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Journal of EnvironmentalScience and Health . Part A:Environmental Science andEngineering and ToxicologyPublication details, including instructions forauthors and subscription information:http://www.tandfonline.com/loi/lesa19

Production andinhibitant‐affectedutilization of butyric acid inanaerobic digestion processChiu‐Yue Lin a , Shinn‐Jyh Wang b &

Rong‐Chung Chang a

a Department of Environmental Engineeringand Science , Feng Chia University , Taichung,Taiwanb Chung Tan Environmental Research Lab. Co.,Ltd , Taichung, TaiwanPublished online: 15 Dec 2008.

To cite this article: Chiu‐Yue Lin , Shinn‐Jyh Wang & Rong‐Chung Chang (1997)Production and inhibitant‐affected utilization of butyric acid in anaerobicdigestion process, Journal of Environmental Science and Health . Part A:Environmental Science and Engineering and Toxicology, 32:4, 1049-1063, DOI:10.1080/10934529709376595

To link to this article: http://dx.doi.org/10.1080/10934529709376595

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J . ENVIRON. SCI. HEALTH, A32(4), 1049-1063 (1997)

PRODUCTION AND INHIBITANT-AFFECTEDUTILIZATION OF BUTYRIC ACID IN ANAEROBIC

DIGESTION PROCESS

Key words : butyric acid, isomerization, anaerobic digestion, oleate,sulfate, ammonia, inhibition.

Chiu-Yue Lin* Shinn-Jyh Wang** Rong-Chung Chang*

*Department of Environmental Engineering and ScienceFeng Chia University, Taichung, Taiwan

**Chung Tan Environmental Research Lab. Co., Ltd, Taichung, Taiwan

Abstract

The fates of both forms of butyric acid (HBu) in anaerobic digesters were

discussed. HBu production was investigated by using glucose as the

substrate in continuous feeding digestion at 35°C at pH 5.7, 6.4 and 6.9

and at the SRTS (solid retention times) of 2.5, 2.0, 1.5, 1.0, 0.5 and 0.25

days. Production of n-HBu increased with increasing SRT. No

production of i-HBu was determined at SRT >2.0 days when the pH was

low. Inhibitant effect on the utilization of HBu were investigated by batch

test at 35°C. The CSTR seed sludge digesters were continuously fed

with n-HBu or i-HBu, and the SRT were 20,14,10,6,5 and 20,14,10,8,7,6

days, respectively. In the batch test, the acclimated sludge (SRT 10 days)

was used . Isomerization between n-HBu and i-HBu was evident from

1049

Copyright © 1997 by Marcel Dekker, Inc.

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1050 LIN, WANG, AND CHANG

the experimental results but the isomerization of i-HBu was readily

affected by oleate, sulfate or ammonia. The relative toxicities were

oleate » sulfate > ammonia for the utilization of both forms of HBu.

INTRODUCTION

Anaerobic wastewater treatment process has been shown to be readily

affected by the appearance of long-chain fatty acids (eg., oléate), sulfate

or ammonia (Hanaki et al.,1987 : Koster and Cramer, 1987 : Winfrey

and Zeikus,1977 ; Choi and Rim, 1991 : Noriega et al., 1991 ; Koster

and Lettinga, 1984 : Koster and Koomen, 1988 ; Kayhanian, 1994). It

has been reported that iso butyric acid (i-HBu) was determined in the

anaerobic digester under unnormal condition (Chang et al., 1982). In

1988, Hill and Holmberg reported that branched i-HBu was an idicator of

digester failure. However, isomerization between the normal and iso-

forms of HBu in anaerobic digestion was found (Gourdon et al., 1988; Lin,

1989; Aguilar et al., 1990; Lin and Hu, 1993) and its pathway was

elucidated (Tholozan et al.,1988 ; Lin and wang, 1992). The fate of

HBu in the anaerobic digester under the appearance of these agents

then seems to be needed to be elucidated. In our work, both forms of

HBu and glucose were respectively used as carbon sources in

mesophilic methanogenic and acidogenic experiments. The purpose of

this research was to investigate the effects of oléate, sulfate or ammonia

on the isomerization and production of HBu.

