degradation of volatile fatty acids in highly efficient anaerobic digestion

Degradation of volatile fatty acids in highly e�cientanaerobic digestion

Qunhui Wanga, Masaaki Kuninobub, Hiroaki I. Ogawaa, Yasuhiko Katoa,*aDepartment of Applied Chemistry, Faculty of Engineering, Kyushu Institute of Technology, Fukuoka, 804-8550, Japan

bKawasaki Giken Engineering and Construction Co. Ltd, 1-Chome 22-11, Mukaino, Minami-ku, Fukuoka, 815-0035, Japan

Received 2 September 1997; received in revised form 18 January 1999; accepted 20 January 1999

Abstract

To improve the e�ciency of anaerobic digestion, we examined the e�ects of C2±C6 volatile fatty acids (VFAs) onmethane fermentation, as well as the behavior of VFAs in anaerobic digestion. The VFA concentrations and

methane production in anaerobic digestion were increased by pretreatment of waste activated sludge (WAS), such asultrasonic disintegration, thermal and freezing treatments. The major intermediate products of anaerobic digestionfor untreated and pretreated WAS, such as acetate, propionate, isobutyrate, butyrate, isovalerate, valerate,

isocaproate and caproate, were used as substrates and the anaerobic degradation of these was carried out under thesame conditions. It was found that decomposition rates of the VFAs (C2±C6) with a straight chain (normal form)were greater than those of their respective isomers with a branched chain (iso form). It was shown that the

decomposition rates of the iso and normal forms of butyrate were greater than those of valerate and caproate. Thiswas caused by the isomerization between butyrate and isobutyrate which occurred during the digestion process.Anaerobic bacteria in digested sludge converted butyrate to isobutyrate and vice versa by migration of the carboxylgroup to the adjacent carbon atom. In addition, inhibition of degradation of the VFAs by acetate in a digester was

also examined. # 1999 Elsevier Science Ltd. All rights reserved.

Keywords: Anaerobic digestion; Methane generation; Solubilization; Pretreatment; Volatile fatty acid; Waste activated sludge

1. Introduction

Anaerobic digestion is one of the major bio-logical waste treatment processes in use today.This process has been popular in the waste treat-ment ®eld because it has many advantages such

as high treatment e�ciency and methane-produ-

cing ability. In the anaerobic digestion of waste

activated sludge (WAS), complex organic ma-

terials are ®rst hydrolyzed and fermented by

rapidly growing and pH-insensitive acidogenic

bacteria into volatile fatty acids (VFAs) [1,2].

The VFAs are then oxidized by slowly growing

acetogenic bacteria into acetate (HAc), molecular

Biomass and Bioenergy 16 (1999) 407±416

0961-9534/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.

PII: S0961-9534(99 )00016-1

www.elsevier.com/locate/biombioe

* Corresponding author.

hydrogen, and carbon dioxide that are suitable assubstrates for the methanogenic bacteria [3±5].

Considerable attention has been focused on therelationships between VFAs concentration andanaerobic fermenter performance [6,7]. It isknown that VFAs are important intermediarycompounds in the metabolic pathway of methanefermentation and cause microbial stress if presentin high concentrations, resulting in a decrease ofpH, and ultimately leading to failure of the diges-ter. Therefore, the concentration of VFAs is animportant consideration for good performance ofa digester. That is, it is necessary to investigatethe optimum conditions and e�ciencies of diges-ters by examining VFAs.

It is widely known and accepted that theoccurrence of VFAs in anaerobic sludgedecreases as the chain length increases. In ad-dition, the presence of formate is usually quitelimited due to its rapid conversion to other com-pounds (e.g. methane and carbon dioxide). Thus,it was reasonable to limit our attention to theC2±C6 VFAs.

In this study, major intermediate products ofanaerobic digestion for untreated and pretreatedWAS, i.e. HAc, propionate (HPr), isobutyrate (i-HBu), butyrate (n-HBu), isovalerate (i-HVa),valerate (n-HVa), isocaproate (i-HCa) and capro-ate (n-HCa) were used as carbon sources in themethanogenic experiments.

