Dark fermentative hydrogen production using macroalgae (Ulva sp.) as the renewable feedstock

Applied Energy 262 (2020) 114574

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Dark fermentative hydrogen production using macroalgae (Ulva sp.) as the renewable feedstock Winny Margaretaa, Dillirani Nagarajana,b, Jo-Shu Changa,c,d, , Duu-Jong Leeb,e,f, ⁎

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a

Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan c Department of Chemical and Materials Engineering, College of Engineering, Tunghai University, Taichung, Taiwan d Center for Nanotechnology, Tunghai University, Taichung, Taiwan e Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 10607, Taiwan f College of Technology and Engineering, National Taiwan Normal University, Taipei 10610, Taiwan b

HIGHLIGHTS

of using macroalgae as the fermentation bioH production was assessed. • Potential process conditions for green macroalgae Ulva sp. were reported. • Optimal H producing bacterial strains were screened and identified. • Suitable • Max H production rate of 812 mL/L/h and H yield of 1.62 were reported. 2

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2

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ARTICLE INFO

ABSTRACT

Keywords: Biohydrogen Macroalgae Ulva sp. Dark fermentation Clostridium sp.

Macroalgae, commonly known as seaweed, are rich in carbohydrates which makes them a potential feedstock for biohydrogen production via dark fermentation. In this study, the green macroalgal biomass Ulva sp. was subjected to mild acid-thermal combined pretreatment for the effective release of fermentable sugars. Among the H2SO4 acid concentrations tested, 4% H2SO4 and 121 °C for 40 min attained the highest hydrolysis efficiency with a reducing sugar yield of 0.21 g RS/g biomass. The concentration of fermentation inhibitors furfural and 5hydroxymethyl furfural were below 1 g/L. Using an initial reducing sugar concentration of 12 g/L and pH 5.5, Clostridium butyricum CGS5 achieved the highest cumulative hydrogen production (2340 mL/L), maximum hydrogen productivity (208.3 mL/L/h), and hydrogen yield (1.53 mol H2/mole RS). In continuous fermentation with 6 h hydraulic retention time, maximum hydrogen productivity increased to 782.45 mL/L/h with a hydrogen yield of 1.52 mol H2/mol hexose. To the best of our knowledge, we report for the first time, biohydrogen production via dark fermentation from green macroalgal biomass Ulva sp. with better yield and productivity.

1. Introduction Over the last few years, the need for energy has increased exponentially, whereas the availability of fossil fuels is reducing. It is estimated that the world energy demand will continue to increase by 36% by the year 2035 [1]. The incessant use of fossil fuels in the transport and energy industries has led to increase in GHG emissions, climate change and an impending energy insecurity. Thus, the development of an environmental friendly and sustainable energy source, such as hydrogen (H2) is of the essence. Biohydrogen holds the promise for a substantial contribution, because it is a highly energy efficient,

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clean burning and pollution-free fuel source [2,3]. It appears particularly suitable for relatively small-scale, decentralized systems, integrated with agricultural and industrial activities or waste processing facilities [4,5]. The most widely used methods for hydrogen production are electrochemical methods like electrolysis or thermochemical methods like steam reforming. However, electrochemical and thermochemical methods are disadvantageous due to the high cost, prohibitive energy demands, utilization of fossil fuel based energy and associated GHG emissions [6]. Consequently, biological methods are preferred, because they are less energy intensive as fermentation occurs mostly at ambient temperature and pressure. Dark fermentation is preferable

Corresponding authors. E-mail addresses: [email protected] (J.-S. Chang), [email protected] (D.-J. Lee).

https://doi.org/10.1016/j.apenergy.2020.114574 Received 22 October 2019; Received in revised form 30 December 2019; Accepted 26 January 2020 0306-2619/ © 2020 Elsevier Ltd. All rights reserved.

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Table 1 The composition of Endo medium. No.

Component

Concentration (g/L)

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

Carbon source NaHCO3 NH4HCO3 K2HPO4 MgCl2·6H2O MnSO4·6H2O FeSO4·7H2O CuSO4·5H2O CoCl2·5H2O L-cystein.HCl Sodium thioglycolate Rezasurin-2127

Based on experiment 6.72 5.24 0.125 0.1 0.015 0.025 0.005 0.000125 0.5 0.5 0.001

Table 5 Production of soluble metabolites during H2 fermentation with various strain using macroalgae hydrolysate at 12 g RS/L.

Concentration (wt% per dry weight)

Carbohydrate Protein Lipid Ash

50.34 25.69 2.31 21.66

Table 3 The composition of macroalgae hydrolysate (using H2SO4 4% v/v). Composition

Concentration (%)

Total reducing sugar (RS) Total nitrogen (TN) Total phosphorus (TP) Acetic acid Propionic acid Hydroxymethyl furfural (HMF) Furfural

21.1 9.8 1.5 0.16 0.31 0.55 g/L 0.18 g/L

Table 4 The amino acid profile of macroalgae hydrolysate (using H2SO4 4%v/v). Amino acid

Concentration (%)

Arginine (Arg) Lysine (Lys) Glutamine (Gln) Asparagine (Asn) Glycine (Gly) Theonine (Thr) Alanine (Aln) Valine (Val) Seline (Ser) Proline (Pro) Isoleucine (Ile) Leucine (Leu) Methionine (Met) Histidine (His) Phenylalanine (Phe) Glutamate (Glu) Aspartate (Asp) Cysteine (Cys) Tyrosine (Tyr) Trypthophan (Try) TOTAL

