ORIGINAL PAPER

Volatile fatty acids distribution during acidogenesis of algalresidues with pH control

Yan Li • Dongliang Hua • Jie Zhang •

Yuxiao Zhao • Haipeng Xu • Xiaohui Liang •

Xiaodong Zhang

Received: 24 December 2012 / Accepted: 23 January 2013 / Published online: 5 February 2013

� Springer Science+Business Media Dordrecht 2013

Abstract The anaerobic acidification of protein-rich algal

residues with pH control (4, 6, 8, 10) was studied in batch

reactors, which was operated at mesophilic(35 �C) condi-

tion. The distribution of major volatile fatty acids (VFAs)

during acidogenesis was emphasized in this paper. The

results showed that the acidification efficiency and VFAs

distribution in the acid reactor strongly depended on the

pH. The main product for all the runs involved acetic acid

except that the proportion of butyric acid acidified at pH 6

was relatively higher. The other organic acids remained at

lower levels. The VFAs yield reached the maximum value

with about 0.6 g VFAs/g volatile solid (VS) added as pH

was 8, and also the content of total ammonia nitrogen

(TAN) reached the highest values of 9,629 mg/l. Low

acidification degrees were obtained under the conditions at

pH 4 and 10, which was not suitable for the metabolism of

acidogens. Hydralic retention time (HRT) required for

different conditions varied. As a consequence, it was

indicated that pH was crucial to the acidification efficiency

and products distribution. The investigation of acidogene-

sis process, which was producing the major substrates,

short-chain fatty acids, would play the primary role in the

efficient operation of methanogenesis.

Keywords Acidogenesis � Algal residues � Mesophilic �Volatile fatty acid � Ammonia

Introduction

With increasing concerns about the energy depletion and

environmental pollution resulting from utilization of fossil

fuel, biodiesel fuel has received considerable attentions in

recent years. But there is a feedstock issue blocking the

development of biodiesel industry. Microalgae, growing

photosynthetically and accumulating lipid during metabo-

lism, will alleviate the tough situation caused by the deficiency

of raw materials (Razon et al. 2011; Demirbas et al. 2011).

With the development of microalgae-biodiesel, algal

residues after lipid extraction from wastewater-grown

algae are biologically converted for energy production. The

lipid extracted algal residues as major by-products still

contain protein and carbohydrate, which makes anaerobic

digestion an efficient way to recover energy in the form of

biogas (Chisti 2007; Sialve et al. 2009; Markou et al. 2011;

Huang et al. 2010).

Biogas as one of the most important gaseous fuels can

be utilized for power production to generate electricity

and heat or for transport and domestic fuel production

(CH4-enriched biogas), which may be an essential sup-

plement to the natural gas shortage (Murphy et al. 2004).

The technology for anaerobic digestion is well devel-

oped. The effects of fermentation conditions such as vari-

ous materials, parameters controlled, inoculum and

substrate concentrations on biogas production have been

studied (Forster-Carneiro et al. 2008; Kaparaju et al. 2009;

Chae et al. 2008).

There are some researches on the anaerobic digestion of

algal residues in single phase. Chisti discussed the recovery

Y. Li � D. Hua � J. Zhang � Y. Zhao � H. Xu � X. Liang �X. Zhang (&)

Key Laboratory for Biomass Gasification Technology of

Shandong Province, Energy Research Institute of Shandong

Academy of Sciences, Jinan 250014, China

e-mail: zhangxd@sderi.cn

Y. Li

e-mail: liyan8297@21cn.com

123

World J Microbiol Biotechnol (2013) 29:1067–1073

DOI 10.1007/s11274-013-1270-z

of energy from the microalgae residues after biodiesel

production, highlighting its potential to meet most of the

energy demands of the preceding process (Chisti 2008).

Using post-transesterified residues of chlorella mono-

cultures, Ehimen investigated the batch anaerobic digestion

of Chlorella residues subjected to two pretreatments, with

average CH4 yields of 222–267.5 ml/g total solids (Ehimen

et al. 2009).

