Improved Hydrogen Production from Galactose Via ...

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Seoul 136-714, South Korea fuel energy sources that are renewable [1,2]. In recent years, biofuels have shown promise as alternative and green energy carriers ...
Arab J Sci Eng DOI 10.1007/s13369-015-1729-3

RESEARCH ARTICLE - CHEMICAL ENGINEERING

Improved Hydrogen Production from Galactose Via Immobilized Mixed Consortia Gopalakrishnan Kumar1 · Periyasamy Sivagurunathan1 · Jong-Hun Park2 · Sang-Hyoun Kim1

Received: 4 February 2015 / Accepted: 8 June 2015 © King Fahd University of Petroleum & Minerals 2015

Abstract In this study, a combined encapsulation and entrapment immobilization strategy was employed to enhance hydrogen production from sewage sludge containing mixed microbial cultures. The results showed that the hydrogen production rate (HPR) and hydrogen yield (HY) of immobilized cells were significantly higher than that of the suspended cells. The peak HPR and HY of 0.76 L/L-d and 1.20 mol/mol galactoseadded attained with the immobilized cell system were comparable to that of the suspended cell system (HPR 0.62 L/L-d and HY 0.88 mol/mol galactoseadded , respectively). The immobilized beads were also found to have efficient hydrogen production upon reuse for more than five cycles, with galactose removal >85 % in all cases. Soluble metabolic product analysis revealed that fermentation followed a butyrate pathway and the major metabolites produced were acetate and butyrate. The peak total energy production rate and yield were 8.6 kJ/L-d and 308 kJ/mol added , respectively. Keywords Hydrogen · Sodium alginate · Chitosan · SiO2 · Soluble metabolic products

1 Introduction Dwindling fossil fuel resources and the environmental pollution have necessitated identification of alternative and clean

B

Sang-Hyoun Kim [email protected]

1

Department of Environmental Engineering, Daegu University, Jillyang, Gyeongsan, Gyeongbuk 712-714, Republic of Korea

2

School of Civil, Environmental and Architectural Engineering, Korea University, Anam-Dong, Seongbuk-gu, Seoul 136-714, South Korea

fuel energy sources that are renewable [1,2]. In recent years, biofuels have shown promise as alternative and green energy carriers; however, there are still many barriers to be overcome before their widespread application is possible [3]. In this spotlight, biohydrogen production from various organic sources including wastewater, agricultural waste and lignocellulose waste was investigated for the sustainable biofuels production [4–6]. Lignocellulose biomass could be a feasible source of bioenergy production; however, the removal of the lignin moiety can be a tedious task that increases the cost of the overall process. In recent years, algal biomass has been extensively investigated as a potential candidate for biohydrogen production [7]. Pretreatment of algal biomass can enable retrieval of sugars as galactose and glucose; however, the galactose fraction is much higher than the glucose fraction due to the galactan nature of algae [8]. Nevertheless, hydrogen fermentation of galactose and glucose– galactose mixtures has been reported for suspended biomass [9]. Accordingly, it would be interesting to investigate the hydrogen fermentation of galactose using immobilized cells because of the advantages of this method. In recent years, the development and application of immobilized cells have increased remarkably due to the advantages of reusability and enhanced performance in biological hydrogen production. When compared to conventional suspended cell systems, immobilization of microorganisms provides more features, such as retention of high biomass concentration, increased resistance to external environmental stress, high metabolic activity and cost-effectiveness [10]. Many reports have described different immobilization techniques, with entrapment of cells or biomass within a porous matrix being extensively studied [11,12]. Indeed, this method is likely to be more advantageous than other methods such as adsorption onto the surface and self-aggregation [13,14].

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Moreover, entrapment facilitates maintenance of higher biomass at lower hydraulic retention times (HRTs) since the microbial growth is greatly affected and generates a strict anaerobic condition in which microbes are attached or surrounded by the matrix. Among the methods of immobilization, entrapment via calcium alginate is easy and low cost; therefore, it has potential for application on an industrial scale [15]. Furthermore, alginate beads can be modified using hybrid or novel materials to improve gas permeability, mechanical strength and density [16]. In this study, a novel hybrid immobilization method (entrapment + encapsulation) based on combination of conventional calcium alginate with the addition of chitosan (for entrapment) and silicon dioxide was proposed to provide mechanical stability to the beads. SiO2 and chitosan were selected to improve mechanical strength and prevent cell leakage [10]. Additionally, the stability in terms of reusability was also investigated by using galactose as a carbon source.