MATERIALS AND METHODS

Production

The seed sludge was obtained from a mesophilic sewage digester and

was acclimated with glucose (20000 mg COD / liter) in four CSTR

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BUTYRIC ACID IN ANAEROBIC DIGESTION PROCESS 1051

digesters (4 liters). Of the temperature, three digesters were controlled

at 35 ± 1 °C and the other without control. The substrate contained

sufficient inorganics (Lin and Hu, 1993). Substrate feeding was in a

continuous mode. The pH for the temperature controlled digesters were

5.7, 6.4 and 6.9 respectively and was uncontrolled for the digester

without temperature control. For each digester the solids retention times

(SRT) were 2.5, 2.0, 1.5, 1.0, 0.5 and 0.25 days.

Before steady state data was measured at each experimental

temperature, the reactors were operated for two to five times the

hydraulic retention times (HRT). Steady state conditions are defined as

the conditions during which product concentration variations are small

(approx. 10%). For each steady state data measurement, an average of

five to six repeated analyses were undertaken over two weeks during

steady state condition. The experiment was conducted at a pH of 7.2±

0.1 which is the optimum range for methane fermentation. The digesters

were monitored for pH, alkalinity, gas production and composition, VFA

distribution, and solids concentration. The gas volumes were corrected

to standard temperature (0 °C) and pressure (760 mmHg) (STP).

Utilization

Experiments were carried out in serum vials with working volume 100 ml

by a batch test and the seed sludges acclimated at SRT 10 days were

used. The seed sludge was obtained from a mesophilic methanogenic

digester and was acclimated in two CSTR digesters (2 liters) with n-HBu

and i-HBu respectively at 35±1°C. Hydrochloric acid was used to adjust

the pH. Sodium oléate, sodium sulfate and ammonium chloride were

used as the inhibitors. Each experimental condition was prepared in

triplicate. The volatile fatty acid (VFA) standards (C2-C6) were obtained

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1052 LIN, WANG, AND CHANG

from HAWANA (Japan) extra-pure chemicals. Table 1 summarizes the

experimental conditions.

Monitoring

The mixed liquor volatile suspended solids (MLVSS) used to express

biomass concentrations were measured according to the Standard

Methods procedure. Gas analyses were done with a gas Chromatograph

equipped with a thermal conductivity detector. VFA were analyzed with

a gas Chromatograph which had a flame ionization detector.

RESULTS AND DISCUSSION

The reliability of the experimental results was examined on COD

recovery from consumed HBu or glucose at various SRT. The COD

recoveries from input HBu and output products (CH4, biomass and VFA )

were 91.3~95.2% and 90,5~96.1% for n-HBu and i-HBu seed sludges,

respectively. The COD recoveries from input glucose and output

products (H2, biomass and VFA) were 89.4~99.4% for the acidogenic

digesters.

The characteristics of the seed sludges under steady-state conditions

at various SRT were: mixed liquor volatile suspended solids (MLVSS)

370~450 and 460~570 mg/liter for n-HBu and i-HBu digesters,

respectively, and 1120-1910 mg/liter for the digesters. The acclimated

seed sludges at the SRT of 10 days were used for the methanogenic

batch test and the VF As in the supernatants were 130+.4 and 79+3

mg/liter for the n-HBu and i-HBu digesters, respectively. Experimental

data of VFA discussed in the following are presented as mean values

(five to six determinations) with coefficients of variation from 0% to 14%.

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BUTYRIC ACID IN ANAEROBIC DIGESTION PROCESS

Table 1 Experimental conditions.

Production(Acidogenesis,temperature controlled at 35°C)

AΒ Glucose,

20000

5.7

6.4 2.5,2.0, 1.5,1.0,0.5,0.25

6.9

1053

Digester Substrate

Concentration

(mg COD /liter)

PH SRT

(days)

Seed Sludge

for Batch Test

Production (Acidogenesis, without temperature control)

Glucose,

20000

2.5,2.0, 1.5. 1.0,0.5,0.25

Utilization(Methanogenesis)

n-HBu, 20000 6 . 8 - 7 . 5 20,14,10,6,5 used 10 days

i - HBu, 20000 7.0 - 7.5 20,14,10, 8, 7, 6 used 10 days

Ε

F

Production of HBu

Figure 1 relates the production of n- and ¡-HBu to SRT at various pH

under condition of temperature control. Both forms of HBu were

produced in the acidogenesis process. Production of n-HBu generally

increased with increasing SRT and greater values were observed in the

greater pH range. The facts coincide with a result reported by Endo et al.

