed. In the formation of acetic acid (3) four moles of hydrogen (498 l of hydrogen) are formed. In the mixed acid formation (4) a maximum of 2.5 mol of gas produced per mole of glucose that is 311 l of hydrogen are formed. In the methane fermentation (5) 3 moles of CO2 and 3 mol CH4 thus arise each 373 l of gas. Eq. (4) can be used for the estimation of the theoretical potential of H2 fermentation. The biogas yield of one gram of carbon is equivalent to 1.865 l theoretically. The cumulative hydrogen production is calculated from experimental data, a modified Gompertz equation was used to fit the kinetics of hydrogen production [5,37,38] in addition to compare

the results obtained in the different tests. This equation is widely used to model gas production data. It expresses as follows:

   e  Rmax HðtÞ ¼ Hmax  exp  exp  ðk  tÞ þ 1 Hmax

ð6Þ

where H(t) (ml g-VS1) is the total amount of hydrogen produced at culture time t (h); Hmax (ml/g-VS) is the maximum amount of hydrogen produced. Rmax (ml g-VS1 h1) is the maximum hydrogen production rate; k (h) is the lag phase before exponential hydrogen production, t is the incubation time (h) and e:exp(1) = 2.71828. Lag phase refers to the initial adaptive phase, during which hydrogen production remains relatively constant prior to rapid growth.

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111

Fig. 2. The diagram of the experimental study.

The quantities Hmax, Rmax, and k have been obtained minimizing the ratio between the square of the difference between observed and calculated values, using the Microsoft Excel’s Solver function. 2.4. Analytical methods The pH was measured according to DIN 38 404-C5 method before and after the experimental procedures using combination electrode (WTW pH-91, Weilheim). The pH measurements during the experiments were performed using a pH indicator (Panpeha, Sigma–Aldrich). Total solid (TS), volatile solid (VS) and ammonia were analysed in duplicates in accordance with DIN 38414-S2, DIN 38409-H1-3, and DIN 38 406-E5-2, respectively. Total carbon (TC), total organic carbon (TOC), total inorganic carbon (TIC) and dissolved organic carbon (DOC) were performed according to DIN EN 1484. The gas samples were taken from the head space of condensation bottles by microliter syringe (1 ml Hamilton Bonduaz). The proportions of hydrogen and methane were analysed using a gas chromatograph (GC) (Hewlett-Packard 6890) fitted with a thermal conductivity detector (TCD) and a 2.0 m  3.2 mm, 80/ 100 mesh Porapack Q column (Supelco) in series with a 2.0 m  3.2 mm, 60/100 mesh Moleculer Sieve 5A column (Agilent). Helium was used as the carrier gas in the column at a flow rate of 45 ml min1. The packed column was maintained at 180 °C for analysis and a thermal conductivity detector was used at 210 °C. The liquid samples harvested during or at the end of the fermentations were centrifuged at 10,000 rpm for 10 min. The obtained supernatants were then filtered through a 0.45 lm syringe filters (Rezist, Whatman) and analysed by HPLC (HP 1100 Series). The HPLC analysis was carried out using an UV–VIS 220 nm (Chrompack) detector proceeded by pre-columns Organic Acid Resin 40  8 and 250  8. The concentrations of the compounds in the samples are determined in comparison with standard samples analysed for the establishment of calibration curves. 3. Results and discussion 3.1. Composition of feedstock The microalgal biomass feedstock (C. vulgaris) was contained 52.5%, 3.25% and 13.4% of proteins, lipids and carbohydrates on a

dry weight basis, respectively. In general, the lipid value is lower than previously reported in the literature. The proportion of proteins, carbohydrates and lipids in C. vulgaris were ranged 51–58%, 12–17% and 14–25%, respectively [39]. Growth conditions such as nutrients stress conditions, salinity, CO2, temperature and light may influence algal biomass composition [40]. The general notion is that high light and high temperature generated a more saturated lipid content and fatty acid composition compared to low light and low temperature conditions [40,41]. H2 and CH4 productions from microalgae vary due to variation in cellular protein, carbohydrate and lipid content, cell wall structure, and process parameters such as the bioreactor type and the digestion temperature [32]. The carbohydrates generated the most hydrogen through biological hydrogen fermentation compared with proteins or lipids [42–44]. The compositional data for C. vulgaris in particular may reflect loss of cellular constituents upon sample preparation and handling as well as growth conditions were not varied to determine the corresponding changes in cellular fractions. The chemical composition of the studied microalgae is given Table 2.

