Appl Microbiol Biotechnol (1992) 37: 808-812 Applied

Microbiology Biotechnology © Springer-Verlag 1992

Effects of free long-chain fatty acids on thermophilic anaerobic digestion I. Angelidaki and B. K. Ahring

Anaerobic Microbiology/Biotechnology Research Group, Department of Biotechnology, The Technical University of Denmark, DK-2800 Lyngby, Denmark

Received 8 January 1992/Accepted 22 May 1992

Summary. Low concentrations of the long-chain fatty acids oleate and stearate inhibited all steps of the anae- robic thermophilic biogas process during digestion of cattle manure. The lag phase increased when the concen- trations of oleate and stearate were 0.2 g/1 and 0.5 g/l , respectively, and no growth was found at concentrations of 0.5 g/1 for oleate and 1.0 g/1 for stearate. The toxic effect of these acids was permanent as growth did not occur when inhibited cultures were diluted to a non-inhi- bitory concentration. No adaptat ion to the fat ty acids toxicity was observed by pre-exposing the cultures to non-inhibitory concentrations and the inhibitory re- sponse was the same as for cultures not pre-exposed to the fat ty acids. Oleate was less inhibitory when added as a neutral oil in the form of the glycerol ester. This indi- cates that it is the free fat ty acid that influences the bac- terial activity.

rial growth and biogas production (Angelidaki et al. 1990).

LCFA can be inhibitory at low concentrations (Nie- man 1954; Demeyer and Henderickx 1967; Henderson 1973; Hanaki et al. 1981; Sheu and Freese 1972; Roy et al. 1985; Rinzema et al. 1989; Koster and Cramer 1987). The inhibitory effect of LCFA is used for food preserva- tion (Kabara 1983) and LCFA have been proposed as dietary supplements for ruminants to suppress methane production (Blaxter and Czerkawski 1966).

In the present paper we compare the effects of glycer- ol trioleate (GTO) to the effect of its free fatty acid, oleate (18: 1), on anaerobic thermophilic digestion. Fur- thermore, the effects of oleate and stearate (18:0) on different steps of thermophilic anaerobic degradation are examined.

Introduction

Anaerobic digestion is widely used for waste treatment. In Denmark, large full-scale biogas plants have been built to treat manure in addition to waste f rom slaugh- terhouses and food industries. Some of these additives have a high lipid content and thus a large potential for biogas production, making the biogas plants more eco- nomically advantageous.

During anaerobic digestion, lipids are first hydro- lysed to glycerol and long-chain fatty acids (LCFA). This step proceeds easily and fast (Hanaki et al. 1981). The resulting LCFA are further degraded to acetate and hydrogen via/~-oxidation (Weng and Jeris 1976). Ace- tate and hydrogen are then finally converted to biogas (Bryant 1979). Over 80°70 of added lipid is degraded to biogas after an adaptat ion period of 55 days when ad- ded to biogas digestors fed with cattle waste (Angelidaki et al. 1990). However, oil can cause inhibition of bacte-

Correspondence to: B. K. Ahring

Materials and methods

Medium and culture conditions. Experiments were performed in 118 ml vials containing 40 ml medium (Angelidaki et al. 1990) sup- plemented with 0.1 g/1 of yeast extract. The vials were closed with butyl rubber stoppers and autoclaved. In all experiments the vials were inoculated with 5°70 (v/v) digested cattle manure. The pH was measured at the beginning and at the end of all experiments and it varied within a range of 6.9-7.2. The vials were incubated at 55°C and all experiments were performed in triplicate.

GTO/oleate experiment. GTO or oleate (5 and 15 mM respective- ly, corresponding to 4.4 and 4.2g/1) were added to the corre- sponding vials and afterwards the mixtures were emulsified by ultrasonification. The medium was inoculated with thermophilic digested manure from a lab-scale reactor digesting cattle manure with approx. 207o (v/v) olive oil added.