(1983) but contrast with a report by Hanaki et al. (1987), who used a

mixture of protein, fats and carbohydrates as the substrate. The peak

production at pH 5.7, 6.4 and 6.9 was at the SRT 1.0, 1.5 and 2.5 days,

respectively. No production of ¡-HBu was determined at SRT >2.0 days

when the pH was small (pH 5.7). These results indicate that the

production of ¡-HBu was dependent on SRT and pH.

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1054 LIN, WANG, AND CHANGO

D/L

)

oen

VF

A

400

300

200

100

0

9000

8000

7000

6000

5000

—

—

—

—

O PH=5.7D PH=6.4Δ ρΗ=δ.9

lfflP-~=-Q-~

/

I0.0 0.5

i-HBu

B ^n-HBu

I I

1.0 1.5SRT (day)

L

/

I

2.0 2

Fig.1 The production of HBu in the acidogenic digestion of glucose at 35°C.

Under the condition of without temperature control, production of both

forms of HBu always decreased with declined temperature but it was

SRT dependent (Fig. 2). Table 2 summarizes the experimental results

of the relationships between HBu production and pH, SRT and

temperature.

Effect of inhibitants on utilization

Effects of oléate, sulfate and total nitrogen ammonia (TAN) on the

utilization of HBu were elucidated. As an example, Fig.3 gives the

degradation of n-HBu with time in the batch test by adding various

concentrations of oléate into the n-HBu acclimated seed sludge. From

the time variation of the plots it is known that the n-HBu degradation

decreased with increasing oléate concentration. Similar patterns of time

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BUTYRIC ACID IN ANAEROBIC DIGESTION PROCESS 1055

400 —

200 —

αOυO)

m 8000 —

6000 —

4000

SRT (day)+ 0.25 Δ 1.5¿0.5 G 2.0O 1.0 O 2.5

15Temperature( C)

Fig.2 Production of HBu under the condition of without temperature control.

Table 2 A summary on the experimental results of the effects ofpH, SRT and temperature on HBu production.

Factors n-HBu production i-HBu productionpH Decreased with decreasing

pH at longer SRT and

decreased significantly when

pH.<_5.7.

Decreased slightly with

decreasing pH but increased

slightly with decreasing pH when

SRT <1.0 day.

SRT Decreased with decreasing

SRT and significantly at SRT

0.25 days.

Produced readily at longer SRT

when pH was high and at shorter

M w a 5 '°w ·Temperature Decreased with decreasing

temperature.

Increased with decreasing

temperature.

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1056 LIN, WANG, AND CHANG

QO

υσ>

HAci-HBu

Δ n-HBu• HCaD Total

Fig.3 Degradation of n-HBu with time in the batch test by adding oléateinto the n-HBu acclimated seed sludge. Oléate concentration:(a) 3, (b) 10, (c) 15, and (d) 20 mg/L .

course of degradation were observed (not shown) after dosing with

sulfate or ammonia. Since the hydrogen partial pressure was very low

(always below 10~4 atm) during digestion, the inhibitions of the three

tested agents on n-HBu degradation might occur from the inhibition of β

-oxidation of hydrogen-producing acetogens (Hanaki et al., 1981) .

Iso HBu produced from n-HBu utilization was also detected (Fig. 4). ¡-

HBu concentration was very low but it increased slightly with the

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BUTYRIC ACID IN ANAEROBIC DIGESTION PROCESS 1057

Ί ' Γ ' \

5 10 15Oléate Concentration (mg/L)

20

I ' I ' \ ' Γ n I ^ I ^ I

0 400 800 1200 1600 2000 2 4 0 0

Sulfate Concentration (mg/L)

Τ Τ Ίί '

0 2000 4000 6000Total Nitrogen Ammonia (TAN) Concentration (mg/L)

Fig.4 The isomerization products from the presences of

the inhibitants: n-HBu formed from i-HBu and ¡-HBu

formed from n-HBu.

increasing oléate concentration which result implies that oléate was

more inhibitory to ¡-HBu utilization (Discussed later). The degradation of

HAc was also inhibited at high oléate concentration which phenomenon

agrees with the observation of Koster and Cramer (1987) . However, low

oléate concentration stimulated the methanogenesis of HAc (Komatsu et

al., 1991).

