ORIGINAL ARTICLE

One-stage and two-stage anaerobic digestionof lipid-extracted algae

Yan Li & Mintian Gao & Dongliang Hua & Jie Zhang &

Yuxiao Zhao & Hui Mu & Haipeng Xu & Xiaohui Liang &

Fuqiang Jin & Xiaodong Zhang

Received: 3 July 2014 /Accepted: 14 September 2014# Springer-Verlag Berlin Heidelberg and the University of Milan 2014

Abstract Waste-grown microalgae are a potentially impor-tant biomass for wastewater treatment. The lipid accumulatedin microalgae could be utilized as feedstocks for biodieselproduction. The algal residues, as major by-products derivedfrom lipid extraction, mainly consist of carbohydrate andprotein, making anaerobic digestion an efficient way to recov-er energy. The conversion of lipid-extracted algal residues intomethane plays dual role in renewable energy production andsustainable development of microalgal biodiesel industry.Therefore, an anaerobic fermentation process for investigationof the methane production potential of algal residues wasconducted in this paper. The effect of inoculum to substrateratios (ISRs) on the methane production by anaerobic diges-tion of Chlorella sp. residue in a single stage was evaluated.The maximummethane yield of 195.6 ml CH4/g volatile solid(VS) was obtained at an ISR of 1:1. The stability and progressof the reaction from algal residues to methane were monitoredby measuring the pH, volatile fatty acids (VFAs), total ammo-niacal nitrogen (TAN), and methane volume. Based on theresults of one-stage experiments, two-stage technology wasproposed and was found to be more suitable for high organicload. The optimum conditions for acidogenesis andmethanogenesis are indicated in this paper.

Keywords Biogas .Methane . Algal residues . One stage .

Two stage

Introduction

The potential of microalgae as a source of biofuels is subject toincreasing academic and industrial research (Brennan andOwende 2010; Ahmad et al. 2011). Therefore, the cultivationof microalgae and subsequent product refinery such as lipidextraction have gained much more attention by many re-searchers (Bellou andAggelis 2012). As a result of microalgaetechnology development, lipid-extracted algal residues(LARs) are the residual biomass from biodiesel productionprocesses that are rich in carbohydrate and protein. LARscould be used as a high-protein animal feed, a source of smallamounts of other high-value microalgal products (Chisti2007), and possibly to produce bioactive compounds andhydrogen (Singh et al. 2005; Yang et al. 2010). The compo-sitions of LARs make anaerobic digestion an efficient way torecover energy, which will also reduce the cost of microalgal-biodiesel production (Sialve et al. 2009).

The technology for anaerobic digestion is well developed.The effects of fermentation conditions such as feedstocks,controlled parameters, inoculum and substrate concentrationson biogas production have been studied (Chae et al. 2008;Forster-Carneiro et al. 2008; Kaparaju et al. 2009). The inves-tigation of algal biomass as feedstock for biogas productionhas also been reported (Marcin et al. 2013). Considering thesubstrates relevant to this paper, there is some research onalgal species such as Spirulina maxima (Samson and Leduy1982), Chlorella vulgaris (Sánchez Hernández and Córdoba1993), Scenedesmus obliquus and Phaeodactylumtricornutum (Zamalloa et al. 2012). In those investigationsfor methane production, intact algae were used; as a result, theresistance of the cell wall limited the hydrolysis step and thefermentation period was prolonged. In addition, the C/N ratioof algal biomass was lower than the empirical value; thus, co-digestion was commonly adopted for nutrient adjustment toenhance the biogas productivity (Zhong et al. 2012). The

Y. Li (*) :M. Gao :D. Hua : J. Zhang :Y. Zhao :H. Mu :H. Xu :X. Liang : F. Jin :X. Zhang (*)Key Laboratory for Biomass Gasification Technology of ShandongProvince, Jinan 250014, Chinae-mail: liy@sderi.cne-mail: xd_zhang77@yahoo.com.cn