2.10 1.43 N/D N/D 1.36 1.03 2.16 1.15 1.31 1.58 1.72 1.95 N/D 0.90 2.07 3.19 0.34 0.62 0.96 N/D 23.87

HLa/SMP (%)

HAc/SMP (%)

HBu/SMP (%)

EtOH/SMP (%)

SMP (g/L)

CH4 CGS5

13.78 16.52

25.61 29.98

53.88 46.51

6.73 7.08

2.83 6.78

over bio photolysis and light fermentation because of the high production rate and yield without the need for light energy [7]. Bio photolysis and light fermentation require light energy and have low light conversion efficiency [6]. Macroalgae are alternative and sustainable sources for biohydrogen production because of their high levels of carbohydrates and low levels of lignin. Macroalgae can be cultivated in non-arable land with minimal nutritional requirements, and hence they can overcome issues regarding land use changes. Macroalgae can be considered as one of the most productive biological systems to mitigate global CO2 emissions and reduce global warming. The photosynthetic efficiency of aquatic organisms is in the range of 6–8%, compared to the 1.8–2.2% of the terrestrial biomass [8]. Compared to microalgae, macroalgae harvesting is easier because they are multicellular and possesses plant-like characteristics [9]. Thus, macroalgal biomass is a potential and untapped resource for biohydrogen production. Based on their pigmentation, macroalgae are characterized as Rhodophytae (red macroalgae), Chlorophytae (green macroalgae) and Phaeophytae (brown macroalgae) with a corresponding carbohydrate content of 25–60%, 30–60% and 30–50% of their biomass, respectively [10]. These carbohydrates are extracted as fermentable sugars by pretreatment and hydrolysis, and used as substrate for biohydrogen production. A majority of the studies reported biohydrogen production from the brown algal biomass Laminaria sp. followed by the red macroalga Gelidium. Till date, very few studies report the utilization of green macroalgal biomass for biohydrogen production. Some studies have used Ulva biomass for bioethanol production [11–13]. The polysaccharides present in green macroalgae are starch, cellulose, mannan and the sulfated polysaccharide ulvan, which upon hydrolysis yields the fermentable monosugars glucose, rhamnose, mannose, xylose, and the sugar acids uronic acid and glucuronic acid [14]. Ulva lactuca biomass can be simply pretreated by a thermal process (121 °C, 25 min) in water attained a reducing sugar yield of 386.0 mg/g biomass powder, with an ethanol yield of 7.8 g/L [13]. van der Wal et al used Ulva lactuca biomass pretreated by thermal-enzymatic process in ABE (acetone-butanol-ethanol) fermentation for butanol production. Clostridium beijerinckii effectively utilized the green macroalgal biomass sugars (glucose, rhamnose, galactose, xylose) and produced 0.35 g ABE/g sugar [15]. Similarly, Ulva lactuca biomass treated by a thermal-acid method (1% H2SO4, 125 °C, 30 min) resulted in a reducing sugar yield of 15 g/L. In ABE fermentation with Clostridium sp., a butanol titer and yield of 4 g/L and 0.29 g butanol/g sugar, respectively was obtained [16]. These studies suggested the suitability of green macroalgae based sugars for fermentation by anaerobic Clostridia, and hence can be applied for biohydrogen production as well. Park et al reported that untreated Ulva lactuca biomass produced a hydrogen yield of 126.9 mL/L in dark fermentation untreated anaerobic sludge [17]. Also, Jung et al showed that untreated green macroalgal biomass Codium fragile can attain a hydrogen yield of 3.70 ± 0.17 mL H2 /g COD in dark fermentation. Thus, to the best of our knowledge, we report for the first time, the thermal-acid pretreatment of Ulva sp. biomass and subsequent use in

Table 2 The composition of macroalgae biomass (Ulva sp.) Component

Strain

2

15

10

5

0

40

3.0

3.0

2.5

2.5

2.0

2.0

30 1.5 20 1.0 10

0 0

5

10

15

HMF Conc. (%)

TRS Conc. (%)

20

50

Hydrolysis Efficiency (%TRS/%CHO)

25

1.5

1.0

0.5

0.5

0.0

0.0

Furfural Conc. (%)

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H2SO4 Conc. (%) Hydrolysis Efficiency (%) HMF Conc. (%) Furfural Conc. (%) TRS (%) Fig. 1. Effect of acid (H2SO4) concentrations on macroalgae hydrolysate.

dark fermentation in biohydrogen production. The pure anaerobic strains Clostridium pasteurianum and Clostridium butyricum were used, because obligate anaerobic fermentation could attain higher hydrogen yields in mesophilic dark fermentation [18]. Optimization of sulfuric acid concentrations for effective biomass hydrolysis was performed, and dark fermentation was conducted both in batch and continuous mode for attaining high hydrogen productivity and yield.

Table 5). 2.3. Hydrolysis of macroalgal biomass by thermal-acid (H2SO4) pretreatment To enhance hydrolysis efficiency and determine efficient hydrolysis conditions for macroalgae, different H2SO4 concentrations, such as 0%, 2.5%, 4%, 5%, 10%, 15%, and 20% (v/v) were used. A one gram of dry macroalgal biomass was mixed with 10 mL dilute sulfuric acid (0 to 20% v/v) in 25 mL glass bottle. The mixture was autoclaved at 121 °C for 20 min. After slowly cooling to room temperature, the macroalgae hydrolysate was neutralized with CaCO3 until pH 7 and the formed solid precipitate was removed by centrifuging several times using highspeed refrigerated centrifuge at 8000 rpm for 20 min and high-speed centrifuge at 13500 rpm for 5 min. The supernatant was further filtered and analyzed by high performance chromatography (HPLC) for reducing sugar content. The supernatant of macroalgal hydrolysate was used as the substrate for biohydrogen fermentation.