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1058 LIN, WANG, AND CHANG

The degradation of i-HBu with time after adding oléate into the i-HBu

acclimated seed sludge was similar to that of n-HBu degradation. The

accumulation of ¡somerized n-HBu was independent of oléate

concentration and always at the concentration range of 210 to 287 mg

COD/liter. The transformation of i-HBu to methane is first via formation

of n-HBu and then by β -oxidation (Tholozen et al., 1988 ; Steib and

Schink, 1989: Lin and Wang, 1992) .

An activity factor Aj (where A{=Vj / Vc, in which V¡ and Vc are the

amounts of HBu degraded in 48 h by inhibitant dosed seed sludge and a

control, respectively) was used to indicate the extent of inhibition. Fig.5

shows the relationships between A{ and inhibitant concentrations.

Utilization activities decreased as inhibitant concentrations increased.

For the same activity level, different inhabitant concentrations caused

various kinds of inhibition for different ¡nhibitants and for both forms of

HBu. It is known that at the same concentration, oléate was more

inhibitory to i-HBu utilization, but ammonia was reversely more inhibitory

to n-HBu utilization. Utilization of HBu ceased when oléate

concentration reached 20 mg/liter. Same inhibition level was observed

for sulfate to the degradation of both forms of HBu.

Stuckey et al. (1980) used a term 50% inhibition to describe a

reduction in gas production by dosing organic chemicals. In the present

paper, the 50% inhibition refers to a 50% reduction in HBu utilization

over 48 h by inhibitant dosage. The results for 50% inhibition of activity

of HBu utilization (A50) read from Fig.5 are summarized in Table 3. A

comparison on the values of A50 indicates that the relative toxicities were

oléate >> sulfate > ammonia for the utilization of n-HBu and i-HBu.

The relative formation of n-HBu from i-HBu and of i-HBu from n-HBu

indicates that only the production of n-HBu was affected more markedly

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BUTYRIC ACID IN ANAEROBIC DIGESTION PROCESS 1059

CO

10080604020

108

80

60

40

20

100

80

60

40

20

0

TAND n-HBuX i-HBu

Sulfate

Oléate

Ί ' I r

5 10 15Oléate Concentration (mg/L)

I I I400 800 1200 1600 2000

Sulfate Concentration (mg/L)2400

0 2000 4000 6000Total Nitrogen Ammonia (TAN) Concentration (mg/L)

Fig.5 The relationships between At and inhibitant concentration.

Table 3 Results for 50% inhibition of activity ofHBu utilization (Aso, mg /liter).

Inhibitants n-HBu i-HBuOléateSulfate

Ammonia

1111403000

81500

>6000

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1060 LIN, WANG, AND CHANG

with increasing inhibitant concentration if the utilization of HBu

progressed (Fig. 4). The isomerization of i-HBu was then known to be

readily affected by the tested inhabitants.

Based on the experimental results, the branched i-HBu is a product

possiblly directly produced from the anaerobic acidogenesis of glucose

or from the ¡somerization of n-HBu in methanogenesis under the

presence of some inhibitants, it seems that this acid is not a proper

indicator of digester failure as suggested by Hill and Holmberg (1988).

CONCLUSIONS

Both forms of butyric acid are produced in the acidogenesis of glucose

and the production depends on temperature, SRT and pH. Isomerization

between iso and normal butyric acid occurs during methanogenesis of

butyric acid and the isomerization of iso form is readily affected by

oléate, sulfate or ammonia. The relative toxicities are oléate » sulfate

> ammonia for the utilization of both forms of butyric acid.

ACKNOWLEDGEMENT

This work was supported by a grant from the National Science Council

(NSC82-0410-E-035-018).

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BUTYRIC ACID IN ANAEROBIC DIGESTION PROCESS 1061

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