As has been done in our previous study, the problems

such as acid accumulation and ammonia inhibition have

occurred as the ISRs B0.5. There was little methane pro-

duced through the process with high organic load. To

improve the process efficiency, a two-stage anaerobic

process was essential for such materials with high organic

load. In such a process, hydrolysis and acidogenesis are

carried out in the first reactor, the effluent of which is

subsequently further treated in the second reactor for

acetogenesis and methane production. Potential toxic

compounds such as ammonia for methanogens, which was

generated in the acidogenesis, could be removed to elimi-

nate the inhibition effect on methanogenesis (Komatsu

et al. 1991).

The enhancement of overall biogas production must be

based on an investigation of the optimum growth condi-

tions and behavior of acidogens in a two-phase process

since they play the primary role in producing major sub-

strates, short-chain fatty acids, for methanogens. It is

important to understand the product spectrum during

anaerobic acidogenesis. The final distribution of the VFAs

generated depends mainly on the nature of the substrate

and the operational parameters, especially for pH Breure

et al. 1984; Horiuchi et al. 2002; Zoetemeyer et al. 1982).

The shift of acid-producing microbial community was

mainly influenced by these factors. There have been several

literatures about acidogenesis on wastewaters from food

industries, pharmaceutical microbial biomass and sewage

sludge (Xu et al. 2011; Penaud et al. 1997; Ponsa et al.

2008). Little acidogenic performance from solid residue

of high content of protein was reported. In addition, there

is an issue that large amount of ammonia was simulta-

neously produced with such material as feedstock. The acid

influent into the methanogenic reactor may not be effi-

ciently converted to methane due to the ammonia toxity to

methanogen.

Therefore, the aim of this study was to investigate the

pH controlled anaerobic acidogenesis process of algal

residues generated by microalgae-biodiesel production.

Main VFAs distribution and acidification efficiency were

fully considered to determine the optimum operating

parameters. The ammonia data during the acidogenesis was

obtained, which was necessary to provide useful informa-

tion for the effective linkage between acidogenesis and

methanogenesis.

Materials and methods

Substrate and inoculum characteristics

The main parameters were listed in Table 1. The algal

biomass (chlorella sp., Tianjian Co., Binzhou, China) after

lipid extraction algal residue was used as substrate,the C/N

ratio of which was 5.3. Anaerobic digested sludge collected

from a wastewater treatment plant(Xiangchi Co., Binzhou,

China) was used as inoculum. The TS and VS contents of

sludge were 7.85 and 86.33 %-TS, respectively.

Acidogenesis

Acidogenesis of algal residue was performed in 1 L bottle.

The mixture of sludge and algal residues were added to get

a final volume of 0.8 L. The inoculum concentration was

set at 20 g VS/L.The outlet of the reactor gap was con-

nected to the airbag and the volume of gas was measured

using a syringe. Then the set-up was placed in water bath at

35 �C. The headspace was flushed with nitrogen. Tests

were run as triplicates to test statistical reliability. Acido-

genesis efficiency (AE) was calculated by the equation:

AE = VFA Output gð Þ=VS Input gð Þ

Keeping a constant inoculum to substrate ratio of 1:3,

pH of 4, 6, 8 and 10 was controlled through the

experiments. The pH was adjusted with 5 M NaOH or

5 M HCl continuously. The control experiment without pH

adjustment was used. Sampling was conducted at 6, 12, 24,

36, 48, 60, 72, 84, 96 and 108 h.

Analytical methods

The compositions of biogas were measured by biogas

analyzer (Geotech, England) pH values were measured

with a pH meter. TS and VS were determined according to

APHA Standard Methords (2005). The free ammonia

concentrations (i.e. unionized NH3) are a function of TAN.