2. Materials and methods 2.1. Dark fermentative H2 producing bacterial strains and medium Dark fermentative H2-producing bacterial strains were isolated from the effluent sludge of a continuous dark fermentation bioreactor [19]. The axenic H2-producing strains used in this study are Clostridium butyricum CGS5 and Clostridium pasteurianum CH4. The 16rRNA gene sequence of these strains have been deposited in the NCBI nucleotide sequence databases under the accession numbers AY540109 (strain CGS5 and EF140981 (strain CH4) [20]. The composition of Endo medium for biohydrogen production is shown in Table 1.

2.4. Fermentation conditions

2.2. Feedstock - green macroalgae Ulva sp.

Each experiment started from the stock culture of Clostridium sp. which was transferred to fresh pre-culture medium. Stock culture and pre culture was grown with Endo medium with added casamino acid (2 g/L) and yeast extract (2 g/L), and sucrose (17.8 g/L) as carbon source. The bacteria were cultivated anaerobically in batch mode at 37 °C, agitation rate 200 rpm, and the initial pH of medium was adjusted to 7.5 using 5 N HCl. Argon was used to achieve and maintain anaerobic conditions. Batch fermentation was carried out by static incubation in sealed serum bottles. The fermentation variables for optimization of H2 production include macroalgae hydrolysate concentration (3, 6, 9, 12 g RS/L), pH control (6.0, 5.5, 5.0) in batch fermentation, and the operated hydraulic

The macroalgal biomass of Ulva sp. was sourced commercially from a local supplier. The wet biomass thus obtained was washed with running water for 5 times to remove salts and extraneous components and dried in the oven at 50 °C until the weight remains unchanged. The dried biomass was then milled to less than 100 mesh size by a grinder. The carbohydrate, protein, lipid and ash content of the biomass was determined. Amino acid content of the macroalgal hydrolysate was measured as described previously [21]. The composition of the macroalgae biomass is shown in Table 2. The composition and amino acid profile of macroalgal hydrolysate is shown in Table 3 and Table 4 (see

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W. Margareta, et al. 100 Glucose Xylose Rhamnose

Sugar utilization (%)

80

60

40

20

0

Sugar utilization (%)

100

Total RS

80

60

40

20

0

500

0

1.0

50

80 0.8 60

0.6

40

0.4

20

0 CH4

Strain

40

30

20

0.2

10

0.0

0

Max. H2 content (%)

1000

60

H2 yield (mole H2/mole RS)

1500

1.2

100

Max. H2 production rate (ml/L/h)

Cumulative H2 production (ml/L)

2000

CGS5

Max. H2 production rate H2 yield Max.H2 content Cumulative H2 production

Fig. 2. Performance of H2 production and substrate consumption with various strains using macroalgae hydrolysate (12 g RS/L) as the carbon source at 200 rpm and with Endo medium.

retention time/HRT = 4, 6, 8 h in continuous fermentation. The optimum macroalgae hydrolysate concentration was used to investigate the performance of pH control, while these optimum results were used to investigate the performance of CSTR system. During fermentation, cell concentration, pH, reducing sugar, H2 production and soluble metabolites were monitored with respect to culture time.

HRT =

V Fo

where V is the working volume of the culture (mL) and Fo is the volumetric feeding rate of the medium (mL/h).

4

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W. Margareta, et al. 100 Glucose Xylose Rhamnose

Sugar utilization (%)

80

60

40

20

0

Sugar utilization (%)

100

Total RS

80

60

40

20

500

1.4

80

1.0 60

0.8 0.6

40

0.4

50

20

0

0.0 Algae powder

Algae hydrolysate

Carbon source

30

20

10

0.2 0

40

Max. H2 content (%)

1.2

H2 yield (mole H2/mole RS)

1000

100

Max. H2 production rate (ml/L/h)

Cumulative H2 production (ml/L)

1500

60

1.6

0

2000

0

Sucrose

Max. H2 production rate H2 yield Max.H2 content Cumulative H2 production

Fig. 3. Performance of H2 production and substrate consumption with various carbon sources at a carbohydrate concentration of 12 g/L. Table 6 Production of soluble metabolites during H2 fermentation with various carbon sources at a carbohydrate concentration of 12 g/L. Carbon source

HLa/SMP (%)

HAc/SMP (%)

HBu/SMP (%)

EtOH/SMP (%)

SMP (g/L)

Macroalgae powder Macroalgae hydrolysate Sucrose

10.39 16.52 12.16

21.28 29.89 25.73

44.21 46.51 52.24

24.12 7.08 9.87

2.15 6.78 6.91

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Glucose Xylose Rhamnose

Sugar utilization (%)

100

80

60

40

20

0

Sugar utilization (%)

100

Total RS

80

60

40

20

0

500

0

1.0

50

80 0.8 60

0.6

40

0.4

20

0 4

8

12

40

30

20

0.2

10

0.0

0

Max. H2 content (%)

1000

60

H2 yield (mole H2/mole RS)

1500

1.2

100

Max. H2 production rate (ml/L/h)

Cumulative H2 production (ml/L)

2000

16

Concentration of macroalgae hydrolysate

Max. H2 production rate H2 yield Max.H2 content Cumulative H2 production

Fig. 4. Performance of H2 production and substrate consumption under various reducing sugar concentrations of macroalgae hydrolysate at 200 rpm on Endo medium.

