mechanism of inhibition caused by long-chain fatty acids in anaerobic digestion process

Mechanism of Inhibition Caused by Long-Chain Fatty Acids in Anaerobic Digestion Process

KEISUKE HANAKI, Department of Civil Engineering, Tohoku University, Aoba, Sendai 980, Japan, and TOMONORI MATSUO and

MICHIHIKO NAGASE, Department of Urban Engineering, The University of Tokyo, Hongo, Tokyo 113, Japan

Summary The inhibitory effect of long-chain fatty acids on the anaerobic digestion process was

examined in batch experiments using synthetic substrates. The addition of long-chain fatty acids caused the appearance of the lag period in the methane production from acetate and in the degradation of both long-chain fatty acids and n-butyrate. Methane production from hydrogen proceeded without lag period although its rate was lowered. Fermentation of glucose was not inhibited. Neutral fat in the whole milk was easily hydrolyzed to long-chain fatty acids, which brought about the inhibition. The addition of calcium chloride reduced the inhibitory effect of long-chain fatty acids, but it did not do so after the culture had been exposed to long-chain fatty acids for more than several hours. The addition of calcium carbonate could not reduce the inhibition because of its insolubility.

INTRODUCTION

Anaerobic digestion of organic sludge is an important operation in wastewater treatment processes. Various kinds of organic materials can be stabilized and, at the same time, converted into methane gas which is used as fuel. Because of this ability of producing methane, the anaerobic digestion has been thought to be a very attractive process of the effective energy recovery.

Sewage sludge comprises lipids, carbohydrates, and proteins. Among them lipids are the most significant substances in the anaerobic digestion, since a larger amount of methane can be produced from lipids than from other components. ' The lipids included in sewage sludge are composed mainly of neutral fats and long-chain fatty acids. Neutral fats are hydrolyzed to long-chain fatty acids and glycerol in the anaerobic digestion as shown in eq. ( 1 ) .

Biotechnology and Bioengineering, Vol. XXIII, Pp. 1591-1610 (1981) 0 1981 John Wiley & Sons, Inc. CCC 0006-3592/81/07 1591-20$02.00

1592 HANAKI, MATSUO, AND NAGASE

CH~OCORI RlCOOH CH20H

CHOCOR2 + 3Hz0+ RzCOOH + CHOH

CH2OCOR3

I I I I (1) R3COOH CHzOH

neutral fat long-chain glycerol fatty acids

R1 , R2, and R3 indicate alkyl groups in this equation. A large portion of chemical oxygen demand (COD) of the neutral fat is conserved in long- chain fatty acids even after the hydrolysis. Long-chain fatty acids are degraded via P-oxidation cycle in the anaerobic d i g e ~ t i o n ~ , ~ as well as in the aerobic processes. These reactions are carried out by “H2-producing acetogenic bacteria” which were proposed by Bryant.4 For example, the degradation of palmitate is shown as follows:

CH3(CH2)14C00- + 14H2O + 8CH3COO- + 7H+ + 14H2 (2) (GI, = + 345.6 kJ/mol)

where GA is the standard free energy change and is calculated from the values of standard free energy of f ~ r m a t i o n . ~ Both propionate and n- butyrate are important intermediates in the anaerobic digestion and also converted into acetate and hydrogen, as shown in eqs. (3) and (4), respectively.

Propionate :

CH3CH2COO- + 3H20 -+ CH3COO- + HC03- + H + + 2H2(3)

(GI, = +76.1 kJ/mol) (ref. 6)

n-Butyrate:

CH3(CH&COO- + 2H20 + 2CH3COO- + H + + 2H2 (4) (GI, = +48.1 kJ/mol) (ref. 6)

The acetate and the molecular hydrogen produced in these reactions can be converted into methane by methanogenic bacteria as shown in eqs. (5 ) and (6).