The pH, dissociation constant and formulae for the calcu-

lation of free ammonia concentrations are available in the

Table 1 Characteristics of algal biomas residue

Parameters Values

Total solid (TS, %) 94.6

Volatile solid (VS, %-TS) 89.3

Total carbon (%) 43.04

Total nitrogen (%) 8.07

Protein (%) 50.4

Carbohydrate 22.5

Lipid (%) 3.1

1068 World J Microbiol Biotechnol (2013) 29:1067–1073

123

literature (Kayhanian 1999). The volatile fatty acids

(acetic, propionic, butyric, valeric, iso butyric and iso valeric

acids) concentrations were determined using an Agilent

7,890 series gas chromatograph (GC) system.

Pretreatments were conducted before VFAs measure-

ments. The samples were centrifuged and then acidified

with 3 % phosphoric acid to a pH less than 2, in order to

convert the fatty acids to their undissociated forms. Then

the samples were diluted with deionized water to assure the

VFAs concentration to be in the range of standard curve

and filtered through 0.22 lm pore-sized filters. The column

of HP-FFAP (50 m 9 320 lm 9 0.5 lm) was selected.

Flame ionization detector (FID) was used and adjusted to

300 �C as operating temperature. Nitrogen was used as

carrier gas with a constant flow rate of 30 mL/min and the

inlet temperature was kept at 250 �C. Oven temperature

was initially set to 60 �C and then increased to 100 �C with

10 �C/min ramping. After 2 min holding time at 100 �C,

the oven temperature was gradually increased to 250 �C at

the rate of 10 �C/min.

Results and discussion

Biogas and methane production

The substrate concentration (ISR = 1:3) has exceeded the

appropriate organic load for batch anaerobic digestion. With

the HRT increasing, the inhibitory products such as volatile

fatty acids and ammonia were accumulated to exert negative

effect on the methanogenesis and then significantly affect the

methane production. As shown in Table 2, at pH 4 and 10, no

methane or little was generated throughout the process

because the pH values were far out of the optimum ranges

suitable for microbial community. Which led to the inhibi-

tion of methanogenesis activity. In comparison, a little more

methane was produced at pH 6 and 8, because at the earlier

stage, the methanogen could convert small amount of VFAs

into methane before being totally toxified.

Distribution of VFAs at different pH

It can be seen from Fig. 1 at pH 4, the main product was

acetic acid with the maximum concentration of 2.1 g/l,

approximately 49 % of the total VFAs, while the propionic

acid concentration was the next of about 0.7 g/l (16.3 %).

The concentrations of other organic acids constantly

remained at low levels. In acidogenesis at pH 4, the con-

centration of acetic acid rose with the time and reached a

stable value after 48 h. The variations of concentrations for

propionic acid, butyric acid, valeric acid, isobutyric acid

and isovaleric acid were not apparent within 108 h. It was

observed that there wasn’t significant increase of the total

VFAs concentration after 48 h.

It is shown in Fig. 2 that acetic acid (8.9 g/l) and butyric

acid (5.8 g/l) are the main organic acids, accounting for 43.5

and 28.6 % of the total VFAs concentration respectively.

The butyric acid was produced in large quantity as climax

community and microbial type shift with the variation of pH

(Zhao et al. 2003). Each VFA concentration except valeric

acid leveled off in 60 h. The concentration of valeric acid

remained constant during the process. As pH was 6, small

amount of methane was generated, which might influence the

fatty acids distribution due to the VFAs consumption of

methanogens. Compared with the result of pH 4, the con-

centration of all fatty acids rose to a higher level.

As indicated in Fig. 3, acetic acid concentration reached

19.6 g/l,exhibiting a sharp rise compared with other ones.

This was mainly attributed to the dissolution of protein to

different extents under alkaline conditions, which was

beneficial for the protein degradation to fatty acids. It

shows that acetate accounts for 56.8 % of total VFAs. The

next important VFAs were propionic acid and butyric acid.

Valeric acid, isovaleric acid and isobutyric acid were found

at lower percentages. The maximum total VFAs concen-

tration and individual VFAs concentration were almost

stabilized after 84 h.