2.5. Analytical methods

column (2.159 mm in inner diameter and 4 m in height) (Shing-Der et al., 2007) was packed with Porapax Q (China Chromatography, Taipei, Taiwan). The operation conditions were flow rate 20 mL/min, oven temperature 50 °C, injector temperature 120 °C, and TCD temperature 140 °C.

2.5.1. Determination of gas products by gas chromatography (GC) The gas products (H2) were analyzed by gas chromatography (Model 9800, China Chromatography, Taipei, Taiwan) using a thermal conductivity detector. The carrier gas for GC analysis was argon and the

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chosen for the pretreatment and hydrolysis of dried and powdered Ulva sp. biomass. The biomass composition of Ulva sp. is shown in Table 1. It can be seen that the high carbohydrate content makes it suitable as a renewable feedstock of reducing sugars for dark fermentative H2 production. Sivagurunathan et al showed that dilute acid pretreatment of the red macroalgal biomass with 1% H2SO4 at 121 °C for 30 min increased the maximum hydrogen production to 52 mL-H2/g-dry biomass from 27 mL-H2/g-dry biomass with only thermal pretreatment [22] Also, sulfuric acid pretreatment enhanced hydrogen production, while treatment with other acids such as hydrochloric acid, phosphoric acid and nitric acid resulted in lower reducing sugar recovery and hydrogen production potential. To optimize the reducing sugar yield, various acid (H2SO4) concentration from 0 to 20% were investigated in this study and the results can be seen in Fig. 1. Among various H2SO4 concentrations examined, best hydrolysis efficiency was obtained with 4% H2SO4. The amount of reducing sugar concentration, hydrolysis efficiency with respect to biomass hydrolysis efficiency with respect to carbohydrates, HMF concentration, and Furfural concentration were 21.1 g/L, 0.21 g RS/g biomass, 0.42 g RS/g carbohydrates, 0.55 g/L, 0.18 g/L, respectively. The inhibitors (HMF and Furfural) concentrations increased correspondingly with H2SO4 concentrations. The HMF and furfural concentrations with 2.5%, 4%, 5% and 10% H2SO4 were 0.47, 0.55, 0.66 and 0.82 g/L and 0.1, 0.18, 0.25 and 0.56 g/L, respectively. To attain optimal sugar recovery with minimal inhibitor formation, sulfuric acid concentration of 4% was chosen for further experiments of mesophilic dark fermentation using macroalgae hydrolysate.

Table 7 Production of soluble metabolites during H2 fermentation under various concentration using macroalgae hydrolysate at different reducing sugar concentrations on Endo medium and 200 rpm. Concentration (g RS/ L)

HLa/ SMP (%)

HAc/ SMP (%)

HBu/ SMP (%)

EtOH/ SMP (%)

SMP (g/ L)

4 8 12 16

16.72 15.25 16.12 15.96

29.10 27.17 28.89 30.11

47.10 49.71 48.51 47.81

7.08 7.87 6.48 6.12

5.87 6.21 6.86 7.35

2.5.2. Determination of reducing sugar and soluble metabolites concentration by high performance liquid chromatography (HPLC) The concentration of reducing sugar and soluble metabolites (volatile fatty acids and alcohol) in the filtered (0.2 mm) supernatant of culture broth were measured by high performance liquid chromatography (HPLC). For reducing sugar, HPLC equipped with refraction index detection (L-2490, Hitachi) and column NH2P-50 4E column (Shodex Asahipax, Japan) was used. The mobile phase was 70% acetonitrile in water at flow rate of 1 mL/min and column temperature was controlled at 30 °C. For soluble metabolites, HPLC equipped with a refraction index detection (RID-2414, Waters) and column ICSep ICECOREGEL 87H3 column (Transgenomic, USA) was used. The mobile phase was 0.008 N H2SO4 at flow rate of 0.4 mL/min and column temperature was controlled at 70 °C. The injection sample volume for both HPLC was 20 μl. To prepare the sample, the fermentation broth was centrifuged at 13500 rpm for 5 min. The supernatant was filtered by 0.22 µm membrane. The concentration of reducing sugar and soluble metabolites (volatile fatty acid and alcohol) were calculated by the standard calibration curve.

3.2. Screening of H2 producing bacteria for effective H2 production using macroalgae hydrolysate The macroalgal biomass was hydrolyzed using 4% sulfuric acid and then the reducing sugars obtained were used as a substrate for biohydrogen fermentation. Clostridium pasteurianum CH4 and Clostridium butyricum CGS5 strains were cultivated in Endo Medium with 200 rpm agitation for mesophilic dark fermentation using macroalgae hydrolysate at a concentration of 12 g/L. The optimal conditions such as initial pH 7.5 and temperature 37 °C for this fermentation was obtained from our previous study (Lo et al., 2008). Among the two strains (CH4 and CGS5) examined, the results (Fig. 2) showed that strain CGS5 utilized macroalgae hydrolysate better than strain CH4. For strain CH4, the H2 production rate, H2 yield, and H2 content were 5.41 mL/L/h, 0.14 mol H2/mole RS, and 11.3%, respectively. Analysis of reducing sugar consumption indicated that CH4 strain can utilize glucose, but cannot utilize xylose and rhamnose, and thus have poor H2 production. This finding is in agreement with the literature, indicating that Clostridium pasteurianum species is unable to use xylose as the carbon source for growth [23] and the xylose utilization effieicncy of CH4 in this study was 5.14%. The capability of Clostridium pasteurianum to utilize rhamnose has not been investigated yet, and from our studies it is clear that C. pasteuranium is unable to metabolize rhamnose. A rhamnose utilization rate of 6.13% was observed with C. pasteuranium and a majority of the rhamnose (about 6 g/ L) remained in the culture medium even after the glucose concentration dropped to < 1 g/L, alleviating the possibility of catabolite repression of rhamnose utilization in the presence of glucose. These results suggested that C. pasteuranium is incapable of rhamnose uptake and utilization. The total sugar utilization efficiency of CH4 remained low at 33.22%, resulting in lower hydrogen production. Thus, CH4 is not a suitable strain for the fermentation of green macroalgae hydrolysate. In contrast, for strain CGS5, the H2 production rate, H2 yield, and H2 content were 73.2 mL/L/h, 1.01 mol H2/mole RS, and 46.9%, respectively. The result indicated that CGS5 strain can utilize all of the