CH3COO- + H20 + CH4 + HC03- ( 5 )

(6)

The values of standard free energy change GA are positive in the reactions of eqs. (2), (3), and (4). These reactions are thermodynamically unfavor- able unless the hydrogen partial pressure is maintained at an extremely low leveL6 Methanogenic bacteria utilize molecular hydrogen in the usual anaerobic digester so rapidly that the hydrogen partial pressure can be

4H2 + C02 + CH4 + 2H20

INHIBITION CAUSED BY FATTY ACIDS 1593

kept low enough to ensure the active performance of the H2-producing acetogenic bacteria. This fact means that the degradation of fatty acids via P-oxidation cycle depends largely on the activity of methanogenic bacteria. Regarding the relationship between the degradation of long- chain fatty acids and the activity of methanogenic bacteria, Heukelekian and Mueller' reported that the long-chain fatty acids were not degraded during the acid-forming phase of the anaerobic digestion where methane was not produced.

McCarty* found that oleate, which is one of the typical long-chain fatty acids, inhibited the anaerobic digestion process. He also mentioned that this inhibition would not occur in the case of continuous feed systems. Besides this report, there have been some investigations which were carried out on the inhibitory effect of long-chain fatty acids. However, the characteristics of this inhibition in the anaerobic digestion have not been clarified yet.

The purpose of the present study is to examine the inhibitory effect of long-chain fatty acids on each elemental reaction in the anaerobic digestion and to reveal the mechanism of inhibition, since long-chain fatty acids are the most prominent potential inhibitors. Furthermore, some tentative ways to reduce the inhibitory effect of long-chain fatty acids were examined in the present study.

MATERIALS AND METHODS

Substrates

Sodium oleate (C18:l) , a fatty acids mixture, and a powdered whole milk were used for substrates. The fatty acids mixture comprised sodium salts of several species of long-chain fatty acids. The powdered whole milk (Type FM-s, Meiji Milk Products Co., Ltd., Japan) contained neutral fat, lactose, and casein as lipid, carbohydrate, and protein, respectively, and their contents are shown in Table I. The individual fatty acids which were contained in the fatty acids mixture and the whole milk are tabulated

TABLE I Composition of the Whole Milk

Contents

Constituent % of dry basis % of COD basis"

Carbohydrate 57.8 43.6 Protein 13.5 12.2 Lipid 25.5 44.2 Ash 3.2 0

a These figures were calculated from experimental COD data of casein and the whole milk and theoretical COD value of lactose.


in Table 11. Acetate and n-butyrate were also used for substrates in the form of their sodium salts.

Seed Sludge

Digested sludge from a digester of Shibaura Sewage Treatment Plant, Tokyo, Japan, was acclimated with the whole milk in a continuous-type reactor of laboratory scale under the constant temperature of 37 k 1°C. The hydraulic detention time and the organic loading were 12-36 days and 0.5-1.5 g of volatile solids/L day, respectively. The mixed liquor of this reactor was used as the seed sludge for batch experiments.

Batch Experiment

The anaerobic degradation process of each substrate was examined by batch experiments at 37 & 1°C using either a laboratory digester system or serum vials. Figure 1 illustrates the laboratory digester system, which consisted of a 2.4-L glass reactor (A) and a gas measuring unit (vessels B and C). Saline-saturated acidified water in the vessel B was replaced by the gas produced in the reactor A. The contents of the reactor were mixed continuously with a magnetic stirrer. Two liters of the seed sludge was transferred into the reactor and the residual air was displaced with N2/C02 mixed gas [70/30 (v/v)]. After incubating for about ten days during which the substrate contained in the seed sludge could be degraded sat- isfactorily, a batch experiment was begun with injecting a concentrated solution of the substrate to the reactor. The mixed liquor of the reactor was drawn with a pipette at certain intervals and served for chemical analyses. At the same time, the amount of gas production was measured

TABLE I1 Fatty Acids Distribution in the Fatty Acids Mixture and the Whole Milk

Percentage Percentage

Fatty Fatty acids Whole acids Whole

Fatty acid" mixtureb milk' Fatty acid" mixtureb milk'

Cl0:O 2 1 ClZ,l 2 0 Cn:o 8 7 c14:I 8 0 c14:o 13 6 c 16: I 20 2 c16:O 16 21 CIK.1 28 39 Cix:o 1 6 C1x:z 0 13

Others 2 4

a In C,:,, m and n indicate chain length of carbon and the number of double bonds, respectively.

Determined by gas chromatography. Data were obtained from Meiji Milk Products Co. , Ltd., Japan.