3.2. Hydrogen production in dark fermentation at thermophilic conditions 3.2.1. Determination of optimum substrate dosage for hydrogen production The investigation of optimum hydrogen production with four different substrates dosages (5 (A1), 10 (A2), 20 (A3) and 30 (A4) g-VS l1) was first carried out with pure cultures of C. vulgaris in the first fermentation set (Set 1). The hydrogen production performances were compared in terms of hydrogen yields allowing a firm basis for a comparison of the substrate dosages. The hydrogen production after 120 h of digestion under thermophilic conditions without any pre-treatment method for the substrate dosage of 5, 10, 20 and 30, were 1.75 ± 1.50, 19 ± 2.94, 8 ± 2.65 and 7 ± 4.11 ml H2 g-VS1, respectively. These results clearly show that better performances were obtained with 10 g-VS l1 compared to substrate dosages (5, 20 and 30 g-VS l1) which produced hydrogen less effectively. Increasing the initial substrate concentration in the bioreactor led to a significant decrease in hydrogen production, while the concentration of the substrate increased from 10 to

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Table 2 Characterization of microalgae C. vulgaris.

a b c

Water Contenta 91.22 (%)

TSa 8.78 (%)

VSb 8.12 (%)

TCa 8.12 (%)

TKNa 84.1 (g kg1)

TOCb 27.37 (g l1)

DOCb 3.67 (g l1)

Carbohydratesb 13.4 (%)

Proteinsb 40.2 (%)

Lipidb 3.25 (%)

Lipidb (4.02 g kg1)

Pectina n.d.c

2-Furfurala <560 (mg kg1)

HMFa <560 (mg kg1)

Cellobioseb n.d.c

Rhamnoseb 40 (g kg1)

Mannoseb 4 (g kg1)

Arabinoseb 3 (g kg1)

Galactoseb 54 (g kg1)

Xyloseb 1.4 (g kg1)

Glucoseb 1.9 (g kg1)

Mga 5.46 (g kg1)

Naa 5.1 (g kg1)

Ka 22.7 (g kg

Pa 13.6 (g kg1)

Mna 0.37 (g kg1)

Caa 2.51 (g kg1)

Sa 6.52 (g kg1)

Sea <200 (lg kg1)

Coa 704 (mg kg1)

Zna 1070 (lg kg1)

Asa n.d.c

Moa <100 (lg kg1)

Fea 3000 (lg kg1)

Cua 3930 (lg kg1)

1

)

Algae substrate. Dry algae. n.d.: non detected.

Fig. 3. Hydrogen production by dark fermentation in batch glass bottles with Chlorella vulgaris used as substrate for different substrate dosages (5 (A1), 10 (A2), 20 (A3) and 30 (A4) g-VS1 l1) as well as pH values during the fermentation. Values correspond to means of three replicated independent values ± confidences intervals (error bars).

20 and 30 g-VS l1, the corresponding cumulative hydrogen yield declined from 19 ± 2.94 to 8 ± 2.65 and 7 ± 4.11 ml H2 g-VS1, respectively. According to Pan et al. [21], the substrate concentration plays a crucial role in hydrogen production, i.e. the higher substrate concentration would result in excess substrate inhibition, simultaneous acid/pH inhibition, and increased hydrogen partial pressures. Fermentative hydrogen production from Chlorella sp. was investigated under mesophilic conditions and the maximum hydrogen yield at the lowest inoculum-substrate ratio was achieved 7.13 ml H2 g-VS1 [45]. The pH regulation in the batch glass bottle experiments was made using NaOH and HCl during the dark fermentation of the microalgae C. vulgaris. The pH rapidly dropped to levels lower than the optimal pH for hydrogen production due to acid metabolites (4.5 for 10 g-VS l1) thereby reducing the final yield reached. In addition the final pH levels were much lower (ranging from 5 to 6) inducing a strong inhibitory effect, not only on the hydrogen production but also on substrate consumption. The experiments lasted for 120 h, and the hydrogen production as a function of digestion time at different substrate dosage and the composition of the biogas yield are shown in Figs. 3 and 4, respectively. The 10 g-VS l1 glucose was used as a reference substrate in this study and the hydrogen production of glucose was measured 230 ± 1.44 ml H2 g-VS1 at thermophilic conditions. 3.2.2. Hydrogen production from enzymatic pre-treated algal biomass The efficiency of enzymatic pre-treatment on fermentative H2 production from microalgae C. vulgaris was evaluated in an exper-