2.5.3. Measurement of hydrogen yield, productivity and sugar consumption The biohydrogen yield was calculated as the amount of H2 produced divided by the amount of sugar consumed during the fermentation. The biohydrogen yield (mol H2 / mol reducing sugar) was calculated using the following equations:

Biohydrogen yield (mol H2/mol RS) =

mol H2 produced mol sugar consumed

Biohydrogen productivity was calculated as the ratio of volume of H2 produced (mL/L) to fermentation time during the exponential growth. The biohydrogen productivity was calculated using the following equations:

Biohydrogen productivity(mL/L/h) =

volume H2 produced fermentation time

Reducing sugar consumption was defined as the ratio between the amounts of sugar consumed during fermentation divided by the initial sugar concentration. The sugar consumption (%) was calculated using the following equations:

Sugar consumption(%) =

(initial RS conc. remaining RS conc.) initial RS conc. 100%

3. Result and discussion 3.1. Effect of acid (H2SO4) concentrations on macroalgae hydrolysis Optimization of Ulva sp. hydrolysis for the release of fermentable sugars was essential for effective reducing sugar recovery and subsequent dark fermentation. A combined thermal-acid pretreatment for

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Sugar utilization (%)

100

Glucose Xylose Rhamnose

80

60

40

20

0

Sugar utilization (%)

100

Total RS

80

60

40

20

1000

500

250

1.4 1.2

200

1.0 150

0.8 0.6

100

0.4 50 0.2

0

0

0.0 Without control

5

pH

5.5

60

50

40

30

20

Max. H2 content (%)

1500

1.6

H2 yield (mol eH2/mole RS)

2000

Max. H2 production rate (ml/L/h)

Cumulative H2 production (ml/L)

2500

0

10

0

6

Max. H2 production rate H2 yield Max.H2 content Cumulative H2 production

Fig. 5. Performance of H2 production and substrate consumption under various pH control using macroalgae hydrolysate (12 g RS/L) and an agitation rate of 200 rpm.

reducing sugars glucose, xylose, and rhamnose with a utilization efficiency of 90.87%, 47.85% and 64.82%, respectively with a total reducing sugar utilization efficiency of 70.43%. Forsberg et al showed that rhamnose was catabolized by Clostridium butyricum strain effectively. The metabolic pathway for the catabolism of rhamnose has been characterized in Clostridium butyricum [24]. L-Rhamnose is metabolized by a pathway mediated by L-rhamnose permease, L-rhamnose

isomerase, L-rhamnulose kinase, and L-rhamnulose-1-phosphate aldolase. The last enzyme cleaves L-rhamnulose-1-phosphate with the formation of equimolar concentrations of dihydroxyacetone phosphate and L-lactaldehyde. The dihydroxyacetone phosphate is further metabolized by glycolysis, while the lactaldehyde is either converted under aerobic conditions to lactate and pyruvate, or under anaerobic conditions, to L-1,2-propanediol by an oxidoreductase, thereby regenerating

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substrate usually plays a crucial role in the efficiency of anaerobic hydrogen production [26,27]. Thus, the effect of macroalgae hydrolysate concentration was investigated. Biohydrogen production was conducted at different macroalgal hydrolysate concentration from 4 to 16 g RS/L (based on the reducing sugar concentration). Fig. 4 indicated that the cumulative hydrogen production and maximum hydrogen production rate increased along with the increase in the concentration of macroalgal hydrolysate for the concentration range of 4 to 12 g RS/L. When the concentration was further increased to 16 g RS/L, the cumulative production, production rate, and hydrogen yield decreased. At high substrate concentration, bacterial growth rate was inhibited by excess substrate (substrate inhibition) and high concentrations of fermentation by-products (Table 7 for the soluble metabolites data). The results clearly indicate that a macroalgae hydrolysate concentration of 12 g RS/L exhibited the best performance in hydrogen production for most of categories compared in Fig. 4, especially for total hydrogen production and hydrogen yield, where the H2 production rate, H2 content, and H2 yield were 75.3 mL/L/h, 45.4%, and 1.00 mol H2/mole RS, respectively. The total reducing sugar utilization decreased slightly from 77.0% to 59.5% when the concentration of macroalgal hydrolysate was increased from 4 to 16 g RS/L. A higher glucose utilization than rhamnose and xylose utilization implied that glucose was catabolized preferably over rhamnose and xylose. In addition, the result indicates that the amount of inhibitors (HMF and furfural) present in the hydrolysate did not affect the fermentation performance of Clostridium butyricum CGS5. The pH decreased correspondingly with H2 production and formation of acidic soluble metabolites, such as lactate, acetate, butyrate, and ethanol (Table 7).