INHIBITION CAUSED BY FATTY ACIDS I595

Fig. 1. Laboratory digester system: (A) reactor; (B, C) gas measuring unit; (M) magnetic stirrer; (G) sampling port for gas; (L) for mixed liquor.

and gases were sampled by a I-mL syringe from both the reactor A and the vessel B to determine their components. Sodium bicarbonate was added to maintain pH between 6.6 and 7.6.

Several numbers of 37-mL serum vials were also used for an another batch experiment.’ In this experiment, after a predetermined amount of substrate was weighed and put into each vial, 20 mL of the seed sludge which did not contain residual substrate was added to each vial. The void of the vial was displaced with N2/C02 mixed gas [70/30 (v/v)] and the vial was sealed with a rubber cap tightly. The vials were incubated without agitation. The amount of gas production was measured by inserting a needle attached to a glass syringe through the rubber cap. The gas in the vial was sampled by a 1-mL syringe for gas analysis and, if necessary, 0.5 mL of the mixed liquor was also sampled by a 1-mL pressure-tight syringe. It was impossible to control pH during this experiment, but its value was between 6.6 and 7.6.

Analytical Method5

The mixed liquor sampled was centrifuged (2500g, 5 min) to separate the solid from the supernatant. Lipids in the solid were extracted by the method of Bligh-Dyer” and lipids in the supernatant were extracted with diethyl ether. The samples were acidified by adding hydrochloric acid before these extractions to make fatty acids un-ionized form. Total lipid content in the extract was determined gravimetrically after evaporating the organic solvent. Gas composition, volatile acids, and long-chain fatty acids were determined by gas chromatography. The analytical conditions are summarized in Table 111. ATP (adenosine triphosphate) was extracted from the mixed liquor in triplicate by the boiling Tris method” and its concentration was assayed by ATP photometer (model 2000, SAI Tech- nology Co.). The firefly lantern extract and Tris were purchased from Sigma Chemical Co. and Calbiochem., respectively.


TABLE 111 Analytical Conditions of the Gas Chromatography

Gas composition Long-chain fatty (N2, CH4, CO2, Volatile acids acids

Subject H2) (c2-c6) (C lo-C 18)

Pretreatment of None Acidified to pH 1-2 None (without

Packing of column" Activated charcoal 20% diethylene Polyester FF sample esterification)

glycol adipate polyester + 2% H3P04

Carrier gas He or N2 N2 N2 Detector TCD FID FID Sample volume 1 mL 5 PL 10 pL

a Purchased from Nishio Kogyo Co., Ltd., Japan.

Indicators of Process Performance

The degradation process of the substrate was evaluated by a cumulative methane production and a cumulative net acid production. The former was calculated from the cumulative gas production and the methane content of the gas and was expressed as miligrams of COD per liter of the mixed liquor. The latter was defined in the batch experiment as follows:

where A is the cumulative net acid production (mg COD/L), M is the cumulative methane production (mg COD/L), Y is the yield coefficient in methane production, and CA is the concentration of volatile acids expressed as COD (mg COD/L). The yield coefficient in methane production was defined as grams of COD of methane produced per gram of COD of intermediates (volatile acids and hydrogen) degraded. Its value was estimated to be 0.96 from an experiment using acetate as substrate. The cumulative net acid production represented the cumulative amount of intermediates, namely volatile acids and hydrogen, which were produced not only by the fermentation of carbohydrates or protein but also by the @-oxidation of long-chain fatty acids. The cumulative methane production and the cumulative net acid production can be expressed as a fractional conversion, which is their proportion to the COD of the substrate added.

RESULTS

Inhibitory Effect of Long-Chain Fatty Acids

The inhibitory effect of the fatty acids mixture on the anaerobic digestion process was examined using 20 serum vials. As the substrate, the


