Anaerobic Degradation of Linoleic (Clsa), Oleic (C18:i) and Stearic (Clsa) Acids and their Inhibitory Effects on Acidogens, Acetogens and Methanogens

Jerald David Anthony Lalman

A thesis submitted in conformity with the requirements For the degree of Doctor of Philosophy

Graduate Department of Civil Engineering University of Toronto

OCopyright 2000 by Jerald Anthony David Lalrnan

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Anaerobic Degradation of Linoleic (Cl82), Oleie (Ci8,i) and Stearic (Cis:o) Acids and their Inhibitory Effects on Acidogens, Acetogens and Methanogens

Jerald David Anthony Lalman Doctor of Philosophy

Department of Civil Engineering University of Toronto

2000

Abstract

Effiuents fiom many food processing industries contain fats and oils in addition to

carbohydrates and proteins. Long chain fatty acids (LCFAs), a hydrolysis byproduct of

fats and oils, are difticult to degrade and are inhibitory to anaerobic organisms. These

acids are degraded via f3-oxidation but the compound initiating the mechanism has not

been clearly identified. Although LCFAs inhibit aceticlastic methanogenesis their effects

on acidogenesis, acetogenesis and hydrogenotrophic methanogenesis have not been well

studied.

This study assessed the degradability of linoieic (C 8:?), oleic (Ci 1) and steark (C

acids and determined their inhibitory effects on anaerobic organisms in 160 mL s e m

bottles. Degradation and inhibition studies were conducted using 10, 30, 50 and 100

r n g ~ - ' LCFA. Inhibition studies using glucose, butyrate and acetate (each at 100 rng-~- ' )

and hydrogen (10.1 kPa) investigated the effects of the three LCFAs on acidogens,

acetogens, acet iclastic methanogens and hydrogenotrophic methanogens.

Unsaturated C 18 LCFAs were degraded to shorter chah LCFAs however, no LCFA

byproducts were detected as intermediates during the degradation of stearic (C18:o) acid.

Palmitic and myristic (CI~:o) acids were produced fiom linoleic CI^:^) acid at al1

concentrations examined and in cultures receiving more than 10 rng-~- l oleic (Ci8:l) acid.

In cultures receiving 100 rng-~' ' Linoleic (C 1 a2) acid both oleic (C 1 !) and palrnitoleic

(C 16:1) acids were detected.

Acidogenesis was af5ected by the presence of LCFAs and synergistic inhibitory

effects of al1 three acids on acetogenesis were observed. Hydrogenotrophic

methanogenic inhibition was observed and aceticlastic methanogens were inhibited at al1

LCFA concentrations examined. in cultures fed with linoleic (C 1 g:2) and oleic (C 18: I )

acids, inhibition of acetate methanogenesis was concentration dependent but for cultures

receiving stearic (C ls:o) acid, the effect was independent of concentration.

In comparison to stearic CI^:^) acid, iinoleic (Ci8:2) and oieic (Cla:~) acids were

degraded faster. Therefore, the design of a full-scaie system will depend on the SRT for

the more slowly degrading LCFA. LCFAs affected glucose and butyrate degradation.

Hence, in effluents containing carbohydrate and LCFAs mixtures, the degradation of

carbohydrate monomers wil be afTected. In comparison to oieic (Cls:~) acid, greater

aceticlastic inhibition was observed for cultures receiving linoleic (Clg:2) acid. Thus, it is

recornmended a two-stage process, acetogenic followed by rnethanogenic, be used to

minimize the inhibition.

... I I I

Acknowledgemen ts

This w-ork is dedicated to my mother. Rosaline Lalman, who passed away while

n ~ i t i n g this thesis. My father. Samuel Lalman. has been a great inspiration throughout

my life and 1 n-ish to thank him for instilling into me the meaning of accomplishrnent. 1

would also like to give my special appreciation to Nin for her precious suggestions. her

stronp encouragement at times of difficulty. love and understanding throughout this

u-hole process. Also. thanks to my brother. Edmund Lalman and my sister. Maria

Crutchley for sharing these troubled times in our lives.