Y. Li :M. Gao :D. Hua : J. Zhang :Y. Zhao :H. Mu :H. Xu :X. Liang : F. Jin :X. ZhangEnergy Research Institute of Shandong Academy of Sciences,Jinan 250014, China

Ann MicrobiolDOI 10.1007/s13213-014-0985-x

number of studies assessing the biochemical methane poten-tial of lipid-extracted microalgae is limited (Chisti 2008;Ehimen et al. 2009; Ehimen et al. 2011; Alzate et al. 2014;Zhao et al. 2014).

To find out the best conditions for the digestion of LARsand to obtain dependable data, the methane potential of thedigestion process was investigated in this article. The experi-ments were performed in one-stage and two-stage set-ups. Theeffect of inoculum to substrate ratios on methane productionin a one-stage reactor was studied in an appropriate loadrange. The operation of a two-stage digestion system wascarried out in terms of a high organic load at which failurewould occur during the process of one-stage digestion.

Materials and methods

Substrate and inoculum characteristics

Dried and disrupted Chlorella vulgaris (Tianjian Co.,Binzhou, China) after lipid extraction was used as substrate.The main parameters were listed in Table 1. The C/N ratio oflipid-extractedChlorella vulgariswas 5.3. Anaerobic digestedsludge collected from a digester operating at 35 °C inXiangchi Corporation was used as inoculum. The total solid(TS) and volatile solid (VS) contents of sludge were 7.85 and86.33 %, respectively.

One-stage experiments

Batch anaerobic digestion of algal residue was performed in1 l bottles. The mixture of sludge and algal residues wereadded to reach final volume of 0.8 l with fresh water supple-ment. The outlet of the reactor gap was connected to theairbag. Then, the set-up was placed in water bath at 35 °Cand shaken at intervals. Headspace was purged with nitrogenevery time the cap was opened. Tests were run as duplicates totest statistical reliability. Inoculum without substrate was usedas control.

The three different ISRs in the test were 2:1, 1:1 and 1:2.They were achieved by keeping a constant inoculum concen-tration (20 g VS/l, an empirical value for seed concentration)and varying the substrate concentrations according to theISRs. For the investigated ISRs, the TS concentrations didn’texceed the maximum empirical value of 10 %. Anaerobicdigestion of algal residues was operated for 15 days.

Two-stage experiments

The preliminary data obtained from one-stage anaerobic di-gestion indicated that there was hardly methane produced atISR of 1:3. Thus, two-stage technology was applied to inves-tigate whether the methane production could be improved or

not. The acidogenesis protocol was identical to that describedfor one-stage digestion, but the ISR was adjusted to 1:3 andthe pH was controlled at 8. Acidogenesis efficiency (AE) wascalculated by the equation:

AE ¼ VFA Output gð Þ=VS Input gð Þ

An Up-flow Anaerobic Sludge Blanket (UASB), made oforganic glass, was used as methanogenic reactor with a work-ing volume of 2.5 l. During the start-up, UASB reactors wereinoculated with granular sludge and acclimated with glucosesolution (1.5 g/l) at an organic loading rate (OLR) of 1.0 gCOD/l·d for 1 month.

Due to the large demand of acidic liquid with long-termoperation of UASB, the component of influent that flew intoUASB simulated genuine volatile fatty acids (VFAs) compo-sitions and content. The UASB reactors were continuously fedwith synthetic medium at an initial OLR of 2.0 g COD/l·d for10 days, and then increased to 3, 4, 6, 8, 10, and 12 g COD /l·dat 10 days intervals. To achieve different OLRs, the hydraulicretention time (HRT) varied with the control of influent vol-ume. The chemical composition of the synthetic medium wasas follows (g/l): acetic acid 0.9, propionic acid 0.225, butyricacid 0.178, valeric acid 0.0225, iso-butyric acid 0.102, iso-valeric acid 0.068. Sodium nitrate and potassium dihydrogenphosphate were supplemented to maintain the COD:N:P at thelevel of 100:4:1. Experiments were run for a total of 60 daysunder controlled room temperature (35 °C).