The purpose of this investigation was toenhance the e�ciency of anaerobic digestion, toevaluate the e�ect of C2±C6 VFAs on themethane fermentation, and to examine the beha-vior of VFAs in anaerobic digestion.

2. Materials and methods

2.1. WAS and pretreatment

The seed sludge (digested sludge) was collectedfrom a mesophilic sewage sludge anaerobic diges-ter in Hiagari, a sewage disposal plant, located inKitakyushu, Japan, and was acclimatized to thesubstrate for 2 months at 35218C. Waste accu-mulated sludge (WAS) thickened by ¯otation wasalso obtained from the same plant. Percentage of

total solids and volatile solids in the sludge were3.3±4.0% and 2.6±3.1%, respectively.

The conditions for pretreatment of WAS wereas follows:

1. ultrasonic disintegration was performed byKubota Insonator at 9 kHz, 200 W

2. thermal treatment was conducted in a Tomyautoclave at 1208C and in hot water bath at608C

3. freezing at ÿ108C.

2.2. Anaerobic digesters and analytical methods

The digester was a 500 ml glass vesselequipped with a sludge sampling port and a gassampling port [8]. Untreated and pretreatedWAS or individual VFA were added to digesterscontaining seed sludge, respectively. After repla-cing the interior air by helium for 5 min, thedigester was set into a shake thermostat kept at36218C to start digestion.

Total solid and volatile solid used to expressbiomass concentrations were measured accordingto the procedure in Standard Methods [9]. Gasanalyses were carried out with gas chromato-graph equipped with thermal conductivitydetector. VFAs were analyzed with gas chro-matograph having ¯ame ionization detector.

The digester liquor was sampled immediatelyby Terumo syringe. These samples were centri-fuged at 13000 rpm for 15 min and supernatantliquors were used to measure VFAs by aShimadzu GC-14A gas chromatography(Shimadzu Seisakusho Ltd, Kyoto, Japan). A3.2 m � 3.2 mm i.d. glass column was packedwith Unisole F-200 30/60. The column tempera-ture was 1308C and the injector port temperaturewas 2008C. The nitrogen ¯ow rate was 30 ml/min. Air and hydrogen pressures were 1.0 kg/cm2

and 0.6 kg/cm2, respectively. 8 set of standardsolutions of HAc, HPr, i-HBu ((CH3)2CHCOOH),n-HBu (CH3(CH2)2-COOH), i-HVa ((CH3)2CH-CH2COOH), n-HVa (CH3(CH2)3COOH),i-HCa ((CH3)2CH(CH2)2COOH) and n-HCa(CH3(CH2)4COOH) were prepared, each contain-ing 100, 300, 500, 700, 1000 mg/l, respectively.

Q. Wang et al. / Biomass and Bioenergy 16 (1999) 407±416408

Fig. 1. Change of the total and individual VFA concentrations and methane generation during anaerobic digestion with various

pretreated WAS: w, HAc; *, HPr; r, i-HBu; R, n-HBu; q, i-HVa; Q, n-HVa; �, t-VFA; W, CH4.

Q. Wang et al. / Biomass and Bioenergy 16 (1999) 407±416 409

3. Results and discussion

3.1. Relationship between VFAs concentration andmethane production by various pretreatments

WAS with and without pretreatment were re-spectively put into anaerobic digesters and weredigested under conditions described inExperimental. Fig. 1 shows change in VFAs con-centration and methane production rate duringdigestion (trace amount of caproate and iso-caproate were also detected, data not shown). Incase of pretreated WAS, generation of methaneincreased drastically during initial 2 days. Itreached a maximum value on 2nd day. The maxi-mal rate of methane generation on ultrasonic dis-integration, heating at 1208C, at 608C andfreezing treatment were 766, 737, 616, and560 ml/l�day, respectively. In contrast, untreatedWAS showed no signi®cant variation in 1st and2nd days readings. The maximal rate of methanegeneration on 1st day was 383 ml/l�day.