It can be observed from Fig. 4 that acetic acid concen-

tration was 10.3 g/l, occupying 67 % in total VFAs of

Table 2 The biogas and methane production profiles at different pH during acidogenesis

Control Biogas (ml) Methane (ml) pH4 Biogas (ml) Methane (ml) pH6 Biogas (ml) Methane (ml)

1d 1760 246 1d 210 0 1d 1775 117

2d 318 4.8 2d 135 0 2d 408 8.2

3d 182 0.36 3d 104 0 3d 295 1.5

4d 58 0 4d 56 0 4d 89 0

pH8 Biogas (ml) Methane (ml) pH10 Biogas (ml) Methane (ml)

1d 1925 258 1d 240 0.5

2d 436 10.8 2d 108 0

3d 240 1.0 3d 73 0

4d 55 0 4d 35 0

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14.8 g/l. However,according to the study by Liu et al. the

output of VFAs produced at pH 10 was higher than the

ones at other pH (Liu et al. 2009). The discrepancy was

caused by the presence of different microbial consortium,

which changed with the variation of substances and envi-

ronment. The retention period of at least 96 h for acido-

genesis at pH 10 is required to achieve a higher acid

concentration. The sequence of organic acids concentra-

tions was quite similar with the data obtained at pH 10, that

is, acetic acid[propionic acid[butyric acid[ isovaleric

acid [ valeric acid [ isobutyric acid.

As presented in Fig. 5, acetic acid and butyric acid as

main products surged at the first 36 h and then became

smooth in the following period, the concentrations of

which arrived at the values of 9.9 and 6.0 g/l, about 45.2

and 27.3 % of total VFAs separately. There was no sig-

nificant increase for total VFAs concentration after 60 h

with the maximum of 21.9 g/l.

Because the pH of control was in the range of 5.85–6.15

after 24 h, the VFAs distribution of control showed little

difference with the one acidified at pH 6, The pH change

rule under the control experiment is shown in Fig. 6. It can

be seen that pH dropped quickly in 24 h, which resulted

from the generation of VFAs. With the production of

ammonium brought by the protein degradation, the pH rose

gradually. The presence of ammonium and VFAs enhanced

the buffering capacity and made pH maintain at a stable

level.

Total ammoniacal nitrogen

The content of protein contained in algal residues exceeded

50 %. Large amounts of ammonium were produced during

acidogenesis, which would affect the following methano-

genesis due to the toxity of free ammonia. Therefore, the

final TAN concentrations at different conditions should be

0 20 40 60 80 100 1200

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

Con

cent

ratio

n (m

g/l)

Time (h)

acetic acid propionic acid butyric acid valeric acid iso butyric acid iso valeric acid

0 20 40 60 80 100 1202000

2500

3000

3500

4000

4500

Con

cent

ratio

n (m

g/l)

Time (h)

Fig. 1 VFAs distribution and total VFAs concentration at pH 4

0 20 40 60 80 100 1200

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

acetic acid propionic acid butyric acid valeric acid iso butyric acid iso valeric acidC

once

ntra

tion

(mg/

l)

Time (h)

0 20 40 60 80 100 120

4000

6000

8000

10000

12000

14000

16000

18000

20000

22000

Con

cent

ratio

n (m

g/l)

Time (h)

Fig. 2 VFAs distribution and total VFAs concentration at pH 6

1070 World J Microbiol Biotechnol (2013) 29:1067–1073

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0 20 40 60 80 100 1200

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

22000C

once

ntra

tion

(mg/

l)

Time (h)

acetic acid propionic acid butyric acid valeric acid iso butyric acid iso valeric acid

0 20 40 60 80 100 1205000

10000

15000

20000

25000

30000

35000

40000

Con

cent

ratio

n (m

g/l)

Time (h)

Fig. 3 VFAs distribution and total VFAs concentration at pH 8

0 20 40 60 80 100 1200

2000

4000

6000

8000

10000

12000

acetic acid propionic acid butyric acid valeric acid iso butyric acid iso valeric acidC

once

ntra

tion

(mg/

l)

Time (h)

0 20 40 60 80 100 120

4000

6000

8000

10000

12000

14000

16000

Con

cent

ratio

n (m

g/l)