The theoretical methane yield of GTO, based on the Buswell equation (Buswell and Neave 1930), is 40 mol CH4/m01 GTO. For calculations of methane production relative to the theoretical maximum, the production from controls without GTO addition was subtracted.

Oleate/stearate experiment. The effect of various concentrations of sodium oleate (0.0-3.0 g/l) or sodium stearate (0.0-1.5 g/l) on biogas production from different carbon sources was tested in batch experiments. The vials were inoculated from a lab-scale di-

gestor fed with cattle waste for 3 months. The effects of oleate and stearate were tested on the following initial carbon sources: 2.46 g/1 (30 raM) sodium acetate, 1.44 g/1 (15 mM) sodium pro- pionate, or 0.55 g/1 (5 ms ) sodium butyrate in addition to the ino- culum. Vials without added substrate served as controls.

When methane production from the carbon source was in- creasing exponentially, sodium oleate or sodium stearate was ad- ded. For vials with butyrate as the initial carbon source, oleate was added when approx, half of the butyrate was degraded. If no oleate or stearate was added, an equal volume of anaerobic dis- tilled water was added.

In order to investigate if adaptation to the LCFA had occurred in vials that had received oleate or stearate at concentrations per- mitting bacterial growth, LCFA and short-chain fatty acids (SCFA) were added again to the same vials after depletion of the initial dose. After 55 days, when activity no longer existed in vials that had initially received 0.2 g/1 of oleate, a new dose of 0.2 g/1 of oleate and acetate was added in order to test if the conversion of oleate to CH4 and CO2 would proceed faster. To the vials that had received 0.1 and 0.3 g/1 of oleate, a higher concentration, 0.5 g/l, was added to test for a possibly higher inhibitory level due to adaptation. For the stearate series, the following concentrations were used (initial/addition): 0.0/0.0, 0.1/1.5 and 0.5/1.5 g/1.

To test whether the inhibition was irreversible or temporary, samples from the vials with 0.5, 1.0, and 1.5 g/1 of oleate were diluted to a concentration of 0.2 g/1 of oleate with medium con- taining 2.46 g/1 (30 mM) of acetate. This concentration would per- mit biogas production if the bacterial culture had not been dam- aged.

Analytical methods. Methane and volatile fatty acids (VFA) were measured by gas chromatography as previously described (Sorens- en et al. 1991). Samples for oil analysis were taken after vigorous shaking of the vials. The oil was extracted using the method of Bligh and Dyer (1959). The neutral oil was separated from the free fatty acids on a silica gel thin-layer chromatography (TLC) plate with n-hexane :ethyl ether :acetic acid (80:20: 1) as eluting solu- tion. The TLC spots were transferred to methylation tubes and an internal standard was added. After saponification with NaOH and methylation with BF3" CH3OH, LCFA were quantified on a Hew- lett Packard gas chromatograph using a flame ionization detector and an SP-2330 fused silica capillary column.

Results

Experiments with GTO/oleate addition

Low methane production was found for the vials with G T O added , and the 20 mM methane p r o d u c e d af te r 25 days of inoculation accounted only for 10% of the me- thane that theoretically could be produced in the vials. Accordingly, VFA production reached a concentration of only 10 mM after approx. 20 days and no further in- crease was f o u n d (Fig. 1).

G T O was hyd ro lysed to oleate at a low rate . The con- cen t ra t ion decreased to app rox . 3 mM (2.6 g / l ) dur ing the first 25 days af te r which the concen t ra t ion s tayed cons tan t for the rest o f the exper imenta l per iod . The co r r e spond ing concen t r a t i on o f oleate reached 6 mM (1.7 g / l ) a f te r 25 days and s tayed at this value (Fig. 1). Oleate was the ma in L C F A p r o d u c e d f rom the hydro ly - sis o f GTO. Traces o f s teara te (18 :0) and pa lmi t a t e (16:0) were also detec ted .