2 Materials and Methods 2.1 Galactose and Seed Inoculum The monosaccharide sugar galactose (Daejung, Korea) was purchased commercially and used as received. The seed inoculum was collected from an anaerobic digester in a local wastewater treatment plant. The characteristics were reported in our previous study [8]. Heat treatment at 90 ◦ C for 30 min in a water bath was applied as pretreatment to avoid the presence of hydrogen consumers (methanogens) and enrich the spore-forming hydrogen producers [17]. 2.2 Preparation of Immobilized Beads Immobilized cells were prepared by adding 20 % w/v of the heat- treated mixed consortia into a solution of sodium alginate (2 %), silicon dioxide (SiO2 - 1 %) and chitosan (1 %) for entrapment and encapsulation [10]. The alginate-cell mixture was extruded into sterile calcium chloride solution (2 %) for cell entrapment in alginate beads. The beads formed (5– 6 mm) were further hardened by stirring in a fresh solution of calcium chloride for 2 h. Finally, the beads were washed three times with sterile distilled water and then dried, after which they were stored in a refrigerator at 7 ◦ C until use.

125, MgCl2 ·6H2 O 100, MnSO4 ·6H2 O 15, FeSO4 ·7H2 O 25, CuSO4 ·5H2 O 5 and CoCl2 ·5H2 O 0.12 [18]. Next, 5 g of immobilized beads or 15 mL of suspended mixed culture was added. The bioreactor was then purged with N2 gas for 3 min to ensure strict anaerobic conditions, after which it was agitated at 100 rpm and 35 ± 0.1 ◦ C. The pH was kept at 6.5 during batch operation. 2.4 Analysis Biogas collected at various time intervals was measured using an airtight glass syringe and then corrected to standard temperature and pressure (0 ◦ C and 1 atm). The H2 , CH4 , N2 and CO2 contents in the biogas were analyzed by gas chromatography (GC, SRI 310, SRI Instrument) using a thermal conductivity detector and a 1.8 m × 3.2 mm stainless steel column packed with mole sieve 5A (SRI Instrument) and a 0.9 m × 3.2 mm stainless steel column packed with Porapak Q (80/100 mesh, SRI Instrument). Organic acids (C1–C6), 5-HMF, levulinic acid and sugars were analyzed by a highperformance liquid chromatography (HPLC, Waters) using an ultraviolet detector (210 nm) and a refractive index detector with a 300 mm × 7.8 mm Aminex HPX-87H ion exclusion column and 0.005 M H2 SO4 as the eluent. Total suspended solids (TSS), volatile suspended solids (VSS) and chemical oxygen demand (COD) were measured according to standard methods [19]. 2.5 Assays The hydrogen production curve was fitted to a modified Gompertz equation (Eq. 1), which provides a suitable model for describing the hydrogen production rates (HPR) [20]     RH × (λ − t) × e + 1 H = P × exp − exp P

(1)

where H = the cumulative hydrogen production (L H2 ), P = the ultimate hydrogen production (L H2 ), R H = the HPR (L H2 /h), l = the lag-phage time (h), t = time (h) and e = the exponential

3 Results and Discussion

2.3 Hydrogen Production Experiment

3.1 Hydrogen Production Performance of Suspended and Immobilized Cells

Hydrogen fermentation was conducted in batch vials using a 150 mL working volume and 2.25 g (15 g/L) of galactose added as substrate. Nutrients were supplied by adding a nutrient solution composed of the following based on a modified version of a previously reported formula (mg/L): K2 HPO4

The cumulative hydrogen production curves obtained from galactose using suspended and immobilized biomass are shown in Fig. 1a, b. The kinetic parameters obtained by fitting the modified Gompertz equation (Eq. 1) are provided in Table 1 with correlation coefficient R 2 values over 0.9.

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(a)

60

600

40 400 30

Biogas H2

200

20

H2 concentration (%)

Production (mL)

50

50 40 30 20

CO2 concentration (%)

60

10

H2 concentration 10

CO2 concentration

0

0

0 0

50

100

150

200

250

300

Fermentation time (h) 100

(b)

60

500

80

400

40

300

30

200

20

Biogas H2

60

40

20

H2 concentration

100

H2 concentration (%)

Production (mL)

50

CO2 concentration (%)

600

10

CO2 concentration 0

0 0

50

100

150

200

250

0

300

Fermentation time (h)

Fig. 1 Hydrogen production by suspended (a) and immobilized cells (b)

The results showed that the cumulative hydrogen production increased significantly in the immobilized cell systems relative to suspended cells. The HPR and hydrogen yield (HY) of 0.62 L H2 /L-d and 0.88 mol/mol galactoseadded were attained from the suspended cell cultures, while 0.76 L H2 /L-d and 1.20 mol/mol galactoseadded were obtained from the immobilized cell cultures, respectively, representing a nearly 30 % improvement in response to immobilized cultures. Kinetic parameters such as hydrogen production potential (P), hydrogen production rate (Rm) and lag phase (λ) are important to hydrogen production [21–23]. The modified Gompertz equation fit the experimental values well (Table 1). The results revealed that the lag phase time for the Table 1 Hydrogen yield, production rate, and lag period estimated by the modified Gompertz equation (Eq. 1) for hydrogen fermentation of galactose with suspended and immobilized biomass