Time (h)

Fig. 4 VFAs distribution and total VFAs concentration at pH 10

0 20 40 60 80 100 120

0

2000

4000

6000

8000

10000

acetic acid propionic acid butyric acid

valeric acid iso butyric acid iso valeric acid

Con

cent

ratio

n (m

g/l)

Time (h)

0 20 40 60 80 100 1202000

4000

6000

8000

10000

12000

14000

16000

18000

20000

22000

24000

Con

cent

ratio

n (m

g/l)

Time (h)

Fig. 5 VFAs distribution and total VFAs concentration of control

World J Microbiol Biotechnol (2013) 29:1067–1073 1071

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elucidated. Variations in TAN concentrations for the test

and control are depicted in Table 3. Comparing the TAN

release at different pH, it was found that TAN concentra-

tion in the effluent at pH 8 was the highest of 9629 mg/l,

corresponding to the maximum VFAs level, while the

lowest value also reached 2054 mg/l. So the effluent of

acidogenesis should be pretreated prior to the methano-

genesis in order to reduce the disturbances on the perfor-

mance of the reactor and a sharp decrease of growth rates

and specific activities of methanogen. The data obtained

should be attached importance to and the ammonium

removal is closely associated with the subsequent operation

of methanogenesis.

It was concluded that the minimum of VFAs concen-

tration was obtained at pH 4, the next was at pH 10, the

third was at pH 6 and the maximum was at pH 8. As the pH

was controlled at 4 and 10, the ability of acidogens to

utilize the substrate decreased, which significantly influ-

enced VFAs production. pH values of six and eight were

relatively beneficial to the acidogenesis, especially the pH

of 8. It could be explained that the protein generally dis-

solved in alkaline condition, which was much easier to be

degraded and greatly contributed to the acidification pro-

cess. Acetic acid is considered to be the major precursor of

methane, from which 70 % of methane was derived

(Chynoweth et al. 2000). The dominant product of acidi-

fication at pH of 4,8 and 10 was acetic acid of above 50 %,

while acetic acid and butyric acid at pH 6 accounted for

43.5 and 28.6 %, respectively.

From the above-mentioned, the highest VFAs yield was

calculated to be 0.6 g VFAs/g VS-added according to the

equation described in experiment as pH was 8. It is likely

that the enzymatic activity of hydrolytic bacteria was

higher than those at other pH values, and also the protein,

as the main component of algal biomass, could readily

dissolve in the alkaline solution, which made it more

available for the acidogens (Li et al. 2010). The main

product at all the pH was acetic acid, the result of which

was similar to the one reported by Chen et al. (2011), who

studied the effect of different pH on the VFAs distribution

during acidogenesis of aquatic biomass. Whether the dis-

tribution of VFAs from acidogenesis of algal residues is

beneficial to the methane conversion or not is still under

investigation.

Conclusion

The strong pH dependency of VFAs production during aci-

dogenesis was investigated in this paper. It was found that at

the pH of 6 and 8, the acidogenesis could performed better

than the one at the extreme pH of 4 and 10 for acidogens. The

VFAs distribution and concentration differed from each

other, although, acetic acid was the dominant products in all

the runs. The practical methane production from the already

obtained results in acidogenic reactor should be verified in

the subsequent methanogenesis.

However, the ammonium was largely generated simul-

taneously with the acidification. Methanogens are more

vulnerable to the ammonia toxicity. If the end products

from acidogenesis directly flowed into the following

methanogenesis, the activity of methanogenic bacteria

might be inhibited. Thus, controlling the ammonium con-

centration could be critical in feeding the acidified effluent

to the methanogenic reactor in the two-stage process. The

effective link between acidogenesis and methanogenesis

should be further studied.

Acknowledgments This study was funded by Twelfth Five-Year

Plan of National Science and Technology (No. 2011BAD14B03),

State ‘‘86300 projects (No. 2012AA101803) and Natural Science

Foundation of Shandong province (ZR2012BL16).

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