Vials with oleate a d d e d were inh ib i ted f rom the be- g inning and less me thane and V F A were p r o d u c e d t han in con t ro l s where the i nocu lum served as the only car-

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~ 6 . , _ - - ~ ~ ~' t- J E

.io 4 ~5 ~

8 3 ' 10 ~

~ 2 ~

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~- 10 20 30 40 50 600 0

Time (days)

Fig. 1. Degradation of glycerol trio]eate (GTO): ~, GTO; ~, oleate; ~, volatile fatty acids (VFA) and --, methane

bon source. The oleate concentration was constant throughout the experimental period (data not shown).

Experiments with oleate/stearate addition

Figure 2 shows me thane p r o d u c t i o n toge ther with a t ime course of acetate, propionate or butyrate degradation, without oleate added. All substrates were converted stoi- chiometrically to methane in accordance with the fol- lowing equations (the contribution from the inoculum was subtracted): 1 mol acetate to 1 mol methane; 1 tool propionate to 7/4 mol methane; and 1 mol butyrate to 5/2 mol methane. When oleate was added at a concen- tration of 0.5 g/1 or more, both methane production and V F A deg rada t i on s topped immed ia t e ly (Fig. 3). No ac-

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30

~ 20

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~ o ~ 4o

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0 4 8 12 162O24 0 4 8 12162O24 Time (days)

Fig. 2a-d. Degradation of short-chain fatty acids in vials without oleate addition: O, methane ; . , acetate; + , propionate; ~, buty- rate. a Control vials without additional carbon source, b With acetate, c With propionate, d With butyrate

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Fig. 3a-d. Degradation of short-chain fatty acids in vials with 0.5 g/1 oleate added: for symbols, see Fig. 2. a Control vials with- out additional carbon source, b With acetate, c With propionate. d With butyrate. Arrows mark the point of oleate addition

60- a

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o

00 - 20 4'0 6'0 Time (days)

Fig. 4. a Methane production from acetate (30 mM) as initial car- bon source, b Acetate degradation. Oleate was added in different amounts at day marked with an arrow: ~, 0.0 g/l; +, 0.1 g/l; ~, 0.2 g/l; O, 0.3 g/l; ×, 0.5 g/l; T, 1.0 g/1

cumulation of acetate or other VFA was observed, indi- cating that the oleate added was not degraded.

Addition of oleate at concentrations f rom 0.1 to 0.3 g/1 gradually increased the lag phase for methane production f rom acetate (Fig. 4a). Measurements of VFA concentrations in vials where methane production was totally inhibited showed that the acetate concentra- tion was nearly constant (Fig. 4b). The concentrations of the other VFA were lower than 0.1 mM during all measurements (data not shown).

Addition of a higher concentration of oleate and the same concentration of acetate after depletion of the ini- tial dose showed that the inhibitory level of oleate was still the same (Fig. 5a). Although an increased biomass

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a

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b

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54 58 62 66 70 74 78 82 Time (days)

Fig. 5. Methane production after a a second addition of oleate and acetate, and b a second addition of acetate and dilution of the cultures to a non-inhibitory oleate concentration. Symbols as be- fore/after concentrations of oleate in g/l: a , , 0.0/0.0; I,, 0.2/ 0.2; +, 0.1/0.5; O, 0.3/0.5; b l , 0.0/0.0; ~,, 0.5/0.2; +, 1.0/ 0.2

concentration would be expected after the initial experi- ment, 0.5 g/1 of oleate again inhibited biogas produc- tion. No lag phase was found for the control vials with- out oleate added while vials with 0.2 g/1 of oleate added showed a lag phase of about 10 days, approx, the same lag phase as after the first addition of oleate.

Dilution of vials where growth had stopped due to addition of oleate in concentrations of 0.5 and 1.0 g/l , to an oleate concentration of 0.2 g/l , did not restore bacterial activity, and no methane production was ob- served f rom the diluted vials even after prolonged incu- bation (Fig. 5b). Control vials exhibited the same me- thane production rate after a new addition of acetate.