I wish to espress my sincere appreciation to my advisor. Dr. David Bagley for his

intelligent supervision. constructive criticisms. inspiration and friendship. My sincere

appreciation estrnds to m). other cornmittee members Dr. Grant Allen. Dr. Don Kirk and

Dr. Brrnr Slecp. n-hose guidance. assistance and friendship are also invaluable. 1 am also

~ratrsful to Dr. Mary Jans Philips and Durga Prasad for their encouragement and ad~vicr. C

1 n.ould like to th& Rajesh Seth. Russell D'Souza and Yale Zheng for sharing their

kno\vledgs and helpful comments.

Financial assistance was providsd by the University of Toronto and the Ontario

Ministr).. of Energy Science and Technology: Singapore-Ontario joint research

programme.

Finall>-. 1 would like to t h d the Department of Civil Engineering for their support

during the four and one-half years of this study.

Table of Contents

Abstract

Acknowledgements

List of Tables

List of Fi, wres

1 . 1 Context

1.2 Research Objectives

1.4 Publications

2.2 Fundamentals of Anaerobic Wastewater Treatment 2.2.1 Hydrolysis 2 - 2 . 2 Acidogenesis 2.3.3 Acetogenesis 2.2 .3 Methanogenesis

2.3 Anaerobic Reactor Technologies Used to Treat Emuents Containing Long Chain Fatty Acids 2.3.1 Introduction 2.3 - 2 LOW Rate Treatment 2.3.3 High Rate Treatment

2.4 Long Chain Fatty Acids 2.4.1 Sources and Treatment 2.4.2 Composition and Structure 2.4.3 Biodegradation of LCFAs 2.4.4 Effects of Hydrogen and Volatile Fatty Acid

A . Hydroger~ B. 1,klafile Fatp Acidr

Page No.

. . 11

5

sii

2.4.5 Inhibitory Effects A . Effecrs on Membrane Fmcîion B. Effecrs on Anaerobic Orgmrisms

2.4.6 Factors AfFecting LCFA Degradation and Inhibitory Eftècts A. Szi bstra~e Molemlar Sb-uctlrre and Conceritratiorr B. Tentperarure Eflects C. Soltrbilzty Efects D. pH EfJecrs E. Coslibsirare ami fiermocjtraniic Ef/ecrs

Summary

MATEMALS AND METEODS

Experimental Plan

Reagents

Batch Reactors 3.3.1 Inoculum Source 3 .3 .2 Operation of Inoculum Reactors

Hydrogen and Methane Measurements

Volatile Fatty Acid (VFA) Measurement

Development of a LCFA Delivery Strategy

Long Chain Fatty Acid (LCFA) Measurement 3.7.1 LCFA Extraction- Method Development 3.7.2 LCFA Extraction- Phase Partitioning Studies 3.7.3 LCFA .4naiysis

Serum Bottle Preparation

Glucose Measurement

Total Suspended Solids (TSS), Volatile Suspended Solids (VSS), Alkalinity and pH hleasurements

BATCH REACTORS

Ex~erimental Results

4.2 Discussion of Results 84

DEGRADATION OF LINOLEIC CI^:^) ACID AND ITS INHIBITORY EFFECTS ON METHANOGENESIS

Experimental Results 5.1 - 1 Linoleic Acid Degradation 5.1.2 Inhibitory Effects of Linoleic (C Acid on

Methanogenesis A. Acetate Degradatiorz B. Hwogen Cor~sumptisrt

i. Hydrogen uptake 1 day after linoleic (C18:2) acid addition

ii. Hydrogen uptake 18 days and 3 5 days after linoleic (C 1 8 . ~ ) acid addition

Discussion of Results 5.2.1 Linoleic Acid Degradation 5 -2.2 Linoleic Acid-Methanogenic Inhibition Studies

Summary 104

DEGRADATION OF OLEIC (Cl~t :~) ACID AND ITS INHIBITORY EFFECTS ON METEIANOGENESIS 105

Experimental Results 10s 6.1 . 1 Oleic (Cls:l) Acid Degradation 105 6.1.2 Inhibitory Effects of Oleic (Cls:l) Acid on Methanogenesis 1 08

A. A ce tare Degrndatiort 1 08 B. Hydrogen Corrs~rmptior~ 110

Discussion of Results 6.2.1 Oleic (C18:l) Acid Degradation 6.2.2 Oleic (C 18:1) Acid-Methanogenic Inhibition Studies