imental system (Set 2) (enzymatic hydrolysis prior to dark fermentation). The experimental results show that enzymatic pretreatment on the microalgae biomass significantly increased the H2 production yields. The H2 fermentations from pre-treated C. vulgaris microalgae with Onozuka R-10 enzyme, Macerozyme R-10 enzyme and a mixture of them were 39 ± 1.79, 62 ± 4.56 and 135 ± 3.11 ml H2 g-VS1, respectively (Fig. 5). The composition of the biogas from Set 2 is displayed in Fig. 6. As a control experiment (Blank), biohydrogen fermentation was also conducted using C. vulgaris biomass without any chemical or enzymatic pre-treatment and the hydrogen yield of control was observed 19 ml/g-VS. Lakaniemi et al. [27] reported that H2 production was 8 ml H2 gVS1 from Dunaliella tertiolecta biomass and 7.9 ml H2 g-VS1 from C. vulgaris biomass without any pre-treatment. The H2 production could be observed in the blank experiment. The tested enzyme mixture effectively enhanced the H2 yield from microalgae by a factor of 7 (135 ± 3.11 ml H2 g-VS1 vs 19 ± 2.94 ml H2 g-VS1in the controls with no enzyme addition). The results mentioned above indicate that enzymatic pre-treatment can efficiently affect hydrolyse of the microalgae biomass; it could also breakdown the structure of the cell walls, making the biomass easier to hydrolyse. Quemeneurr et al. [19] stated that the enzyme mixture effectively triggered the H2 yield from wheat straw, probably through the release of additional carbohydrate units such as glucose. According to Liu et al. [46], a high hydrolysis efficiency of the microalgal biomass can be achieved using appropriate enzymatic pre-treatment. The maximum dark hydrogen yield of 276.2 ml H2 g-VS1 was obtained from the pre-treated

N. Wieczorek et al. / Applied Energy 132 (2014) 108–117

113

Fig. 4. Biogas composition for Set 1.

Fig. 5. Hydrogen production from pre-treated microalgae C. vulgaris and pH values during the fermentation. Values correspond to means of three replicated independent values ± confidences intervals (error bars).

Fig. 6. Biogas composition for Set 2.

mixed biomass of Chlorella pyrenoidosa and cassava starch [47]. Moreover, poor biohydrogen production was observed when the microalgal biomass was not pre-treated with enzymes, suggesting the need of biomass pre-treatment [46].

3.3. Methane production in dark fermentation at mesophilic conditions Methane production potential from residues after H2 production process and from C. vulgaris biomass was studied using dark

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fermentation (Set 3). The maximum methane productivity and the higher rate of methane productivity were observed at enzymatic pre-treated C. vulgaris (C1). When the algal biomass does not result from any cell disruption process, the cell wall can strongly protect the cell, which leads to reduced cell biodegradability. Therefore, the enzymatic pre-treatment result in to increase the cell biodegradability and also methane production. At Set 3 experiments, the final methane productivity was 414 ± 2.45, 245 ± 2.64, 362 ± 1.79 and 74 ± 2.48 ml CH4 g-VS1 for pre-treated C. vulgaris (C3) and without pre-treatment on C. vulgaris (C4), pre-treated residues following H2 production (C1) and without pre-treatment on residues after H2 production process (C2), respectively (Fig. 7). The methane yields obtained from C. vulgaris in this study can be compared with literature data. Methane conversion of C. vulgaris without pre-treatment reached 245 ± 2.64 ml CH4 g-VS1 under 21 days at 37 °C. The methane productivity ranged between the values obtained by Alzatea et al. [48] during the anaerobic digestion of 3 different microalgae (188–387 ml CH4 g-VS1). In addition, the methane productivities for 6 different algae were tested by Mussgnug et al. [49] that ranged between 218 and 387 ml CH4 g-VS1. Methane yields obtained from C. vulgaris ranged from 147 to 240 ml CH4 g-VS1 [50]. By day 6, the CH4 productivities for