Addition of stearate caused inhibition at concentra- tions higher than 0.3 g/1 with all carbon sources tested, inoculum alone, acetate, propionate and butyrate (Fig. 6). An increased lag phase of varying length, f rom 5 to 30 days, was found with 0.5 g/1 of stearate added with all carbon sources. No methane production was found with 1.0g/1 or more added. Measurements of VFA showed, as with the oleate experiment, no further de- gradation of VFA in vials where stearate was added at concentrations of 1.0 g/1 or more (data not shown).

D i s c u s s i o n

The present experiments showed that relatively low con- centrations of the free LCFA oleate and stearate inhi- bited the degradation of acetate, propionate and buty- rate in biogas reactors fed manure. For both LCFA, the toxic effect was irreversible as activity could not be res- tored by dilution. The toxic effect of oleate was slightly higher than that of stearate, with initial inhibitorY con- centrations of 0.1-0.2 g/1 for oleate and approx. 0.5 g/1 for stearate. These results are in agreement with those of

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d

!0 20 40 60 Time (days)

Fig. 6. Methane production from a organic material in the inocu- lure as the initial carbon source; b acetate (30 mM); C propionate (15 mM); d butyrate (5 mM). Additions of different amounts of stearate are marked with arrows: , , 0.0 g/l; +, 0.1 g/l; ~, 0.3 g/ 1; O, 0.5 g/l; , , 1.0 g/l; x , 1.5 g/1

Galbraith et al. (1971), who found that oleate was the most inhibitory of ten fatty acids tested against pure cul- tures.

Both oleate and stearate affected growth by increas- ing the lag phase, which implies that a shock load of LCFA can make a biogas reactor inactive for longer pe- riods. In the experiment with GTO (Fig. 1), oil was slowly degraded over a period of 25 days, after which degradation ceased at a relatively low level. At the same time the concentration of the free fatty acid oleate in- creased to 6 mM (1.7 g/l) by day 25. The s a m e experi- ment with ol@ate showed, however, immediate and total inhibition. These results indicated that the free LCFA was the toxic component of the lipid.

The delayed inhibitory effect of GTO may be due to gradual accumulation of inhibitory concentrations of oleate f rom the hydrolysis of GTO. Rapid GTO hydro- lysis and relatively slower oleate degradation would re- sult in the accumulation of free oleate and inhibition. This is in accordance with earlier investigations with me- thane-producing bacteria (Prins et a1.\1972) where the free carboxyl group of the fat ty acids was required for toxicity. This suggests that the response of a biogas process to addition of neutral lipids may depend upon the degree of adaptat ion to lipids, whereas' the addition of free LCFA above a certain concentration may result in failure of the process. Adaptat ion to oil may be due to development of an acetogenic bacterial population that can degrade LCFA, as they are released f rom the neutral oil by hydrolysis at a rate adequate to prevent accumulation to inhibitory concentrations.

Henderson (1973) explained the toxic effect of free LCFA as surface-active fatty acids adhering to the bac- terial cell wall, thus impeding the passage of essential nutrients through the membrane. I f this is the mecha- nism of inhibition, the toxic effect should depend on the

ratio between cell mass and LCFA. This would imply that after successful degradation of some SCFA and LCFA, the bacterial population should be able to consume a larger amount, as the bacterial biomass in the system would have increased during the consumption of the first dose. In the present experiments, however, increased tolerance was not observed after depletion of the original dose (Fig. 5a). The same effect was observed by Koster and Cramer (1986) and they concluded that the toxic effect is concentration-dependent, which is supported by our results.

For operation of large-scale biogas plants treating manure and organic industrial wastes, as practised for some years in Denmark, these results have important practical implications: organic industrial wastes often contain high amounts of lipids. Our results indicate that such wastes should be introduced gradually and fed con- tinuously to biogas reactors to allow adaption and main- tenance of a bacterial population capable of LCFA de- gradation and to prevent accumulation of high concen- trations of LCFA.

Acknowledgements. This work was supported by grants from The Danish Energy Council, no. 1383/89-1, and The Nordic Minister- ial Council.

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