Summary 115

DEGRADATION OF STEARIC (Cls:o) ACCD AND ITS iNHIB ITORY EFFECTS ON METHANOGENESIS

Experimental Results 7.1.1 Stearic (Cls:o) Acid Degradation

7.1.2 Inhibitory Effects of Stearic (Cis:,) Acid on Methanogenesis A. Acetate Degradation B. Hydrogen Consrrmptio?l

7.2 Discussion of Results 7.2.1 Stearic (C18:0) Acid Degradation 7.2.2 Stearic (C ls:o) Acid-Methanogenic Inhibition Studies

7.3 Summary

8.0 COMPARISON OF LCFA DEGRADATION STUDIES

8.1. Products of Linoleic CI^:^), Oleic and Stearic (C18:o) Acids Degradation

8.2 Possible Pathways for Linoleic (Cls:i) Acid Degradation

9.0 INBIBITORY EFFECTS OF LINOLEIC (Cls:r), OLEIC (Cis:,) AND STEARIC (Cls:o) ACIDS ON ACIDOGEKIC, ACETOGENIC AND METHANOGENIC ACTWITI'

9.1 Experimental Results 9.1.1 Acidogenic Inhibitory Effects-Glucose Degradation 9.1 . 2 -4cetogenic Inhib itory Effects-Butyrate Degradation 9. 1.3 Methanogenic Lnhib itory Effects-Hydrogen Consumption

9.2 Discussion of Results 9.2.1 Glucose Degradation 9.2.2 Butyrate Fermentation 9.2.3 Hydrogen Consumption

9.3 Summary

10.0 SUIM31ARY OF METHANOGENIC STUDIES AND LCFA INHLBITION MECBANISM

10.1 Methanogenic Studies 10.1 . 1 Aceticlastic Methanogens 10.1 .1 Hydrogenotrophic Methanogens

10.2 LCFA Inhibition Mechanism

11.0 SUMlMARY AND CONCLUSIONS

11.1 Summary 1 1.1.1 LCFA Degradation 1 1 - 1 2 LCFA Inhibition

A. A cidogem B. Acefogens C. Methmiogerzs

i. Aceticlastic methanogens ii. Hydrogenotrophic methanogens

1 1.2 Conclusions

12.0 ENGINEERING SIGNIFKANCE AND SUGGESTIONS FOR FUTURE RESEARCH

13.0 REFERENCES

Appendix A: LCFA Free Energy Cakulations

Appendix B: Example of LCFA Mass Balance Calculations

Page No.

Table 2.1 : Table 2.2: Table 2.3: Table 2.4: Table 2.5:

Table 2.6

Table 2.7: Table 2.8: Table 2.9: Table 2.1 O: Table 2.1 1 : Table 2.12: Table 2.13 : Table 2.14: Table 2.15: Table 2.16: Table 2.17:

Table 3.1 : Table 3.2:

Table 3.3

Table 3.3:

Table 3.5:

Table 3.6:

Table 3.7: Table 3.8: Table 3.9: Table 5.1 :

Table 5.2:

Table 5.3:

Table 5.4:

Some hydrolytic reactions and fiee energies 10 Some acidogenic reactions and fiee energies 13 Some acetogenic reactions and fiee energies 14 Some methanogenic reactions and fiee energies 15 Design parameters reported for anaerobic reactors treating effluents containing fats and oils Selected properties of linoleic (C ~ g : ~ ) , oleic (C 18: 1 ) and steark (Clg:~) acids Main edible oil categories EdibIe oils containing Iinoleic (Cl*:?) acid EdibIe oils containing oleic (CI 8: 1 ) acid Edible oils containing stearic (C18:o) acid Hydrogenation of linoleic (ClgI2) acid by rumen bacteria LCFA oxidation reactions Organisms associated with LCFA degradation Fatty acids aqueous solubilities and dissociation constants Free energy changes for C 18 LCFAs oxidation to acetate LCF.4s Gibbs free energy of formation values Change in fiee energy of some relevant j3-oxidation and methanogenic reactions 58 Experimental design matrix 60 Linoleic (C I S : ~ ) , oleic and stearic (C I R O ) acids degradation studies 6 1 Acidogenic inhibition studies conducted with linoleic (CIH:~) , oleic (Cl8:l) and stearic (&O) acids Acetogenic inhibition studies conducted with linoleic (C 1 8 : ~ ) . oleic (Ci8:1) and stearic (C lx:o) acids Aceticlastic rnethanogenic inhibition studies conducted with linoleic (C18:I), oieic (C18:I) and steark (Clrt:~) acids Hydrogenotrophic methanogenic inhibition studies conducted with linoieic (C 18:2), oleic (Clg:l) and stearic (Ci8:o) acids LA/O.LVSA interaction studies Basal medium characteristics Dispersing agent solubilities Maximum concentration of byproducts formed during linoleic ( C l 4 acid degradation Initial acetate degradation rates for varying linoleic (C 1 8 : ~ ) acid concentrations First order rate constants for hydrogen removal 1 day afier adding LA First order rate constants for hydrogen removal 18 days af-ler adding LA