biomass C1, C2, C3 and C4 accounted for 64%, 99%, 98% and 83% and of the final productivities, respectively. Theoretically, proteins, carbohydrates and lipids yield 851, 415 and 1014 ml CH4 g-VS1, respectively [39]. The species that can reach higher lipid content have a higher methane yield [51]. The experimental conditions and the corresponding methane conversion yield from microalgae were evaluated by Sialve et al. [39] who reported that the methane yield varies from 90 to 450 ml CH4 g-VS1 depending on the species and culture conditions. Six different strains of Chlorella were tested that C. vulgaris showed the highest methane production (361 ± ml CH4 g-VS1) without enzymatic pre-treatment [34]. Microalgae Chlorella spp. were being considered of great research interest for biogas production by Prajapati et al. [52] who observed that relatively higher biogas yield of 464 ± 66 ml biogas g-VS1 added with 57% (v/v) CH4 content was obtained for C. pyrenoidosa biomass during 30 day digestion. In our study, CH4 yield (414 ± 2.45 ml CH4 g-VS1) from enzymatic pre-treated C. vulgaris biomass was higher than in glucose control (339 ml CH4 g-VS1), as well as CH4 production from pre-treated residues following the H2 production process remained above that of the glucose controls. Enzymatic pre-treatment supported the CH4 productivity which increases almost 60%.

Fig. 7. Fermentative methane production from C. vulgaris and residues following the H2 production process. Values correspond to means of three replicated independent values ± confidences intervals (error bars).

Fig. 8. Biogas composition for Set 3.

Table 3 Fermentative hydrogen and methane production performances from microalgae C. vulgaris. Enzymatic pretreatment

Temperature Model simulationa (°C) Hmax Rmax k R2 (ml) (ml h1) (h)

5 (g-VS 11) Microalgae 10 (g-VS 11) Microalgae 20 (g-VS 11) Microalgae 30 (g-VS 11) Microalgae

No

60

19.26

0.666

3

0.99 1.75 ± 3.75

No

60

1.13

0.123

3

1.00

19 ± 2.94

No

60

6.78

0.263

3

1.00

No

60

5.84

0.166

3

0.97

Blank A

No

60

–

–

Set 2 B1

Yes

60

38.76

4.39

3

1.00

39 ± 2.13

Yes

60

61.2

2.54

3

1.00

Yes

60

135.49

3.14

No

60

–

Yes

35

362

No

35

53

Yes

35

414

No

35

245

No

35

–

60 35

Set 1 A1 A2 A3 A4

10 (g-VS 11) Microalgae B2 10 (g-VS 11) Microalgae B3 10 (g-VS 11) Microalgae Blank B – Set 3 C1

Digestate B3 (250 ml) C2 Digestate B Blank (250 ml) C3 10 (g-VS 11) Microalgae C4 10 (g-VS 11) Microalgae Blank C –

Two-stage combine fermentation process Yes Stage 1 10 (g-VS 11) (B3) Microalgae Stage 2 Yes (C1) a b c d

Calorific valuec (kJ g-VS1)

Theoretical H2 yield (ml g-VS1)

Theoretical CH4 yield (ml g-VS1)

Theoretical calorific valued (kJ g-VS1)

0.00

311

–

3.36

–

0.21

311

–

3.36

8 ± 2.65

–

0.09

311

–

3.36

7 ± 4.11

–

0.08

311

–

3.36

0.87

–

–

–

–

0.42

311

–

3.36

62 ± 4.56

–

0.67

311

–

3.36

3

0.94 135 ± 3.11

–

1.46

311

–

3.36

–

–

3.83 ± 1.34

0.15

–

–

–

4.46

5

1.00 –

362 ± 1.79

13.00

–

373

13.15

1.59

5

1.00 –

74 ± 2.48

1.87

–

373

13.15

5

1.00 –

414 ± 2.45

14.86

–

373

13.15

5

1.00 –

245 ± 2.64

8.80

–

373

13.15

–

–

–

163 ± 3.31

5.80

–

–

–

–

–

–

–

311

–

16.51

–

–

–

–

–

373

–

–

–

16.6 6.13

H2 yieldb (ml g-VS1)