immobilized cultures (8.4 h) is much larger than those of the suspended cell cultures (1.3 h). This could be because of the initial mass transfer problem associated with the immobilized cells, as well as adaptation to the new environment by the bacteria as explained in some other studies [10,11]. P, Rm and λ values of 254 mL, 5.1 mL/h and 1.3 h were reported for suspended cells, while values of 313 mL, 6.3 mL/h and 8.4 h were calculated for the immobilized cell cultures, respectively. The average hydrogen content also varied significantly and was quite stable in the case of immobilized cells (Fig. 1). The enhancement of hydrogen production activity of the immobilized cells over suspended cells was likely due to variations in the final pH and the distribution of the soluble metabolic products. 3.2 Soluble Metabolites Distribution and B/ A Ratio The production of VFAs or solvents during the anaerobic fermentation process is often considered a crucial signal in monitoring the efficiency of hydrogen producing cultures and determining the metabolic pathway [24–27]. The VFA production observed in the present study is presented in Table 2. Butyric and acetic acids were the main volatile fatty acids produced during fermentation, with a total organic acid production of 14.7 and 13.5 g COD/L being observed for the suspended and immobilized cell systems, respectively. As shown in Table 2, a high amount of propionic acid (3424 mg COD/L) and lactic acid (1230 mg COD/L) was observed in suspended cell systems, which resulted in lower hydrogen production performance than for immobilized cells. In general, these metabolites (propionic acid and lactic acid) are usually considered undesired products during hydrogen fermentation [28–30]. The formation of propionic acid is an undesired reaction during hydrogen fermentation because it consumes the produced hydrogen. Moreover, propionic acid has been reported to inhibit production performance. Production of n-butyric acid was dominant in all experimental sets, with more than 40 % being generated in both groups. The ratio of butyrate to acetate (B/A) is a unique and important key indicator in the process of hydrogen fermentation, as these soluble metabolites are primarily produced

Parameters

Units

Suspended biomass

Immobilized biomass

Biogas

mL

530.0±8.7

636.7±20.1

Hydrogen

mL

254.9±5.8

313.5±11.3

RH

mL/h

5.1±0.2

6.3±0.2

P

mL

257.9±6.1

313.8±11.5

Lag phase (λ)

h

1.3±0.4

8.4±0.5

HPR

mL/L-d

614.4±27.7

756.7±25.2

HY

mol/mol galactoseadded

0.88±0.03

1.08±0.04

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Arab J Sci Eng Table 2 Volatile fatty acids production of the suspended and immobilized biomass

13488±716

HAc

2824±125

2124±145

Hpr

3424±212

1768±95

i-Hbu

5670±324

6034±246

n-Hbu

1435±106

1670±124

HLa

1230±86

768±68

HFo

88±10

124±38

B/A ratio (mol basis)

1.0±0.5

1.5±0.6

during the acetogenesis process, which is an intermediate step in the anaerobic fermentation process. A higher butyrate concentration has been reported to correspond to a higher HPR or yield in other studies [31,32]. In the present study, the difference in B/A ratio primarily influenced hydrogen production performance in both groups. As explained previously [33–35], the increased butyrate formation significantly affected the HY. In the present study, the B/A ratio was positively influenced the hydrogen production. The higher production performances were achieved with immobilized cells (B/A = 1.5) than suspended cells (B/A = 1.0). This could be explained by the amount of the butyrate and acetate produced. Specifically, if the production of butyrate is greater than the acetate production, the reaction is thermodynamically favorable [35]. Thus, the results revealed that the B/A ratio is directly related to the production performance. 3.3 Effluent Characterization Table 3 shows the effluent characterization of different parameters analyzed once the fermentation was over. VSS, which represent the biomass, differed greatly between suspended (3.4 g/L) and immobilized cells (1.5 g/L). This was mainly because the biomass is freely available in the suspended cell systems, while it is packed inside the beads in the immobilized cell system. Moreover, the beads were reused for five cycles in the immobilized cell system, resulting in loss of bioTable 3 Effluent water quality analysis Suspended biomass

Immobilized biomass

T- COD (g/L)

2.3±0.6

2.0±0.4

S-COD (g/L)

1.0±0.5

0.9±0.3

VSS (g/L)

3.4±0.7

1.5±0.3

TSS (g/L)

6.5±0.8

2.4±0.4

pH

4.8±0.3

5.2±0.4

ORP (−mV)

318±24

376±38

Sugar removal (%)

86±7

90±5

123

1.5

400 HPR HY 200

1.0

HY(mol/mol added)

14671±863

600

0.5

0

0.0 0

1

2

3

4

5

6

7

8

Repeated batch

Fig. 2 Hydrogen production rate (HPR) and yield (HY) in repeated batch operation with immobilized cells

mass. Similarly, pH, ORP, TSS, and total and soluble COD also varied greatly between systems (Table 3). Overall, sugar removal efficiencies of 86 and 90 % were observed in the suspended and immobilized cell systems, respectively. 3.4 Stability and Efficiency of the Immobilized Cells The stability in terms of reusability was evaluated in repeated batch experiments with the immobilized cells (Fig. 2). The results showed that the immobilized cells efficiently produced a similar amount of HPR (0.76 L/L-d) and HY (1.2 mol/mol galactoseadded ) with a range of