Bioresource Technology 124 (2012) 500–503

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Bioresource Technology

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Short Communication

Volatile fatty acids production from marine macroalgae by anaerobic fermentation

Thi Nhan Pham, Woo Joong Nam, Young Joong Jeon, Hyon Hee Yoon ⇑Department of Chemical and Bio-Engineering, Gachon University, Seongnam, Gyeonggi-do 461-701, South Korea

h i g h l i g h t s

" Volatile fatty acids (VFAs) were produced from macroalgae by anaerobic fermentation with a mixed culture." The highest VFAs product concentration of 15.2 g/L was obtained from 50.0 g/L of alkaline pretreated Laminaria japonica in three days of anaerobic

fermentation." The VFA productivity varied with different types of macroalgae, probably depending on the type of polysaccharides in the macroalgae." VFAs productivity was enhanced up to 56% by the alkaline pretreatment.

a r t i c l e i n f o

Article history:Received 3 February 2012Received in revised form 13 July 2012Accepted 21 August 2012Available online 31 August 2012

Keywords:MacroalgaeVolatile fatty acidsAnaerobic fermentationLaminaria japonicaPachymeniopsis elliptica

0960-8524/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.biortech.2012.08.081

⇑ Corresponding author. Tel.: +82 31 750 5356; faxE-mail addresses: hhyoon@kyungwon.ac.kr, hhyoo

a b s t r a c t

Volatile fatty acids (VFAs) were produced from the marine macroalgae, Laminaria japonica, Pachymeniop-sis elliptica, and Enteromorpha crinite by anaerobic fermentation using a microbial community derivedfrom a municipal wastewater treatment plant. Methanogen inhibitor (iodoform), pH control, substrateconcentration, and alkaline and thermal pretreatments affected VFA productivity. Acetic, propionic,and butyric acids were the main products. A maximum VFA concentration of 15.2 g/L was obtained from50 g/L of L. japonica in three days at 35 �C and pH 6.5–7.0. Pretreatment with 0.5 N NaOH improved VFAproductivity by 56% compared to control. The result shows the applicability of marine macroalgae as bio-mass feedstock for the production of VFAs which can be converted to mixed alcohol fuels.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Marine alga biomass is a potential feedstock for the productionof biofuels and chemicals (Brennan and Owende, 2010; Nigam andSingh, 2010; Roesijadi et al., 2010). Macroalgae have been utilizedfor biological conversions such as anaerobic digestion (Hanssenet al., 1987; Qin et al., 2005; Vergara-Fernandez et al., 2008) andethanol fermentation (Adams et al., 2009; Borines et al., 2011).Thermo-chemical conversions of macroalgae have also been inves-tigated (Ross et al., 2008; Yoon et al., 2011).

In the present study, volatile fatty acids (VFAs) were producedfrom marine macroalgae. Acetic, propionic, and butyric acids areintermediates of the acidogenic and acetogenic stages of anaerobicdigestion. Compared with biomethane production, a higher

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: +82 31 750 5364.n@gachon.ac.kr (H.H. Yoon).

productivity can be expected under conditions conducive to VFAsproduction since this process occurs within two to three days ascompared to 15–20 days for methane production. The VFAs canbe converted to a mixed alcohol fuel (e.g., isopropanol, 2-butanol,and 3-pentanol) by hydrogenation with a catalyst (Holtzappleand Granda, 2009). The VFAs fermentation process uses a mixedculture and does not require a sterilization process. VFA productionfrom municipal solid wastes (Chan and Holtzapple, 2003), agricul-tural residue (Fu and Holtzapple, 2010), and food waste (Lim et al.,2008) has already been carried out; however, VFAs productionfrom marine algae for the mixed alcohol fuels production has notbeen reported.

Laminaria japonica, Pachymeniopsis elliptica, and Enteromorphacrinita are brown, red, and green algae, respectively. They growwidely throughout Asia and were used as model marine biomassin this study. The basic factors surrounding the VFAs fermentationprocess, such as concentration of methanogen inhibitor, iodoform,pH control, and substrate concentration were investigated. To en-hance the VFAs productivity, physicochemical pretreatment wasalso applied and evaluated.