Table 8 Production of soluble metabolites during H2 fermentation under various pH control using macroalgae hydrolysate (12 g RS/L) in Endo medium and 200 rpm. pH control

HLa/SMP (%)

HAc/SMP (%)

HBu/SMP (%)

EtOH/SMP (%)

SMP (g/ L)

Without control 5.0 5.5 6.0

16.12 15.25 18.11 17.79

28.89 30.37 29.34 30.71

48.51 46.89 45.36 44.64

6.48 7.48 7.29 6.87

6.86 5.17 6.68 6.41

NAD to produce biohydrogen [24]. Thus, Clostridium butyricum CGS5 was chosen for the dark fermentative H2 production using green macroalgal hydrolysate. The pH decreased correspondingly with H2 production and formation of acidic soluble metabolites, indicating that hydrogen evolution was accompanied by formation of acidic soluble metabolites. Using macroalgae hydrolysate as the substrate, butyrate appeared to be the dominant soluble metabolite, as the ratio of butyrate to soluble metabolite products (HBu/SMP) was around 0.5. The second abundant metabolite was acetate, followed by lactate and ethanol (Table 4). The metabolite composition indicates that biohydrogen production with the pure isolates (Clostridium pasteurianum and Clostridium butyricum strains) belonged to butyrate-type fermentation, which is in accordance with previous findings [20]. Furthermore, the total volatile fatty acids or TVFA (soluble metabolites without ethanol)/SMP ratio was essentially around 0.95. The predominance of TVFA in total soluble metabolites suggests that H2 production was metabolically favorable since acidogenic pathway was predominant over solventogensis [25].

3.5. Effect of pH control on H2 production

3.3. H2 production using non-hydrolyzed macroalgae powder

The results shown earlier indicate that an initial pH of 7.0 was preferable for bio hydrogen production, whereas hydrogen evolution stopped when the pH was lower than 5.0. This is mainly because at this low pH the cells cannot grow. Therefore, pH control seems to be a key operational parameter to improve hydrogen production performance. Dark fermentation was conducted at three different pH values, namely, 6.0, 5.5, and 5.0, to identify the optimal pH for bio hydrogen production with Clostridium butyricum CGS5. For comparison, a control experiment without pH control was also carried out. The pH range was chosen because optimal pH for anaerobic H2 production reported in literature was essentially within the range of 5.5–6.5 [28]. Fig. 5 shows that cell growth and hydrogen production were strongly inhibited at pH 5.0. Operation at pH 5.5 attained the highest total hydrogen production and hydrogen yield of 208.3 mL/L/h and 1.39 mol H2/mole RS, respectively. The H2 content was in the range of 40–51% for all tests, with the highest value of 50.2% at pH 5.5. Operation at pH 5.5 resulted in a rapid decrease in pH because the bacteria completely consumed carbon sources after 10 h cultivation and produced acidic soluble metabolites products. The major soluble metabolites producing during H2 fermentation using macroalgae hydrolysate were butyrate, acetate, lactate, and ethanol (Table 8). Therefore, higher hydrogen productivity and yield compared were achieved at pH 5.5, compared to pH 5.0 and 6.0. This optimal pH is the same as that proposed in our previous study when using sucrose as carbon source [29]. This indicates that the change in the type of carbon source did not affect the optimal pH for the Clostridium butyricum CGS5 in dark fermentative hydrogen production, even when a complex substrate like macroalgal hydrolysate was used.

In this study, mesophilic dark fermentation with H2 producing bacteria using macroalgae powder was investigated. For comparison, a control experiment using sucrose and macroalgae hydrolysate was also carried out. The mono sugar concentration in macroalgae powder (without hydrolysis) was 0.44 g/L (glucose 0.13 g/L, xylose 0.06 g/L and rhamnose 0.25 g/L). The results (Fig. 3) show that cell growth and hydrogen production were low with untreated macroalgae powder as a fermentation substrate, where the hydrogen production rate, hydrogen yield, and hydrogen content were 1.74 mL/L/h, 0.20 mol H2/mole RS, and 15.4%, respectively. Poor bio hydrogen production was observed when the microalgal biomass was not pretreated with chemicals or enzymes, suggesting the need of biomass pretreatment. The results indicated that Clostridium butyricum CGS5 could not utilized polysaccharides efficiently. Also, the comparison between macroalgae hydrolysate and sucrose as a fermentation substrate for dark fermentation was investigated. For macroalgae hydrolysate (Fig. 3), the hydrogen production rate, hydrogen yield, and hydrogen content were 73.2 mL/L/h, 1.01 mol H2/ mole RS, and 46.9%, respectively, while for sucrose, the hydrogen production rate, hydrogen yield, and hydrogen content were 84.9 mL/ L/h, 1.32 mol H2/mole RS, and 48.3%, respectively. The results showed H2 production using macroalgae hydrolysate was slightly lower, but still comparable with the results using sucrose. Several previous studies pointed out that Clostridium butyricum CGS5 prefer hexose over pentose as the carbon substrate for cell growth and H2 production, most likely due to a more efficient hexose metabolism [23]. Nevertheless, the results still indicated that macroalgae hydrolysate can be an alternative feedstock for bio hydrogen production in the future. The soluble metabolites are shown in Table 6.

3.6. BioH2 production via continuous fermentation

3.4. Effect of macroalgae hydrolysate concentration on bioH2 production

Mass production of biohydrogen is usually achieved by continuous bioreactors. Continuous fermentation has many advantages over other fermentation methods including high productivity and better control

Organic loading, such as the composition and concentration of 9

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Fig. 6. Continuous H2 fermentation with Clostridium butyricum CGS5 at different HRT using macroalgae hydrolysate (12 g RS/L) as the carbon source, Endo medium, 200 rpm, and pH control 5.5.