Based on the results of anaerobic digestionusing WAS as carbon source in our experiment,the concentration of individual VFA accumulatedin digestion process was found to be in thefollowing order: HAc>HPr>i-HVa>i-HBu>

(n-HVa, n-HBu)>i-HCa>n-HCa. This orderseemed to be independent of WAS whether itwas pretreated or not. To explain this order,characteristics of individual VFA degradationwere investigated.

3.2. Degradation of various VFAs

Experimental set up of VFAs degradation wassimilar to that of anaerobic digestion usingWAS. The major intermediate products of an-aerobic digestion such as HAc, HPr, i-HBu, n-HBu, i-HVa, n-HVa, i-HCa and n-HCa wereused as the carbon source for seed sludge in 8digesters respectively. They had an initial concen-tration of about 700 mg/l each. The results of in-dividual VFA degradation are presented in Fig.2.

Under the same conditions, VFAs degradationrates were classi®ed into four groups; the orderbeing n-HBu>(HAc, n-HCa, n-HVa, i-HBu)>(HPr, i-HVa)>i-HCa. Decomposition

Fig. 2. Degradation curves for individual VFA during anaero-

bic digestion (with seed sludge well acclimatized): w, HAc; *,

HPr; r, i-HBu; R, n-HBu; q, i-HVa; Q, n-HVa; r, i-HCa;

W, n-HCa.

Fig. 3. The determination of rate constant for VFA degra-

dation: w, HAc; *, HPr; r, i-HBu; R, n-HBu; q, i-HVa; Q,

n-HVa; r, i-HCa; W, n-HCa.


rates of normal-form of VFAs (C4±C6) weregreater than their respective iso form. Moreover,decomposition rates of iso- and normal-forms ofbutyrate were greater than those of valerate andcaproate, respectively.

VFAs degradation rate constants were deter-mined (Fig. 3). All VFA degradation reactionsobeyed ®rst order kinetic.

3.3. Reciprocal isomerization of i-HBu and n-HBu

To elucidate the reason why degradation rateof n-HBu was the highest compared to that ofother VFAs and degradation rate of i-HBu washigher than that of i-HVa and i-HCa (iso-formsof valerate and caproate), anaerobic degradationof n-HBu and i-HBu as substrate was examinedin detail. The substrates and products weremeasured (Fig. 4). As the results, n-HBu andHAc were produced with the decrease in i-HBu

[Fig. 4(a)] and reversely i-HBu and HAc wereproduced with decreasing n-HBu [Fig. 4(b)].

The production of HAc is a result of n-HBuand i-HBu degradations. However, occurrence ofn-HBu in i-HBu digestion and vice versa demon-strated the isomerization between n-HBu and i-HBu during the anaerobic digestion. The ratio ofconversion of n-HBu into i-HBu seemed to begreater than that of the reverse conversion (Fig.4). This result almost agreed with that reportedby Matthies and Schink [10], where they used ananaerobic bacterium strain WoG13 which fer-mented glutamate. But this could not be saidwith full con®dence as decreasing rate of n-HBuproduced from i-HBu was so fast that it couldnot be detected for such a small concentration.

To clarify the reciprocal isomerization betweeni-HBu and n-HBu, n-HBu and i-HBu anaerobicdegradation was further examined with no

Fig. 4. Time course of isobutyrate (a) and butyrate (b) degra-

dation (with seed sludge well acclimatized): (a): w, HAc; r, i-

HBu; R, n-HBu; (b): w, HAc; r, i-HBu; R, n-HBu.

Fig. 5. Time course of isobutyrate (a) and butyrate (b) degra-

dation (with no acclimatized seed sludge having low activity):

(a): w, HAc; r, i-HBu; R, n-HBu; (b): w, HAc; r, i-HBu;

R, n-HBu.


acclimatized seed sludge having low activity.Because the degradation rates of both substratesand products were slow, reciprocal isomerizationof i-HBu and HBu were con®rmed (Fig. 5).

The isomerization of n-HBu to i-HBu and thereverse were reported for the ®rst time withstrictly anaerobic bacterium strain WoG13recently isolated by Matthies and Schink [10].13C-NMR experiments proved that this isomeri-zation was accomplished by migration of the car-boxyl group to the adjacent carbon atom.Isomerase activity depended strictly on the pre-sence of coenzyme B12 (Fig. 6).