T.N. Pham et al. / Bioresource Technology 124 (2012) 500–503 501

2. Methods

2.1. Feedstock

Air-dried macroalgae samples (L. japonica, P. elliptica, and E. cri-nita) were supplied by Wando Fisheries Cooperative, Wando, Kor-ea. The macroalgae were cultivated and harvested at the inshorearea of Wando and shredded to particles of around 1.0 mm in size.The samples were used as received.

(g/

L) 10

(a)

2.2. Inoculum preparation and VFAs fermentation

Anaerobically digested sludge was obtained from a municipalwastewater treatment plant in Seongnam, Korea. A continuousanaerobic fermentation was carried out in a 1-L bioreactor to pre-pare an adapted inoculum culture. Part of the fermentation brothwas removed daily and replaced with a fresh feed that containeddried macroalgae substrate and fermentation medium as used inVFA fermentation. The VFAs concentration in the fermention brothwas maintained at 3–5 g/L. The inoculums were taken from thecontinuous fermentor. The VFA fermentation medium contained(g/L of medium) NH4HCO3, 4.0; KH2PO4, 2.0; MgSO4�7H2O, 0.2;NaCl, 0.02; MoNa2O4, 0.021; CaCl2, 0.02; MnSO4, 0.026, and FeCl2

0.008. VFAs fermentation experiments were carried out in a 300-ml anaerobic flask with a 100-mL working volume. The flask wassealed using a rubber stopper. The VFA fermentation was carriedout in a shaking incubator at 35 �C and 150 rpm. The concentrationof macroalgae substrate (g of dried alga/L) and inoculum size was30.0 g/L and 10%, respectively, unless specified otherwise. Iodo-form (CHI3) (20 g/L ethanol) was used as methanogen inhibitor at30–70 ppm (v/v). CaCO3 powder (1.0 g/g of substrate) was addedto control the pH between 6.5 and 7.0. Liquid and gas samples weretaken daily. The liquid sample was centrifuged at 4300g for 10 minfor acid analysis. The flask was purged with nitrogen whenever theflask was open to the atmosphere.

Time (d)

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Laminaria japonicaPachymeniopsis ellipticaEnteromorpha crinita

g/L

)

5

2.3. Pretreatment and fermentation with pretreated macroalgae

For alkaline pretreatment, 5.0 g of macroalgae was soaked in30 ml of 0.5 N NaOH at room temperature for 24 h. After the pre-treatment, the mixture was neutralized with 0.5 N HCl. For thermalpretreatment, 5.0 g of macroalgae was mixed with 80 ml of de-ion-ized (DI) water and autoclaved at 120 �C for 20 min.

VFA fermentations were performed by the addition of nutrientmedium and sludge inoculum into the pretreated sample solutionas described above. DI water was also added to adjust the substrateconcentration. The initial substrate loading in the pretreatmentwas used as the substrate concentration in the VFAs fermentationwith the pretreated macroalgae sample.

L. japonica P. elliptica E. crinitaVF

A c

once

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tion

at

4 da

ys (

0

1

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4Acetic acidPropionic acidButyric acidValeric acid

(b)

Fig. 1. VFAs production from macroalgae; (a) Total VFAs production, (b) Compo-sition of VFA at four days of fermentation; Substrate concentration, 30 g/L. Eachdata point represents the average value of duplicate measurements.

2.4. Analysis

Liquid samples were analyzed for acetic, propionic, and butyricacids by a HPLC (Shimatzu) equipped with a UV detector. A Biorad-HPX-87H column was used at 45 �C with 0.005 M H2SO4 as mobilephase at a flow rate of 0.6 mL/min. The VFAs concentration wascalculated as sum of those three acids, since other acids, such asformic acid, lactic acid, valeric acid, caproic acid, and heptanoicacid amounted to less than 1% of the total VFAs. Gas samples wereanalyzed for methane by a gas chromatography with a thermalconductivity detector (GC-TCD; Agilent). A capillary column (HPPLOTQ) was used. Helium was used as carrier gas. The tempera-tures of the injector, column and detector were 150, 35, and260 �C, respectively.