fatty acids mixture of 250-2000 mg/L (as oleate) was used, and in some cases, acetate, n-butyrate, or glucose was also added to make the inhibitory effect clear. All vials were inoculated with the same seed sludge. The experimental conditions are shown in Table IV. Figure 2(a) shows the cumulative net acid production versus incubation time in the case where the fatty acids mixture was added alone. The cumulative net acid production represented the p-oxidation of the fatty acids mixture, and it was accompanied by a lag period which became longer with the increase of the concentration of the fatty acids mixture added. This result means that the fatty acids mixture was inhibitory to the p-oxidation of itself. Figure 2(b) shows the cumulative methane production versus incubation time in the case where acetate was added with the fatty acids mixture. The lag period that appeared in the methane production became longer with increasing the concentration of the fatty acids mixture added, although there appeared no lag when only acetate was used as the substrate. This result shows that the fatty acids mixture inhibited methane production from acetate. The degradation of n-butyrate to acetate was also inhibited by the fatty acids mixture in a manner similar to that described above [Fig. 2(c)]. The effect of the fatty acids mixture on the degradation of glucose was also examined. The time course of the cumulative net acid production is shown in Figure 2(d). The cumulative net acid production reached about 2000 mg COD/L within a day irrespective of the concentration of the fatty acids mixture added. Since the p-oxidation of the fatty acids mixture did not take place in a few days of incubation in the case where high concentration of the fatty acids mixture was added as shown in Figure 2(a), the cumulative net acid production observed here means the degradation of glucose. This result shows that the fermentation of glucose was not inhibited by the addition of the fatty acids mixture.

To investigate the inhibitory effect of the fatty acids mixture on methane production from molecular hydrogen, hydrogen gas was injected into two vials which had been incubated for ten days in the experiment described above. The conditions of these two vials were as follows: to one only acetate had been added and its degradation had been completed (control),

TABLE 1V Experimental Conditions ( I ) "

The fatty acids mixture added (mg/L as oleate)

fatty acids mixture 0 250 500 lo00 2000 Substrate added together with the

None 0 615 1230 2460 4920 Acetate 2000 mg/L 2140 2755 3370 4600 7060 n-butyrate 2000 mg/L as acetate 5320 5935 6550 7780 10240 Glucose 2000 mg/L 2140 2755 3370 4600 7060

a Values in the table are total COD (mg/L) of the substrates added to the vials. Initial MLVSS was 2500 mg/L.


Incubation Time (days) Incubation Time (days)

Fig. 2. Inhibitory effect of the fatty acids mixture: (a) cumulative net acid production when the fatty acids mixture was added alone; (b) cumulative methane production when acetate was added together; (c ) degradation of n-butyrate when it was added together; (d) cumulative net acid production when glucose was added together. (3) No addition of the fatty acids mixture; (A) 250 mgiL; (0 ) 500 mg/L; (0) 1000 mg/L; (A) 2000 mgiL addition of the fatty acids mixture.

and to the other both acetate and 2000 mg/L of the fatty acids mixture had been added and the methane production from acetate was inhibited at that time (under inhibition). The hydrogen consumption and the methane production of these vials versus incubation time after the addition of hydrogen gas are shown in Figure 3. Although the hydrogen consumption rate was smaller in the vial under inhibition than in that of the control because of the inhibitory effect of the fatty acids mixture, hydrogen was readily utilized without lag period even in the vial under inhibition. The agreement between the hydrogen consumption and the methane production in COD basis shows that methane was formed from hydrogen and not from acetate in these vials.

The degree of the inhibition was evaluated by a length of the lag period before the initiation of active methane production, which was determined schematically as shown in Figure 4. Figure 5 shows the relationship between the length of the lag period and the concentration of the fatty acids


400k 200 1 2

0 Incubation Time after Hydrogen . .

Addition (days)

Fig. 3. Inhibitory effect of the fatty acids mixture on the methane production from hydrogen: (0) methane production and ( 0 ) hydrogen consumption in the control vial; (b) methane production and (A) hydrogen consumption in the vial under inhibition.

mixture added. The addition of either n-butyrate or acetate somewhat intensified the inhibitory effect of the fatty acids mixture.

The difference in the degree of inhibitory effect between the fatty acids mixture and oleate was examined using six vials. The concentration of substrate added to each vial is shown in Table V. The relationship between the length of the lag period in methane production and the concentration of the fatty acids mixture or oleate added is presented in Figure 6. This figure shows that oleate was less inhibitory to the methane production than the fatty acids mixture.