In the present study, reciprocal isomerizationbeteen n-HBu and i-HBu was observed under an-aerobic conditions when seed sludge was used. Itwas suggested that bacterium with the enzymesimilar to anaerobic bacterium strain WoG13and its enzyme existed in the seed sludge mighthave caused the isomerization.

Because of isomerization and b-oxidation reac-tion, the decreasing rate of n-HBu concentrationwas larger than that of HAc (Fig. 2). But thetime necessary for complete degradation of thatproduct (i.e. HAc and i-HBu from n-HBu) waslonger than that of HAc (data were omitted).

3.4. Degradation of i-HVa and n-HVa

Similarly, n-HVa and i-HVa were added todigested sludge to conduct anaerobic degradationexperiments. Reciprocal isomerization wasobserved between n-HBu and i-HBu, but in caseof n-HVa and i-HVa, isomerization was notobserved, even when digestion sludge having lowactivity was used. i-HVa molecule is not formedirrespective of shifting of the carboxyl group inn-HVa molecule to the next carbon atom,because chemical formula of i-HVa being(CH3)2CHCH2COOH. Only HAc and HPr wereproduced with the decrease of n-HVa. This resultsuggested that the degradation of n-HVaoccurred only via b-oxidation [Fig. 7(b)].

If the degradation route of i-HVa was also b-oxidation, then HAc and acetone (CH3COCH3)or isopropyl alcohol (CH3CH(OH)CH3) wouldalso have been produced. But only HAc wasdetected [Fig. 7(a)]. There could have been a

Fig. 6. Scheme of the rearrangement of [3-13C] butyrate to

[2-13C] isobutyrate catalyzed by cell suspensions of strain

WoG13. The labelled C atom is marked by a star [10].

Fig. 7. Time course of isovalerate (a) and valerate (b) degra-

dation (with seed sludge well acclimatized): (a): w, HAc; q, i-

HVa; (b): w, HAc; *, HPr; Q, n-HVa.


possibility that other substances could not bedetected by our assay method. Therefore degra-dation route of i-HVa could not be perfectly elu-cidated.

Degradation of long chain fatty acids of odd-numbered carbon atoms like n-HVa begins from

the carboxyl end, HAc and HPr being the ®nalproducts formed by b-oxidation. The n-HVa de-composition rate was not slow, but HPr (formedfrom n-HVa) decomposition rate was slower(Figs. 2 and 8). This could be possible as HPrdegradation route involves quite unusual enzymereactions and its oxidation is thermodynamicallyunfavorable in anaerobic digestion [11±14].

3.5. Degradation of i-HCa and n-HCa

The production of i-HBu, n-HBu and HAc ac-companied the degradation of n-HCa [Fig. 9(b)].This suggested that n-HCa was converted intoHAc and n-HBu by b-oxidation, followed by iso-merization of n-HBu into i-HBu.

In case the degradation route of i-HCa was b-oxidation, then with decrease in concentration,HAc, i-HBu and n-HBu (by isomerization of i-HBu) should have been produced. But very smallamounts of i-HBu and n-HBu were detected(Fig. 9(a)) because degradation rate of i-HCawas very slow leading to immediate degradationof i-HBu and n-HBu.

The results mentioned above indicated that n-HBu, n-HVa and n-HCa were rapidly degradedby b-oxidation. Furthermore, n-HBu was con-verted by isomerization, hence increasing itsdegradation rate.

Based on the results of VFAs degradation(Figs. 2 and 9), it was possible to explain theresults of anaerobic digestion with untreated andpretreated WAS (Fig. 1). Decomposition rate ofHAc was relatively faster than the other VFAs(excluding n-HBu, Fig. 2), but in case of anaero-bic digestion using WAS, concentration of accu-mulated HAc was the highest. This was due tothe fact that most of the organic constituents inthe WAS were converted into VFAs prior tobeing converted into methane. Long chain fattyacids, having even or odd-numbered carbonatoms, produced HAc by b-oxidation. The HPrconcentration was also found to be high (Fig. 1),as it was ®nally produced by b-oxidation fromlong chain fatty acids of odd-numbered carbonatoms like n-HVa but its decomposition rate wascomparatively slower.