10

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40

20

0

1.6

50

800

1.5 40

600 30 400 20

1.4 1.3 1.2 1.1

200

10

0

0 Batch

HRT 4 hr

HRT 6 hr

H2 yield (mole H2/mole RS)

60

1.7

60

Max. H2 content (%)

Total RS utilization (%)

80

1000

Max. H2 production rate (ml/L/h)

100

1.0 0.9

HRT 8 hr

Max. H2 production rate H2 yield Max.H2 content Total RS utilization

Fig. 7. H2 production, substrate consumption, and soluble metabolites during H2 fermentation under batch and continuous fermentation using 12 g RS/L macroalgae hydrolysate as carbon source, Endo medium, 200 rpm, and pH control 5.5.

over growth rates. The continuous culture was carried out at a progressively decreasing HRT from 8 to 4 h using macroalgae hydrolysate. Figs. 6 and 7 show that the H2 content in biogas was stably maintained at around 50–55% for over 8 days of operation. The pH varied between 5.5 and 5.8. The hydrogen production rate increased from 428.9 mL/L/h to 812.4 mL/L/h and the hydrogen yield decreased from 1.62 mol H2/mole RS to 0.98 mol H2/mole RS when the HRT was shortened from 8 to 4 h. The productivity significantly increased with a decrease in HRT due to the increase in the volumetric organic loading rate when a sufficient amount of H2 producing biomass was retained in the bioreactor. However, decreasing HRT increases the flow rate of fresh medium. When the flow rate is increasing, it will reflect in the production cost (higher substrate cost). Inverse correlation between H2 yield and HRT has been observed in many dark fermentation cases reported in the literature [30,31]. The results clearly indicate that HRT 6 h exhibited the best performance in hydrogen production for most of categories, where the H2 production rate, H2 content, and H2 yield were 782.5 mL/L/h, 51.6%, and 1.52 mol H2/mole RS, respectively. The soluble metabolites in the continuous culture consisted mainly of butyric acid, which accounted around 45% of total soluble metabolites (Fig. 7), followed by acetic acid, lactic acid, and ethanol. This composition is quite similar to that obtained in batch cultures. Under this optimal operation conditions, the maximum hydrogen production rate and hydrogen yield obtained from this work is better than those reported in related studies. Hence, Clostridium butyricum CGS5 isolate is a potential candidate for practical biohydrogen generating processes from macroalgae hydrolysate. Table 9 summarizes the studies reported in literature for biohydrogen production based on macroalgal biomass. As mentioned in Section 1, most studies use brown or red macroalgae for biohydrogen production. This could be due to the abundant availability of both these macroalgae; red and brown seaweed contribute to about 57% and 43% of the total cultivated aquatic plants [32]. Red macroalgae is cultivated for the extraction of carrageenan, and the farmed brown macroalgae kelp is a known food source [33]. Farming of these macroalgae mainly takes place in South east Asian countries. On the other hand, green macroalgae, particularly Ulva sp. or the sea lettuce is the major algae forming green algal blooms due to eutrophication and massive shoring

of these algae is a potential environmental risk [34,35]. Thus, disposal of the massive macroalgal biomass that is shored could be handled in an environmental friendly way by using them for dark fermentation. We showed the feasibility that certain Clostridial species such as Clostridium butyricum presents high potential for simultaneous utilization of Ulva biomass derived sugars. Rhamnose and xylose were not metabolized by Clostridium pasterianum, indicating that the choice of the fermentative bacteria highly influences the hydrogen productivity and yield. Thus, biohydrogen production from Ulva sp. can establish effective coastal ecology management, pollution control and energy generation. 4. Conclusions Macroalgae has high carbohydrates content and lower lignin content, which makes them a preferable candidate for biofuels production. The green macroalgae Ulva sp. was used as a third generation sustainable feedstock for biohydrogen production in this study. Ulva sp. biomass consisted of 50.3% carbohydrates, 25.7% protein, 2.31% lipids, and 21.7% ash. The major reducing sugars released upon hydrolysis were glucose and rhamnose, followed by galactose and xylose. So it was essential to choose a Clostridial strain that could effectively metabolize rhamnose. The incapability of Clostridium pasteurianum CH4 to uptake and metabolize rhamnose was observed clearly in this study, and the species also showed poor xylose utilization. Clostridium butyricum CGS5 could uptake and metabolize rhamnose and xylose simultaneously, indicating the suitability of this strain for fermentation of mixed sugars from renewable biomass. Thus, the best-macroalgae hydrolysate based H2 producer was Clostridium butyricum CGS5 with 12 g RS/L concentration of reducing sugar and pH control 5.5 and attained H2 production rate, H2 yield, and H2 content of 208.2 mL/L/h, 1.39 mol H2/mole RS, and 50.3%, respectively. Continuous fermentation strategy (HRT 6 h) was done to achieve a higher productivity of 782.5 mL/L/h and H2 yield was 1.52 mol H2/mol RS. Under this optimal operation conditions, the maximum hydrogen production rate and hydrogen yield obtained from this work is better than those reported in related studies. Even though many studies reported biohydrogen production from brown and red macroalgae, we report for the first time effective hydrolysis and fermentation of the green macroalga Ulva sp. for 11

12

Ulva sp. (12 g RS/L)

Padina tetrastromatica, thermal-acid treatment: 1% H2SO4, 100 °C, 2 h. Laminaria japonica, thermal-acid pretreatment: 1% H2SO4 at 121 °C for 30 min Arthrospira platensis and Laminaria japonica combined biomass, thermal-acid pretreatment: 2.5% dilute H2SO4 at 135 °C for 15 min Ulva sp., thermal acid treatment: 4% H2SO4, 121 °C, 40 min

Thermally treated Laminaria japonica at 121 °C and 30 min

Laminaria japonica, thermal-acid pretreatment: 4.8% H2SO4, 93 °C, 23 min Laminaria japonica powder

Thermally treated Laminaria japonica at 170 °C and 20 min.