To know the behavior of long-chain fatty acids and the biological condition of sludge, total lipid content in solid and ATP were determined during the degradation of the fatty acids mixture. The concentration of the fatty acids mixture added and that of the initial MLVSS were 1000 mg/L (as oleate) and 2910 mg/L, respectively. The result is shown in Figure 7. A large portion of the fatty acids mixture added was adsorbed by the solid within a day, although the fatty acids mixture was added in the form of its soluble sodium salt. ATP began to decrease immediately after the addition of the fatty acids mixture and reached about &th its

Length of the Lag Period

Incubation Time

Determination of length of the lag period in methane production. Fig. 4.


r

The Fatty Acids Mixture Added (mg/l asoleate)

Fig. 5 . Relationship between the length of the lag period and the concentration of the fatty acids mixture added: (0 ) addition of the fatty acids mixture alone; addition of (0) acetate and (a) n-butyrate together with the fatty acids mixture.

initial value on the third day. Methane production proceeded actively after ten days of incubation, which was accompanied with the decrease of total lipid content in the solid. ATP was not restored even after the fatty acids mixture had been degraded.

Hydrogen content of the gas in a vial to which oleate had been added was analyzed to estimate the thermodynamical condition for the H2-producing acetogenic bacteria. The hydrogen content was as low as 0.005% even when the methane production was inhibited by oleate.

Degradation of Neutral Fat

The degradation of neutral fat in the whole milk was examined using the laboratory digester system. The initial concentration of the whole milk and that of MLVSS were 4000 mg/L (5840 mg COD/L) and 2190 mg/ L, respectively. Total lipid content in the solid, total long-chain fatty

TABLE V Experimental Conditions (2)"

Vial No.

Substrate concentration 1 2 3 4 5 6 ~~~~~ ~ ~~~

The fatty acids mixture (mg/L as oleate) 250 500 lo00 0 0 0 Oleate (mg/L) 0 0 0 250 500 1000 Acetate (mg/L) 2000 2000 2000 2000 2000 2000

a Initial MLVSS was 1780 mg/L.


Long-chain Fatty Acid Added (mg/l asoleate)

Fig. 6. Difference in inhibitory effect between oleate and the fatty acids mixture: (*) oleate; (3) fatty acids mixture.

acids both in the solid and in the supernatant, cumulative methane production, and cumulative net acid production were examined and their time courses are shown in Figure 8. Total long-chain fatty acids means a sum of the concentration of the individual fatty acids. Long-chain fatty acids accumulated in the solid rapidly within a day. On the other hand, they did not accumulate so much in the supernatant. This result shows that the neutral fat in the whole milk was easily hydrolyzed to long-chain fatty acids and they were adsorbed by the solid; at the same time, it shows that the degradation of long-chain fatty acids did not proceed

- 1 I -

Incubation Time (days)

Fig. 7. Total lipid content in the solid and ATP during the degradation of the fatty acids mixture: (0) total lipid content in the solid; ( 0 ) ATP in the mixed liquor; (3) cumulative methane production.


5 c c Incubation Time (days) .P .P

3 3- Incubation Time (days)

00

Degradation of the whole milk: ( 0 ) total lipid content in the solid; (3) total long- chain fatty acids in the solid and ( A ) in the supernatant; (.) cumulative net acid production; ( g ) cumulative methane production.

5 5

Fig. 8.

immediately after the hydrolysis of the neutral fat. This retardation of the degradation of long-chain fatty acids was probably brought about by their own inhibitory effect to the H2-producing acetogenic bacteria that degraded long-chain fatty acids. The cumulative net acid production increased stepwise as shown in Figure 8. The second step after the eighth day arose from the degradation of long-chain fatty acids. On the other hand, the first step probably represented the fermentation of carbohydrate and protein in the whole milk.

The methane production was not inhibited markedly in the above case, but it was often retarded when the initial food-to-microorganism ratio was high. One typical example of the retardation of the methane production is shown in Figure 9. In this case, the initial concentration of the whole milk and that of MLVSS were 4000 and 1880 mg/L, respectively.

These results show that long-chain fatty acids produced by the hydrolysis of neutral fat inhibited both P-oxidation of themselves and methane production, although neutral fat itself was not inhibitory.


C C I+ so 1.0- fk

P --

/ - - r/ I

I I ‘ a a - 0 10 20 ’% 5 5 Incubation Time (days) 00

Fig. 9. Inhibition in the degradation of the whole milk: (0 ) cumulative net acid production; (c) cumulative methane production.