On the other hand, since the decomposition

Fig. 8. Time course of propionate degradation (with seed

sludge well acclimatized): w, HAc; *, HPr.

Fig. 9. Time course of isocaproate (a) and caproate (b) degra-

dation (with seed sludge well acclimatized): (a): w, HAc; r, i-

HBu; R, n-HBu; r, i-HCa. (b): w, HAc; r, i-HBu; R, n-

HBu; W, n-HCa.


rates of normal-form of VFAs were greater thanthose of their respective iso-forms, n-HBu, n-HVa and n-HCa were not accumulated in diges-tion using WAS. The decomposition rate of i-HVa was slow, so it followed accumulation con-centration of HAc and HPr, in magnitude (Fig.1). The decomposition rate of i-HCa was theslowest, but it did not accumulate due to lowproduction in normal digestion using WAS.

3.6. VFA relationships in anaerobic digester

HAc was produced when concentration of i-HBu, n-HBu, i-HVa, n-HVa, i-HCa, n-HCa orHPr decreased (Figs. 4 and 9). Hence HAc wasparticularly important product being the precur-sor of methane producing reaction.

Reactions and standard Gibbs free energy(pH 7.0, at 258C, substrates and products are

1 mol/l concentration, or 1 atm) of some VFAsdegradations are presented in Table 1. HPr andn-HBu degradation reactions (ii, iii) would nothave taken place from the viewpoint of positivestandard Gibbs free energy. However, whetherreactions occurred or not in practice would bedetermined by substrates and products concen-tration. If reactions (ii, iii) and the reaction con-suming H2 (M) were conjugated, the reactionwould become feasible thermodynamically(ii+M, iii+M). Therefore, if HPr and n-HBuwere expected to degrade, HAc concentrationand H2 partial pressure should have been main-tained at a lower level, which could have beenpossible in digesters, by methanogenic consump-tion.

To clarify the correlation among VFAs and toinvestigate the inhibition of HAc on HPr and n-HBu degradation, two sets of experiments wereperformed by injecting di�erent concentration ofHAc and HPr into the digesters as shown inTable 2.

Table 1

Standard free energy and equation of each fatty acid degradation

Reaction DG8 ' (kJ) at 258C

(i) CH3COOÿ+H2O4HCOÿ3+CH4 ÿ31.0(ii) CH3CH2COOÿ+3H2O4CH3COOÿ +HCO3

ÿ+H++3H2 +76.1

(iii) CH3CH2CH2COOÿ+2H2O4 2CH3COOÿ+H++2H2 +48.1

(iv) CH3CH2CH2CH2COOÿ+2H2O4 CH3COOÿ+CH3CH2COOÿ +H++2H2 +25.1

(M) 4H2+HCOÿ3+H+4CH4+3H2O ÿ135.6(ii+M) 4CH3CH2COOÿ+3H2O4 4CH3COOÿ+HCOÿ3+H++3CH4 ÿ102.4(iii+M) 2CH3CH2CH2COOÿ+HCOÿ3+H2 O44CH3COOÿ+H++CH4 ÿ39.4

Fig. 10. Degradation curves of HPr for runs made with di�er-

ent mixtures of HAc and HPr: *, Run 1 (HAc=0 mg/l); q,

Run 2 (HAc=760 mg/l); R, Run 3 (HAc=1400 mg/l); r,

Run 4 (HAc=2000 mg/l); w, Run 5 (HAc=2700 mg/l).

Table 2

Initial acid concentrations of HAc and HPr in experimental

runs 1±8

Condition HAci HPri Run

HPriV750 mg/l 0 734 1

760 760 2

1400 710 3

2000 759 4

2700 725 5

HAciV750 mg/l 782 0 6

764 760 7

752 2200 8


In the 1st set, approximately the same concen-tration of HPr and increasing concentration ofHAc were injected. HPr was added about 750 mg/l in each digester and the concentration of HAcadded (HAci) varied between 0 and 2700 mg/l. Inthe second set of runs the procedure wasreversed, HAc concentration was kept constantabout 750 mg/l and HPr concentration varied inthe range of 0±2200 mg/l.