Table 5.5 :

Table 6.1 :

Table 6.2:

Table 7.1 : Table 7.2:

Table 7.3:

Table S . 1 :

Table 8.2: Table 8.3: Table 8.4: Table 8.5:

Table 9.1 :

Table 9.2:

Table 9.3:

Table -4.1 : Table A.2:

Table -4.3 : Table A.3: Table AS: Table B. 1 :

First order rate constants for hydrogen removal 35 days afier adding LA Initial acetate degradation rates for varying oleic ( C I R : ~ ) acid concentrations First order rate constants for hydrogen removal in the presence of oleic (C 18: 1) acid Stearic (C1g:O) acid degradation rates Acetate degradation rates for varying stearic (Clg:o) acid concentrations First order rate constants for hydrogen removal in the presence of stearic (Cig:~) acid Byproducts detected in cultures receiving 100 rng .~ ' ' LCFA Byproducts detected in cultures receiving linoleic (C18:2) acid Byproducts detected in cultures receiving oleic (Cl s: 1) acid Initial LCFA degradation rates Free energy values for fi-oxidation of linoleate (&:2), oleate (Cls: ,) and stearate (C ,s:o) to palmitate (C 16:0) Glucose degradation rates for individual and mixed LCFA su bstrates Butyrate degradation rates for i ndividual and mixed LCFA substrates First order rate constants for hydrogen removal in the presence of individual and mixed LCFAs Constants -4 and B for AG; calculations Calculation of free energy of formation for gaseous LCFAs Estimation of constants for Antoine's equation Estimation of Henry's constant Estimation of ionized LCFA free energy of formation Mass balance calculations for cultures receiving 100 mg^“ Linoleic acid at time 17.3 days

List o f Figures

Figure 2.1 : Anaerobic conversion processes Fisure 2.2: Structure of a polysaccharide Figure 2.3: Structure of a tripeptide Figure 2.4: Structure of a triacylglycerol Figure 2.5: Operating HRT and COD ranges for aerobic and

anaerobic biological treatment technologies Figure 2.6: Anaerobic treatment process classification Figure 2.7: The anaerobic contact process flow schematic Figure2.8: Anaerobicfilterflowschematic Figure 2.9: UASB flow schematic Figure 2.10: tinoieic (CIR:~) acid molecular structure Figure 2.1 1 : Oleic (Cis:r) acid molecular structure Fisure S. 1 2. Steark (C acid molecular structure Fisure 2.13 : Proposed model of fatty acid transport in Escherichia

col; Figure 2.14: Postulated mechanism of the interaction of linoleic (C 1 4

acid with active site of A"-cis, A' l-trans-isomerase and the conversion of substrate to cis-9,trans- 1 1 -octadecadienoic acid

Figure 2.15: P-Osidation cycle showinz individual enzyme reactions Figure 2 16: Two possible pathways of linoleic (cis 9, cis 12))

acid P-oxidation Figure 2.1 7: Two possible pathways of oleic (Cl (cis 9)) acid

(3-osidation Fisure 2.18: Possible biohydrogenation pathway for linoleic (C1s:z)

acid by rumen bacteria Figure 2.19: Proposed a-linolenic (Clp:3) acid hydrogenation mechanism Figure 2.20: Free energy vs hydrogen concentration for some

Figure 3.1 : Figure 3 .2 : Figure 3.3: Figure 3.4:

Figure 3.5 :

Figure 3.6: Figure 4.1 : Figure 4.2: Figure 4.3 : Figure 4.4: Fisure 5.1 :