The C, H, and N contents in the macroalgae were analyzed withan elemental analyzer (Elementar). Approximately 3 mg of samplewas burned at 1145 �C with a sulfanilamide standard. The volatilecontent was measured using thermo-gravimetry (TGA; TA Instru-ments). Approximately 5 mg of sample was heated to 550 �C at arate of 10 �C/min in air. Volatile content was measured by the massloss between 105 and 550 �C. Ash was determined by the residualmass. The protein content was determined by the Kjeldahl methodwith a Kjeldahl coefficient of 6.25 (Analytical Methods of KoreanFood Standards Codex, 2011a,b). The lipid content was measuredby the acid decomposition method with HCl for the decompositionand ether as an extractant (Analytical Methods of Korean FoodStandards Codex, 2011a,b). The total carbohydrate content was cal-culated by subtracting the lipid, protein, ash, and moisture con-tents from the volatile matter content.

3. Results and discussion

3.1. VFAs production from different macroalgae

Figure 1(a) shows that the VFAs concentration produced from30 g/L of L. japonica, P. elliptica, and E. crinita reached 6.3, 4.4, and3.6 g/L, respectively, in four days of anaerobic fermentation. Acetic,propionic, and butyric acids were major VFAs (Fig. 1(b)). Aceticacid exhibited the highest percentage; however, the proportionof each acid varied with the type of macroalgae. Although valericacid was also detected, its percentage in the total VFAs was lessthan 1%. A higher VFAs productivity was expected for the macroal-gae sample having a higher volatile matter content and/or carbo-hydrate content; however, no significant relationship betweenVFA productivity and content of volatile matter and/or carbohy-drate, protein, and lipid was observed (Fig. 1, Table 1). This result

Table 1Compositions of biomass sample (as-received).

L. japonica P. elliptica E. crinita

TGA analysis (wt.%)Moisture 4.4 9.6 4.3Volatile matter 59.9 66.9 60.0Ash 35.7 23.5 35.7

Elemental analysis (wt.%, dry)C 27.76 31.95 29.16H 4.16 4.49 4.15N 1.74 2.06 2.85

Compostion analysis (wt.%)Carbohydrate 10.0 53.8 41.2Protein 48.4 12.1 17.0Lipid 1.5 1.0 1.8

Time (d)

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Control30 ppm inhibitor50 ppm inhibitor70 ppm inhibitor

Fig. 2. Effect of methanogen inhibitor (iodoform) addition on VFAs fermentation;Substrate, 30 g/L Laminaria japonica. Each data point represents the average value ofduplicate measurements.

Time (d)0 1 2 3 4 5 6

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No control pHpH controlled at 6.5- 7.0

Fig. 3. Effect of pH control on VFAs fermentaion. Calcium carbonate (1.0 g/g ofsubstrate) was added as a neutralizing agent; Substrate, 30 g/L Laminaria japonica.Each data point represents the average value of duplicate measurements.

(a)

0 10 20 30 40 50 60Max

imum

VF

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5

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ControlNaOH 0.5 NThermal

Substrate concentration (g/L)

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5

10

15

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ControlThermal NaOH 0.5 N

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Fig. 4. Effect of pretreatments on VFAs product concentration; (a) Maximum VFAsconcentration obtained at different substrate loadings, (b) Time course of VFAsconcentration at 50 g/L of substrate concentration. The Laminaria japonica samplewas pretreated with 0.5 M NaOH at room temperature for 24 h, or thermallypretreated at 120 �C for 20 min. Each data point represents the average value ofduplicate measurements.

502 T.N. Pham et al. / Bioresource Technology 124 (2012) 500–503

suggests that the constituents of the polysaccharides in the macro-algae affected the VFAs fermentation rate. Like cellulosic biomass,some types of structural polysaccharides in macroalgae can be re-calcitrant to fermentation because of their resistance for hydrolysis(Roesijadi et al., 2010).