The degradation curves of HPr for runs 1±5are shown in Fig. 10. HAc was absent in the 1strun, but HPr degradation proceeded at a fastrate with 97.3% reduction in HPri within 64 h.760 mg/l HAc was added in the 2nd run andHPr degradation rate was almost the same as inthe 1st run. More than 1400 mg/l of HAc wasadded in the 3rd±5th run. From Fig. 10 it wasapparent that HPr degradation was inhibited.The time required for achieving over 90% ofdegradation increased to 76 h in the 4th run(HAci=2000 mg/l) and to 88 h in the 5th run(HAci=2700 mg/l). In other words, for the sameconcentration of HPr, lower the HAc concen-tration, faster was HPr degradation. Within 64 hreductions of HPr were 97.3% (runs 1 and 2),86.5% (run 3), 74.2% (run 4) and 63.0% (run 5),respectively. These results demonstrated that pro-duct inhibition depended on HAc concentrationin the reactor. When HAc level was higher than1400 mg/l, HPr degradation was inhibited. Theseresults agreed approximately with those ofHeijnen [15], Gorris [16], Fukuzaki [17], Mawson

[18] and Alonso [19]. Few of these authorsreported product inhibition occurred even whenHAc concentrations were lower than 480±600 mg/l, thus our results were only qualitatively similar.

About 750 mg/l HAc was added in runs 6±8,while HPri concentration was varied between 0and 2200 mg/l. Fig. 11 evidently showed at samepH and temperature conditions, HAc degra-dation rate depended only on its own concen-tration in the reactor and was independent of thepresence of HPr.

The relationship between HAc and n-HBu inanaerobic digester was also investigated by inject-ing various concentrations of HAc and n-HBuinto the digesters. HAc in the reactor had practi-cally no in¯uence on the degradation of n-HBu,when concentration of HAc was lower than2000 mg/l (data not shown).

4. Conclusion

The VFAs in anaerobic digestion were pro-duced by pretreatment of WAS such as ultra-sonic disintegration, thermal and freezingtreatments. When their concentrations did notexceed the inhibition levels for methane fermen-tation, they were used e�ciently as good sub-strates by methanogenic bacteria. Therefore boththe rate and volume of methane production wereincreased signi®cantly, as compared with those ofthe untreated WAS digestion.

Under the same conditions, the degradationrates of the VFAs were classi®ed into fourgroups; the order being n-HBu>(HAc, HCa,HVa, i-HBu)>(HPr, i-HVa)>i-HCa. Thus, n-HBu, n-HVa and n-HCa were present only inlimited quantities in anaerobic digestion ofuntreated or pretreated WAS in this investi-gation. All VFA degradation reactions obeyed®rst order kinetics. The degradation rates of i-HBu, i-HVa and i-HCa were slower than thoseof their normal forms, which might have beendue to the structural di�erences between the twoforms.

It was shown that reciprocal isomerizationbetween n-HBu and i-HBu occurred during theanaerobic digestion. The degradation rate of

Fig. 11. Degradation curves of HAc for runs made with

di�erent mixtures of HAc and HPr: *, Run 6 (HPr=0 mg/l);

R, Run 7 (HPr=760 mg/l); Q, Run 8 (HPr=2200 mg/l).


n-HBu was highest as it is degraded not only byb-oxidation but also by isomerization.

HAc was degraded into CH4 and CO2 directlyby methanogens. However, VFAs with morethan 4C chain could not be used directly bymethanogens. These VFAs were converted intoHAc prior to being converted into methane, thusaccumulating HAc to high concentrations in an-aerobic digestion. The HPr concentration wasalso high in anaerobic digestion because it was®nally produced from VFAs of odd-numberedcarbon atoms by b-oxidation, but its decompo-sition rate was comparatively slow.

The inhibition of HPr degradation by HAcdepended on the HAc concentration in the diges-ter. When the HAc concentration was greaterthan 1400 mg/l, the HPr degradation rate mark-edly decreased.

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degradation of volatile fatty acids in highly efficient anaerobic digestion

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