Analytical methods

The composition of biogas was measured using a double-channel infrared technique with a biogas analyzer (GA2000,Geotech), which showed the desired methane content. pHvalues were measured with a pH meter. Total ammoniacalnitrogen (TAN, NH3 and NH4

+) was analyzed according toNessler’s colorimetry with 5B-2(N) ammoniacal nitrogenanalysis meter (Lianhua Technology Company, China).COD was measured by a COD analyzer (Lianhua TechnologyCompany, China). The free ammonia concentrations (i.e.,unionized NH3) are a function of the total ammoniacal

Table 1 Characteristicsof lipid-extracted algalresidue

Parameter Value

Total solid (TS, %) 94.6

Volatile solid (VS, %-TS) 89.3

Total carbon (%) 43.04

Total nitrogen (%) 8.07

Protein (%) 50.4

Lipid (%) 3.1

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nitrogen concentration, pH, and dissociation constant, andformulae for the calculation of free ammonia concentrationsare available in the literature (Kayhanian 1999). TS and VSwere determined according to APHA (2005).

The VFA (acetic, propionic, butyric, valeric, iso-butyricand iso-valeric acids) concentrations were determined usingan Agilent 7890 series gas chromatograph (GC) system. Thecolumn of HP-FFAP (50 m×320 μm×0.5 μm) was selected.Flame ionization detector (FID) was adjusted to 300 °C as theoperating temperature. Nitrogen was used as the carrier gas,with a constant flow rate of 30 ml/min, and the inlet temper-ature was kept at 250 °C. Oven temperature was initially set to60 °C and then increased to 100 °C with 10 °C/min ramping.After a 2 min holding time at 100 °C, the oven temperaturewas gradually increased to 250 °C at the rate of 10 °C/min,with 2 min holdings.

Results and discussion

One-stage anaerobic digestion

Biogas and methane production

Figure 1 shows the cumulative methane production, as afunction of time with different ISRs used, corrected takingthe control production into account. As indicated in Fig. 1,methane production started immediately on the first day ofdigestion. Methane production almost reached a maximum onday 7 and leveled off thereafter at all ISRs, because thesubstrate was almost completely consumed by the bacteriaconsortium. The methane production was proportional to theVS load applied. As can be seen from Fig. 1, the cumulativemethane volumes after 15 days of digestion for the ISRs of1:2, 1:1 and 2:1 were 850, 3,066 and 1,565 ml, respectively.

The methane yield was calculated for each ISR by dividingthe final methane volume by the VS weight of substrateadded. Table 2 indicates that the methane yield after 15 daysof digestion reached high levels of 195.6 ml CH4/g VS and191.6 ml CH4/g VS as the ISRs were 2:1 and 1:1 (initialsubstrate VS loads of 8 g VS and 5.3 g VS), respectively,but decreased when the substrate load (32 g VS for ISR of 1:2)was further increased. The result gave a methane yield higherthan that found in batch experiments with Microcystis spp.carried out by Zeng, who obtained a yield of 132.44 ml CH4/gVS at an ISR of 1:1 (Zeng et al. 2010). However, compared tothe methane yields of 300 ml/g VS (Alzate et al. 2014) and380 ml/g VS (Zhao et al. 2014), it was lower mainly becauseof different composition of algae and digestion conditions. Ascan be seen in Table 2, the methane yield decreased from195.6 ml/g VS to 26.6 ml/g VS with the ISR varying from 2:1to 1:2. The same conclusion was previously achieved by otherresearchers using different substrates (Raposo et al. 2006;Medina and Neis 2007).With an ISR of less than 1:1, methaneyields significantly decreased and reached the lowest level of26.6 ml CH4/g VS at an ISR of 1:2, with yields approximatelyone-tenth of those obtained at other ISRs (Table 2). Combinedwith Fig. 2, though, the pH value for the whole process at anISR of 1:2 was in an appropriate range, which was the contri-bution of the coexistence of VFA and ammonium. The yielddecrease was perhaps due to the weak methanogenic activityin the digesters, resulting from the inhibition caused by am-monia (Sawayama et al. 2004).