3.2. Effect of a methanogen inhibitor and pH control on the VFAsproduction

VFAs are intermediates in the anaerobic digestion of biomass tomethane. Hence, in order to produce a high amount of VFAs, meth-ane formation from VFAs was inhibited to maximize the VFAsproduct. Fig. 2 shows the effect of the methanogen inhibitor, iodo-form (CHI3). VFAs productivity increased with the addition of iodo-form. Without the methanogen inhibitor, methane started to formafter two days (Supplementary Fig. A1 in Electronic Annex). Nomethane was detected in the presence of the methanogen inhibi-tor. The lower VFAs productivity in the presence of 70 ppm of iodo-form suggests that higher concentration of this inhibitor adverselyaffected the activity of microorganisms for VFAs fermentation.

In the presence of calcium carbonate, a pH of 6.5–7.0 was main-tained, whereas in the absence of calcium carbonate, the final pHreached 5.4–6.2, depending on the VFAs concentration (Supple-mentary Fig. A2 in Electronic Annex). A higher VFAs concentrationwas obtained with pH control (Fig. 3).

3.3. Effect of pretreatment

Figure 4(a) shows the maximum VFA concentrations obtainedwith pretreated macroalgae at different substrate concentrations.

At low substrate concentration, the pretreatment effect was mini-mal. However, at high substrate concentration, the VFAs produc-tion was enhanced by the pretreatment. For instance, at 50 g/L ofL. japonica substrate concentration, the maximum VFAs productconcentration increased from 11.4 g/L to 15.2 g/L with an alkalinepretreatment and to 13.5 g/L with a heat treatment. Fig. 4(a) alsoshows that a higher VFAs product concentration was obtained byincreasing the substrate concentration to up to 50 g/L. The yield

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of VFAs (g VFAs/g substrate) was in the range of 30.7–41.2%,decreasing slightly as substrate concentration increased (Supple-mentary Fig. A3 in Electronic Annex). VFAs productivity (g VFAs/(L d)) increased significantly as the substrate concentration in-creased (Supplementary Fig. A3 in Electronic Annex). This resultindicated that substrate inhibition and/or product inhibition wasminor under the conditions used. However, at more than 50 g/Lof substrate in the fermentor, significant mixing problems occurreddue to gelation of the fermentation broth.

Pretreatment also improved the fermentation rate and thus VFAproductivity. As shown in Fig. 4(b), the maximum VFAs concentra-tion was observed at three days for the pretreated sample, while itwas observed after five days for the untreated sample (control).Although the thermal pretreatment effect was not as high as thatof the alkaline pretreatment, it has the advantage of being a simpleand clean process. Although the highest VFAs concentration wasobtained with alkaline pretreated L. japonica, similar pretreatmenteffects with P. elliptica and E. crinita samples were observed (Sup-plementary Figs. A4 and A5 in Electronic Annex).

It was reported that 32.4 g/L of carboxylic acid was obtainedwith the acid productivity of 1.69 g/(L d) from sugarcane bagasseand chicken manure (Fu and Holtzapple, 2010). A lower VFAs con-centration of 15.2 g/L was obtained in the present study; however,the VFA productivity of 5.1 g/(L d) was higher. Therefore, macroal-gae could be a feasible feedstock for VFAs production, although fur-ther studies, such as those involving a fed-batch process for ahigher product concentration should be carried out. In addition,the fermentation conditions and the types of macroalgae can resultin different VFAs compositions and thus can affect the quality ofthe final mixed alcohol fuels.

4. Conclusions

VFAs were produced from the macroalgae, L. japonica, P. ellipti-ca, and E. crinita by anaerobic fermentation with a mixed culture.The VFA productivity varied with different types of macroalgae.The inclusion of a methanogen inhibitor was a key factor in theVFAs fermentation process. The VFAs product concentrationincreased up to 56% by alkaline pretreatment, depending on thetype of macroalgae. The results show the applicability of marinemacroalgae as feedstock for the production of VFAs which can beconverted to mixed alcohol fuels.

Acknowledgements

This work was financially supported by the Korean Ministry forFood, Agriculture, Forestry and Fisheries (20111001212-00).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.biortech.2012.08.081.

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