1.13 ± 0.38

1.72 ± 1.12

135 ± 3.11 –

CH4 yieldb (ml g-VS1)

13 ± 3.71

24 ± 0.98

– 362 ± 1.79

14.46

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Substrate concentration

Model simulation data were calculated by modified Gompertz equation. The data are the mean values of triplicate tests (the ‘±’ denotes standard deviation of triplicates). Computed with calorific value of 35.8 kj l1 and 10.8 kj l1 for methane and hydrogen, respectively. Theoretical calorific value based on glucose.

115

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CO2 was produced in all bottles indicating degradation in all substrates (Fig. 8). The proportion of methane in the biogas ranged from 58 to 82% for the Set 3. Singh and Gu [11] reported that biogas produced from microalgae by anaerobic digestion mainly consists of a mixture of methane (55–75%) and CO2 (25–45%). One of the most crucial factors impacting CH4 proportion in the biogas is the pH, which controls the speciation of the carbonate system and the release of CO2. If the pH is high, due to high alkalinity from NH3 release, then the gas content will shift more to CH4. During the fermentative experiments, there was no difference between initial pH and final pH.

3.4. Cumulative hydrogen and methane production from microalgae C. vulgaris The biogas produced from microalgae C. vulgaris by dark fermentation contained only H2, CO2 and CH4. Table 3 shows the cumulative H2 production fitted well to the Gompertz Eq. (2), with determination coefficients R2 over 0.98 on average. Cumulative methane production from microalgae C. vulgaris and residues resulted from H2 production process matched to Gompertz Eq. (2) with the highest R2 value (Table 3). In addition, these cumulative methane yields were statistically similar to theoretical methane production. The methane yields could be increased from 245 ± 2.46 to 414 ± 2.45 ml CH4 g-VS1 by using enzymatic pretreatment. According to results, microalgae offer a significant potential for biogas production, commercial productions have a lot of difficulties in implementation. On the other hand, two-stage combined process (combination of dark fermentation for H2 production and CH4 fermentation from residues following H2 production process) (14.46 kJ g-VS1) is not advantageous in comparison to direct CH4 production from C. vulgaris (14.86 kJ g-VS1) in terms of energy production. Depending on the technological status and conversion yields, methanogenic digestion and ethanol fermentation can most efficiently convert microalgal biomass to energy carriers [32]. However, the main aim of the study is the hydrogen production by dark fermentation at thermophilic conditions. The highest theocratic energy yields reported in the literature have been 14.4 as methane kJ g1 dry weight and 1.2 as hydrogen kJ g1 dry weight [32]. In this study, the energy production from H2 fermented from C. vulgaris 1.46 kJ g-VS1 is higher than the reported one in the literature.

4. Conclusion There is a great interest in microalgal biomass as a renewable energy source due to their availability to accumulate substantial quantities of lipids. The various valuable products such as biodiesel, bioethanol, biohydrogen and biomethane can be obtained from various algae feedstocks. The present study shows that a maximum H2 yield of 135 ± 3.11 ml H2 g-VS1 was observed at the substrate concentration of 10 g dry algae l1 with enzymatic pre-treatment at thermophilic conditions and a seven-fold increase in H2 production yields was obtained with enzymatic pre-treated microalgae substrate C. vulgaris compared to no pre-treatment. The energy yield from the H2 fermentation was 1.46 kJ g-VS1 which is higher than the reported values in the literature. The energy production of the two-stage combined fermentation process was 14.46 kJ g-VS1 and less than the reference value (14.86 kJ g-VS1) of the singlestage methane fermentation process. It was also observed that during the CH4 fermentation process high turnover rates had been achieved, whereas the H2 production from microalgae showed potential. This potential can be realized by optimizing the biological variables of the process.

Acknowledgement The authors are grateful to Dr. Dorothea Rechtenbach and Jörn Heerenklage for their support and advice. The authors wish to thank the E. ON Hanse AG for the financial support and the provision of microalgae.

Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.apenergy.2014. 07.003.

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