It was observed that the operating time of 15 days was alittle long for the digestion. The methane production wasalmost finished in 10 days. The practical period for digestioncould be shortened from the perspective of methane produc-tion and energy conservation. Taking the methane yield andappropriate organic load into consideration, an ISR of 1:1 wasthe optimum for anaerobic digestion with algae alone, theorganic loading of which was 2 g VS/(l·d).

pH variation

The variation of pH over the period of digestion is shown inFig. 2 and ranges from 6.7 to 7.7. When measured at days perunit, the pH climbed and gradually reached a relatively steadystate with increasing time. It was obtained (not listed in Fig. 2)

0 2 4 6 8 10 12 14 16

500

1000

1500

2000

2500

3000

3500

Cum

ulat

ive

met

hane

vol

ume

(ml)

Time (d)

ISR=1:2 ISR=1:1 ISR=2:1

Fig. 1 The cumulative methane production for different ISRs

Table 2 Performances of reactors at ISRs of 1:2, 1:1 and 2:1

ISR TS loadedsubstrate (g/l)

VS loadedsubstrate (g/l)

Methane yield(ml CH4/g VS-add)

1:2 72.4 40 26.6

1:1 45.6 20 191.6

2:1 35.5 10 195.6

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that the pH decreased during the first few hours of day 1. Organicacid production was generally initiated from the onset of thereactor operations and resulted in these expected pH drops. Theshort-chain fatty acids as important intermediates were thenquickly converted to methane by methanogens. Ammoniumwas simultaneously produced and drove the pH upwards.

Poor performance of methane production was obtained atan ISR of 1:2. At this ISR, the pH stayed in the narrow rangebetween 6.7 and 7.2, which was compatible with the normalgrowth of anaerobic microorganisms. However, as can be seenfrom Fig. 3, VFAs were accumulated to a high level, but pHdidn’t drop accordingly, due to the simultaneous presence of ahigh level of ammonium (Fig. 4). The stability in pH can beattributed to the high buffering capacity of the reactor.

TAN production

Total produced ammoniums of different ISRs are indicated inFig. 4. An increment on TAN was observed at the experimen-tal ISRs. This fact was predictable, since it is widely knownthat some organic nitrogen (mainly protein) is converted toammonium during anaerobic digestion (Koster and Lettinga1988). The final ammoniacal nitrogen concentrations in-creased from 2,115 to 13,200mg/l as the ISR values decreasedfrom 2:1 to 1:2.

Ammonia, known as one of the inhibitors of methanogens,may be thought of as a problem in anaerobic digestion, espe-cially those digestions using protein-rich feedstocks as sub-strates (Nielsen and Angelidaki 2008). Ammonia could mainlyinfluence the anaerobic digestion by affecting acetate-utilizingmethanogenic archaea, hydrogen-utilizing methanogens andsyntrophic bacteria. Though the inhibitory concentrations ofammonia varied due to different experiments, a TAN of 1.7–5 g/l, corresponding to 0.4–1 g/l free ammonia, is the mostacceptable concentration inhibiting anaerobic digestion

(Hashimoto 1983). As the ISRs were 2:1 and 1:1, the observedTAN concentrations in this study appeared not to directly affectthe process. This suggested that the initial and final ammoniawere too little to inhibit anaerobic digestion.