Untreated Laminoria japanica

Untreated green macroalgae Codium fragile powder

Gelidium amansii, Acid-thermal treatment: 0.5% H2SO4, 15 min, 161–164 °C, detoxified by activated carbon treatment Laminaria japonica, electric pretreatment: 58.5 V for 30 min

Batch, Endo medium, 200 rpm agitation, pH 5.5, substrate: 12 g RS/L Continuous, 6 h HRT, Endo medium, 200 rpm agitation, pH 5.5, substrate: 12 g RS/L

Batch, 36 °C, 120 rpm, Initial pH 7, 400 mg/L Fe2+, substrate: 10 g-VS/L Batch, 37 °C, 30 rpm, initial pH 6, C/N ratio 26.2, substrate concentration 20 g VS/L

Batch fermenter, Initial pH 7, cultivation pH < 5.5, 35 °C, substrate at 20 g COD/L Batch fermenter, Initial pH 7, cultivation pH < 5.5, 35 °C, substrate at 20 g COD/L Batch fermenter, Initial pH 7, cultivation pH < 5.5, 35 °C, substrate at 20 g COD/L Batch fermenter, Initial pH 8, cultivation pH < 5.5, 35 °C, substrate at 20 g COD/L Batch fermenter, Initial pH 7.5, cultivation pH 5.5, 35 °C, substrate at 20 g COD/L Batch, nutrient medium, 120 rpm agitation, 35 °C, Initial pH 7, cultivation pH 6, 2% substrate Batch, nutrient medium, 37 °C, pH 6.

Batch, Nutrient medium, pH 7, 36 °C, 150 rpm agitation Batch, Nutrient medium, Initial pH 7, 36 °C, 150 rpm agitation 1 g vs/L substrate Batch, Endo medium, pH 7, 35 °C, 150 rpm agitation. Batch, Nutrient medium, pH 7.5, 35 °C, substrate 15 g RS/L Batch, Nutrient medium, pH > 5.3, 35 °C, 150 rpm agitation 15 g/L substrate –

Fermentation conditions

782.45

208.28

4.14

–

51.61

50.32

–

–

28.4

–

– –

–

–

–

–

–

–

–

–

– –

–

510

34.3

70

This study This study

1.52 mol H2/mol hexose

[46]

[45]

[44]

[43]

[42]

[41]

[40]

[40]

[40]

1.39 mol H2/mol hexose

85.0 mL H2/g VS

19.47 mL H2/g VS

78 ± 2.9 mL/0.05 g VS

71.4 mL H2/g TS or 0.92 mol H2/mol hexose 83.45 ± 6.96 mL H2/g biomass

159.6 mL H2/g DCW

106.9 mL H2 /g COD added

5.53 ± 0.20 mL H2 /g COD added

3.70 ± 0.17 mL H2 /g COD added

DCW

[39]

102.7 mL g

[38] −1

[17]

[22]

[37]

[36]

Reference

37.0 mL H2/g dry biomass

0.89 mol H2 /mole RS

52.8 ± 0.2 mL H2/g TS

–

1.59 mL/h

15.8 mL H2/g TS

H2 yield

28 mL H2/g TS

–

H2 content (%)

2.81

8.56 mL/h

H2 production rate (mL/L/h)

Footnotes: TS – total solids, VS – volatile solids, RS – reducing sugars, DCW – dry cell weight, COD – chemical oxygen demand, HRT – hydraulic retention time

Clostridium butyricym CGS5

Clostridium butyricym CGS5

Heat treated anaerobic sludge

Heat treated digester sludge

Heat treated sewage sludge

Heat treated anaerobic sludge Electricity treated anaerobic sludge Heat treated anaerobic sludge Heat treated anaerobic sludge Heat treated anaerobic sludge Heat treated anaerobic sludge Heat treated anaerobic sludge Heat treated anaerobic sludge

Laminaria japonica: microwave pretreatment at 160 °C

Irradiation treated digested sludge Heat treated anaerobic sludge Heat treated granular digested sludge Heat treated sewage sludge

Laminaria japonica, microwave – acid pretreatment: 140 °C and 2450 MHz with 1% H2SO4 for 15 min Gelidium amansii, acid-thermal pretreatment: 1% H2SO4, 121 °C, 30 min, 19.4 g RS/L which equals 28,000 mg/L COD Laminaria japonica, thermal pre-treatment at 120 °C for 30 min

Substrate and pretreatment

H2 producing bacteria

Table 9 Comparison of macroalgae-based biohydrogen production performance between our results and related studies.

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biohydrogen production. [18]

CRediT authorship contribution statement

[19]

Winny Margareta: Data curation, Formal analysis. Dillirani Nagarajan: Data curation, Formal analysis. Jo-Shu Chang: Supervision, Writing - original draft. Duu-Jong Lee: Conceptualization, Writing - original draft, Writing - review & editing.

[20] [21]

Declaration of Competing Interest [22]

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

[23] [24]

Acknowledgment

[25]

The financial supports from Ministry of Science and Technology (MOST) of Taiwan (No. 107-2221-E-002-098-MY3 and No. 108-2811-E002-547) is highly appreciated.

[26]

Appendix A. Supplementary material [27]

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.apenergy.2020.114574.

[28]

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[29]

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