Reduction of the Inhibitory Effect of Long-Chain Fatty Acids

The inhibitory effect of long-chain fatty acids may be reduced by adding calcium ion because it can precipitate long-chain fatty acids. Calcium chloride (CaC12.2H20) was added to examine whether the inhibitory effect of the fatty acids mixture can be reduced. The experimental conditions are shown in Table VI. The amount of CaCI2 added was about five times that amount stoichiometrically required to precipitate the fatty acids mixture added. Figure 10 shows the cumulative methane production in this experiment. A prominent lag period appeared in the methane production when CaC12 was not added. On the other hand, methane production took place without retardation when CaClz was added, and its rate was com- parable to that in the vial to which only acetate was added.

The reduction of the inhibitory effect of oleate by the addition of CaClz was also examined. The experimental conditions are shown in Table VII. The cumulative methane production in each vial is shown in Figure 11. This result shows that the inhibitory effect of at least 4000 mg/L of oleate could be remarkably reduced by the addition of a sufficient amount of CaC12.

In the experiments mentioned so far, CaC12 was added to each vial before the seed sludge was inoculated to the vial. Now the effect of CaC12

TABLE VI Experimental Conditions (3)”

Vial No.

Substrate concentration I 2 3

The fatty acids mixture (mgiL as oleate) 1000 1000 0 Acetate (mg/L) 1000 1000 1000 CaClz (mmol/L) 0 8.7 0



40001

0 10 20 30 40 50 t 0 Incubation Time (days)

Fig. 10. Reduction of the inhibitory effect of the fatty acids mixture by the addition of CaClz: (0) addition of acetate alone; (3) addition of acetate and the fatty acids mixture; (A) addition of acetate, the fatty acids mixture, and CaCI2.

was examined in the case where it was added after the sludge had already been exposed to the fatty acids mixture. Four exposure periods, namely 5 min and 4, 8 , and 24 h, were selected as shown in Table VIII. The cumulative methane production in each vial is shown in Figure 12. The inhibitory effect was reduced remarkably when the exposure period was 5 min. The lag period became longer with increasing the exposure period of the sludge to the fatty acids mixture. Addition of CaC& could not at all reduce the inhibitory effect of the fatty acids mixture when the exposure period was 24 h.

Calcium carbonate (CaC03), which is insoluble in water, was added to reduce the inhibitory effect of the fatty acids mixture. Substrates added to three vials are shown in Table IX. CaC03 was added to the vial before the seed sludge was inoculated to it. The cumulative methane production in each vial is shown in Figure 13. CaC03 could hardly reduce the inhibitory effect of the fatty acids mixture because of its insolubility.

The reduction of the inhibitory effect of long-chain fatty acids derived

TABLE VII Experimental Conditions (4)"

Vial No. Substrate concentration 1 2 3 4 5

Oleate (mgiL) 0 1000 2000 4000 lo00 Acetate (mglU 2000 2000 2000 2000 2000

0 CaC12 (rnrnollL) 21 21 (llj" 21 (6)b 21 O)b

a Initial MLVSS was 1560 rng/L. The ratio of added CaCI2 to that stoichiometrically required is shown

in parentheses.

INHIBITION CAUSED BY FATTY ACIDS I605

TABLE VIII Experimental Conditions (5)"

Vial No.

Condition I 2 3 4 5

The fatty acids mixture added loo0 1000 1000 lo00 lo00

CaClz added (rnmol/L) 8.1 8.7 8.7 8.1 0 Exposure period before the 5 min 4 h 8 h 24h -

(rng/L as oleate)

CaC12 addition


from hydrolysis of neutral fat in the whole milk was examined by adding CaCI,. The experiment was carried out using two vials. Substrate added to each vial is shown in Table X. The cumulative methane production and the cumulative net acid production in each vial are shown in Figure 14. When CaCl, was not added, the net acid production increased stepwise and the rate of methane production decreased after two days of incubation. The addition of CaC1, eliminated these phenomena which were brought about by the inhibitory effect of long-chain fatty acids.

DISCUSSION

Sodium salts of long-chain fatty acids were used as primary substrates in the present study. Regarding the inhibitory effect of sodium ion,


Fig. 11. Reduction of the inhibitory effect of oleate by the addition of CaClz: (0 ) addition of acetate alone; (3) addition of acetate and 1000 mg/L of oleate; addition of acetate and CaC12 together with ( 2 ) loo0 mg/L, (A) 2000 mg/L, and (A) 4000 mg/L of oleate.