When the ISRwas 1:2, an accumulation of TAN of 4,300mg/l at day 2 was far above the threshold concentration (4,000 mg/l)reported in literature (Hashimoto 1983); therefore, the relativelylowermethane yield at an ISRof 1:2was ascribed to the presenceof a large amount of NH4

+-N. Ammonia exerts negative effectson microbial cells and the concentration must be monitored inorder to prevent toxic amounts from being reached.Methanogens have been proven to be particularly sensitive toammonia (Sasaki et al. 2011; Procházka et al. 2012). In terms ofanaerobic digestion, a low C/N ratio can compromise the effi-ciency of the process, especially when high input and/or longretention times are applied.

For ISRs of 1:1 and 2:1, the maximum free ammonia wasabout 148 and 102 mg/l, respectively. The TAN concentrationreached 13,200 mg/l at the ISR of 1:2, corresponding to240 mg/l free ammonia. It was found from Fig. 3 that VFAswere accumulated to a high level at ISR of 1:2, which waspossibly due to the poor methanogenesis, inhibited by

0 2 4 6 8 10 12 14 166.0

6.2

6.4

6.6

6.8

7.0

7.2

7.4

7.6

7.8

8.0pH

Time (d)

ISR=1:2 ISR=1:1 ISR=2:1

Fig. 2 pH variation for different ISRs during anaerobic digestion

0 2 4 6 8 10 12 14 16

0

1000

2000

3000

4000

5000

6000

7000

8000

0 2 4 6 8 10 12 14 1610000

15000

20000

25000

30000

35000

40000 ISR=1:2 ISR=1:1 ISR=2:1

Vola

tile

fatty

aci

ds (V

FAs)

con

cent

ratio

n (m

g/l)

Time (d)Time (d)

Fig. 3 VFA variation for different ISRs

0 2 4 6 8 10 12 14 160

2000

4000

6000

8000

10000

12000

14000

Amm

onia

cal n

itrog

en c

once

ntra

tion

(mg/

l)

Time (d)

ISR=1:2 ISR=1:1 ISR=2:1

Fig. 4 NH4+-N variation for different ISRs during anaerobic digestion

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ammonia. Because the inhibitory effect of ammonia is asso-ciated with many factors such as pH, temperature and accli-mation of microorganisms, the free ammonia concentrationfor inhibition couldn’t be determined in this study.

VFA production

VFAs, as one of the most important parameters for the accu-rate control of anaerobic digestion, have a direct correlationwith digester performance. The variations in VFA concentra-tion during the course of the digestion at different ISRs areshown in Fig. 3. The initial values of VFAs increased with theamount of algae added. As indicated in Fig. 3, the VFAconcentrations showed a declining tendency at ISRs of 1:1and 2:1, and low levels of VFAswere detected after 10 days ofdigestion, which means that the conversion of VFAs intomethane was proceeding and gradually terminated. The de-crease of VFA concentrations also indicated that the produc-tion of VFAs was relatively slower than consumption bymethanogenesis, which corresponded to the high output ofmethane. For the ISR of 1:2, unstable operation occurred asindicated in Fig. 3. VFAs weren’t completely consumed, butaccumulated to a level as high as 36,240 mg/l with a smallamount of methane produced. Combined with the pH valueand ammoniacal nitrogen concentration, it could be explained

that methanogenic activity was significantly affected due tothe inhibition of free ammonia, despite the pH being relativelystable and appropriate for anaerobic digestion.

Two-stage anaerobic digestion

Bad performance of biogas production occurred during theabove-mentioned results in one-stage operation, and wasmainly due to system overload with an ISR of 1:2. Accumu-lation of high concentrations of VFAs and ammonia duringthe initial stage of anaerobic digestion subsequently affects themethanogenic stage in single-stage anaerobic reactors. Toovercome these problems, a two-phase anaerobic digestionsystem at ISR of 1:3 was adopted to permit different bacterialenrichment in two different reactors, by providing optimalgrowth conditions for initial acidogens and later methanogens(Cirne et al. 2007). The inhibition caused by the accumulationof intermediates could be eliminated through stage separation.