1


Fig. 12. Effect of CaC12 addition after the culture was exposed to the fatty acids mixture. The exposure period before the CaC12 addition was (*) 5 min, (.) 4 h, (2) 8 h, and ( A ) 24 h. (3) No CaCI2 addition.

McCarty' reported that the concentration higher than 3500 mg/L of sodium ion was inhibitory to the anaerobic digestion process. Because the concentration of total sodium ion, including that from the sodium salt of long-chain fatty acids and from other sources, was less than 1000 mg/L in the present study, the toxicity of sodium ion may be negligible.

The adsorption of the fatty acids mixture by solid, accor panied by a

5000,

4000

3000

2000

1000

0 Incubation Time (days)

Fig. 13. Reduction of the inhibitory effect of the fatty acids mixture by the addition of CaC03: ( 0 ) addition of acetate alone; (<)) addition of acetate and the fatty acids mixture; (a) addition of acetate, the fatty acids mixture, and CaCO3.


TABLE IX Experimental Conditions (6)”

Vial No.

Substrate concentration 1 2 3

The fatty acids mixture (mgiL as oleate) 1000 1000 0 Acetate (mgiL) 2000 2000 2000 CaC03 (mmol/L) 20 0 0

a Initial MLVSS was not determined, but probably between 1800 and 2500 mg/L.

marked decrease of ATP (Fig. 7), suggests that the fatty acids mixture which adsorbed by solid was toxic to the bacteria. The inhibitory effect of the fatty acids mixture and other long-chain fatty acids was probably brought about by their toxicity.

Several investigators reported the toxicity of long-chain fatty acids. In a rumen, where methane is produced exclusively from only hydrogen and not from acetate,I2 long-chain fatty acids were found to be inhibitory to the methane production in v ~ v o ’ ~ , ’ ~ and in vitro.I5 It was also reported that long-chain fatty acids were toxic to the pure culture of methanogenic bacteria which utilized only hydr~gen.’~.’’ Methane production from hydrogen did not stop completely, although its rate decreased as the result of the inhibition in their experiments. These results observed by previous investigators agree with the result of the present study (Fig. 3).

The toxicity of long-chain fatty acids to acetate-utilizing methanogenic bacteria and H,-producing acetogenic bacteria has not been clearly con- firmed before the present study. H2-producing acetogenic bacteria cannot degrade long-chain fatty acids and n-butyrate if the hydrogen partial pressure is not low enough. So it might be possible that the degradation of these acids was inhibited not because long-chain fatty acids were toxic to the H2-producing acetogenic bacteria but because their toxicity to the H,-utilizing methanogenic bacteria caused an accumulation of molecular hydrogen. However, this mechanism of inhibition was unlikely in the present study, since the hydrogen content in the produced gas was extremely low, as low as 0.005%. Long-chain fatty acids were probably toxic directly to H2-producing acetogenic bacteria.

TABLE X Experimental Conditions (7)a

Vial No.

Substrate concentration 1 2

The whole milk (mg/L) 6000 6000 CaC12 (mmol/L) 8.7 0

a Initial MLVSS was 1850 rng/L.


1.0

0.8

0.6

0.4

0.2

0 5 10 15 20 Incubation Time (days)

Fig. 14. Effect of CaClz addition on the degradation of the whole milk: cumulative net acid production under the condition of (*) CaC12 addition and (0) not; cumulative methane production under the condition of (A) CaClz addition and (A) not.

It was reported that long-chain fatty acids were toxic also to some species of bacteria which ferment carbohydrates and were not to some specie^.'^ The fermentation of glucose was not inhibited by long-chain fatty acids in the present study. This result suggests that there existed a sufficient amount of bacteria which could ferment glucose and were not susceptible to the toxicity of long-chain fatty acids in the anaerobic cul- .ture.

Some investigators reported that the inhibitory effect of long-chain fatty acids could be reduced by the addition of calcium salt.8*'8 The result of the present study agreed with these reports and it should be noted that the addition of CaCI, could not reduce the inhibitory effect sufficiently once the culture had been exposed to long-chain fatty acids for more than several hours before the addition of CaCI2. It is also important that CaC03 could not reduce the inhibition because of its insolubility. As carbonate ion (C032-) exists in high concentration in the anaerobic digester, a con- siderable amount of added calcium ion may be precipitated in the form of CaC03 as well as calcium salt of long-chain fatty acids. This phenom- enon may diminish the effectiveness of calcium ion in the reduction of the inhibitory effect of long-chain fatty acids.

Lipid content of a sewage sludge accounts for 28% of the total organic solids' and its concentration in a thickened sludge can be as high as 10,000 mg/L, while about 1000 mg/L of lipid caused the severe inhibition as shown in the present study. In a practical continuous operation, the concentration of long-chain fatty acids in a digester does not become so high under the normal condition because long-chain fatty acids can be degraded continuously. However, an overloading or a shock loading of the raw sludge to the digester may cause an accumulation Of IOng-chain fatty

INHIBITION CAUSED BY FATTY ACIDS I 609

acids, and the process may be severely retarded in this case. Feeding of the raw sludge at constant rate and under the constant environmental conditions is important to prevent the digester from the inhibition caused by the accumulation of long-chain fatty acids. The concentrations of long- chain fatty acids or total lipid in a digester and of raw sludge should be monitored. Furthermore, the control of them will improve the stability of this process.

CONCLUSIONS

Long-chain fatty acids were found to be inhibitory to the several kinds of essential reactions in the anaerobic digestion because of their toxicity to the bacteria. The methane production from acetate and the degradation of both long-chain fatty acids and n-butyrate were inhibited so strongly that a long lag period appeared before these reactions began. The rate of methane production from hydrogen was lowered by long-chain fatty acids, although the reaction proceeded without a lag period. The fermentation of glucose was not inhibited by long-chain fatty acids. Neutral fat in the whole milk was hydrolyzed easily to long-chain fatty acids which caused the inhibition.

An addition of soluble calcium salt reduced the inhibitory effect of long- chain fatty acids. However, its addition was ineffective after the anaerobic culture had been exposed to long-chain fatty acids. Insoluble CaC03 could not reduce the inhibition.

The authors are grateful to Dr. J . Matsumoto and Dr. T . Noike for their instructive advice and discussions. This work was supported by grants-in-aid for Scientific Research, Ministry of Education, Japan.

References 1. D. P. Chynoweth and R. A. Mah, Adv. Chem. Ser., 105, 41 (1971). 2. J . S . Jerk and P. L. McCarty, J. Wafer Pollur. Control Fed., 37, 178 (1965). 3 . C. Weng and J . S. Jerk, Wafer Res. , 10, 9 (1976). 4. M. P. Bryant, J. Animal Sci.. 48, 193 (1979). 5 . H. D. Brown, Ed., Biochemical Microcalorimefry (Academic, New York, 1969). 6. R. K . Thauer, K. Jungermann, and K. Decker, Bacferiol. Rev. , 41, 100 (1977). 7. H. Heukelekian and P. Mueller, Sew. f n d . Wastes, 30, 1108 (1958). 8. P. L. McCarty, Public Works, 95, 91 (November 1964). 9. T. L. Miller and M. J . Wolin, Appl . Microhiol., 27, 985 (1974).

10. E . G . Bligh and W. J . Dyer, Can. J. Biochern. Physiol., 37, 911 (1959). 1 1 . J . W. Patterson, P. L. Brezonik, and H. D. Putnam, Environ. Sci. Technol., 4, 569

12. R. E. Hungate, Ann. Rev. E d . Sysf., 6 , 39 (1975). 13. J . W. Czerkawski, K. L. Blaxter, and F. W. Wainman, Br. J. Nufr . , 20, 349 (1966). 14. J . W. Czerkawski. K. L. Blaxter, and F. W. Wainman, Br. J . Nufr . , 20, 485 (1966). 15. D. I . Demeyer and H. K. Henderickx, Biochim. Biophys. Acra, 137,484 (1967).

(1970).


16. R. A. Prins, C. J . Van Nevel, and D. 1. Demeyer, Antonie van Leeuwenhoek J .

17. C. Henderson, J . Agric . Sci . Cumb., 81, 107 (1973). 18. H. Galbraith, T. B. Miller, A. M. Paton, and J . K. Thompson, J . Appl . Bacteriol.,

Microbiol. Serol., 38, 281 (1972).

34, 803 (1971).

Accepted for Publication December 12, 1980

mechanism of inhibition caused by long-chain fatty acids in anaerobic digestion process

Documents