Acidogenesis

The strong pH dependency of VFA production duringacidogenesis was investigated in our previous research (Liet al. 2013). It was found that at a pH of 8, acidogenesis couldoccur better than at other pH values. The VFA yield was

a b

Fig. 5 VFA distribution and total VFAs concentration at pH 8

Table 3 Performance of UASBreactors under different OLRs

* 2 g/l of COD for influent wasadopted in all the experimentswith various OLRs

OLR (g/l·d) HRT (h) COD ofinfluent (g/l)

COD ofefluent(g/l)

COD removalefficiency (%)

Methane yield(ml CH4/g COD)

2 24 2 0.041 97.9 336

3 16 2 0.051 97.4 332

4 12 2 0.11 94.5 330

6 8 2 0.17 91.5 323

8 6 2 0.24 88.0 324

10 4.8 2 0.29 85.5 318

12 4 2 0.33 83.5 319

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calculated to be 0.6 g VFAs/g VS-added, according to theequation described in experiment.

As indicated in Fig. 5b, the total VFA concentrationreached 37,200 mg/l when the pH of the acidogenic reactorwas controlled at a value of 8. This was mainly attributed tothe dissolution of protein to different extents under alkalineconditions, which was beneficial for protein degradation tofatty acids. However, according to the study by Liu et al., theoutput of VFAs produced at pH 10 was higher than at otherpHs (Liu et al. 2009). The discrepancy was caused by thepresence of a different microbial consortium, which changedwith the variation of substances and environment. As shownin Fig. 5a, acetate accounts for 56.8 % of total VFAs. The nextimportant VFAs were propionic acid and butyric acid. Valericacid, iso-valeric acid and iso-butyric acid were found at lowerpercentages. The maximum total VFA concentration and in-dividual VFA concentration leveled off after 108 h.

Methanogenesis

Of the several intermediate steps in LARs based anaerobicdigestion, methane formation from VFAs is often the criticalpathway that limits the overall reaction rate, because thesubstrate could be readily degraded. The efficient conversionof VFAs was closely correlated with the enrichment anddiversity of methanogens; therefore, methanogenesis was arate-limiting step during the whole process. In this study, theauthors operated UASB reactors at different organic loadingrates using an artificial medium containing acetic acid,propionic acid, butyric acid, valeric acid, iso-butyric acidand iso-valeric acid.

A quite stable performance of UASB reactors under fluc-tuating organic loads was observed (Table 3). Parawira et al.(2006) reported that the UASB can provide a stable processconversion rate up to an OLR of 6.1 g COD/l·d. In the presentstudy, the OLR reached 12 g COD/l·d with specific VFAscompositions, which provided a high COD removal efficiency(>80 %) throughout the operational periods. Based on theinfluent COD of UASB, the methane yield for the test was319 ml CH4/g COD.

Conclusion

In this study, anaerobic digestion of LARs was carried outusing one-phase and two-phase systems. Themaximummeth-ane yield was obtained at an ISR of 1:1 in a one-stage reactor.The methane production decreased or even failed when theorganic load was 2:1 and 1:2. At a high organic load of ISR1:3, the two-stage system seems to be very effective for theimprovement of methane production. To upgrade yields in atwo-stage system, more investigations would be needed

concerning the system set-up, microbial consortium in boththe hydrolyser and the methanizer, etc., in order to optimizeconditions in both reactors. However, if the two-stage systemis operated in a straightforward manner, like the one-phasesystem, the latter would be the best choice.

Acknowledgments This study was funded by National High Technol-ogy Research and Development Program of China (863 Program) underGrant No. 2012AA101803, Key Projects in the National Science &Technology Pillar Program during the twelfth 5-year Plan Period(No.2014BAD02B04), and the Natural Science Foundation of Shandongprovince (ZR2012BL16, ZR2012CL04).

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