P-oxidation and methanogenic reactions Percent LCFA estracted into chloroforni Percent LCFA extracted into chloroform:rnethanol Percent LCFA extracted into hexane Percent recovery for 5.625 mg^" C6 to C1g in hexane and hexane MTBE: 5 mins shaking at 200 rpm Percent recovery for 5.625 r n g - ~ - ' CU to CIR in hexane and hexane MTBE: 15 mins shaking at 200 rpm Linoleic (C 1 8 : ~ ) acid extraction partitioning studies Reactors schematic Reactor B glucose byproduct degradation profiles Reactor B gas production profiles Acetate profiles for glucose degradation in serum bottles Linoleic (Cl gT2) acid (LA) degradation profiles

s i i

Figure 5 . 2 :

Figure 5.3:

Figure 5.1:

Figure 5.5:

F ipre 5.6:

Figure 5.7: Figure 5.8:

Figure 5.9:

LCFA concentration profiles for cultures receiving 100 mg^" linoleic (C18:2) acid (LA) Palmitic (Cl6:O) acid profiles in cultures receiving linoleic (C I 8:2) acid (LA) Myristic (Cl.4:o) acid profiles in cultures receiving linoleic (CW) acid (LA) Oleic (CIs:l) acid (OA) profiles in cultures receiving linoleic acid (LA) Effect of linoleic (Cig:2) acid (LA) concentration on acetate production Linoleic acid degradation study mass balance Acetic acid removal in the absence of linoleic (CIEJ:~) acid (LA) Acetic acid removal in the presence of linoleic (c18:i)

acid (LA) Fisure 5.10: Hydrosen removal in the absence of linoleic (Clg:~)

acid (LA) Figure 5.1 1 : Hydrogen removal in the presence of linoleic ( C I P : ~ )

acid (LA) Figure 5.12: Hydrogen rernoval in the presence of linoleic (Cin,~) acid

(LA) 18 days after LA addition F e 5 . 3 : Hydrogen removal in the presence of linoleic ( C ~ M ) acid

Figure 5.14

Figure 6.1 :

Figure 6.2:

Figure 6.3 :

Figure 6.4:

Figure 6.5: Figure 6.6:

Figure 6.7:

Figure 6.8:

Figure 6.9:

Figure 7.1 :

Figure 7.2:

(LA) 35 days after LA addition Proposed pathway for formation of palmitoleic (C16.1) acid from linoleic acid Oleic (CIg:l) acid (OA) profiles in cultures receiving oleic (Cis:~) acid Palmitic (CI6:()) acid profiles in cultures receiving oleic (Cr*:[) acid (OA) Myristic (Ci4:o) acid profiles in cultures receiving oleic (Crx:~) acid (0.4) Effect of oleic (C1g:l) acid (0.4) concentration on acetate production Oleic acid (0.4) degadation study mass balance Acetic acid removal in the absence of oleic (Ci8:l) acid (OA) Acetic acid removal in the presence of oleic (Cix:~) acid (OA) Hydrogen removal in the absence of oleic ( C I K : ~ ) acid (OA) Hydrogen removal in the presence of oleic acid (OA) Stearic (C18:0) acid (SA) degradation profiles in cultures with stearic acid Acetate production profiles for cultures fed with stearic (C 1 RO) acid (SA)

F i g r e 7.3 Figure 7.4

Figure 7.5

Figure 7 .6 :

Figure 7.7:

Figure 8.1 : Fipre 9.1 :

Figure 9.2:

Figure 9.3

Figure 9.3:

Figure 9.6:

Figure 10.1 :

Stearic (CIR:O) acid (SA) degradation mass balance Acetic acid removal in the absence of stearic (Cis:o) acid (SA) Acetic acid removal in the presence of stearic (Cig:o) acid (SA) Hydrogen removal in the absence o f stearic (Clg:~) acid (SA) Hydrogen removal in the presence of stearic (CI 8:0)

acid (SA) Proposed C 18 LCFAs degradation pathways Glucose degradation profiles for cultures with and without diethyl ether Glucose degradation profiles for cultures receiving individual and mixed LCFA substrates Butyrate degradation profiles for cultures with and without diethyl ether Butyrate degradation profiles for cultures receiving individual and mixed LCFA substrates Duplicate control cultures hydrogen profiles with and without diethyl ether Hydrogen profiles for cultures receiving individuai and mixed LCFAs Possible LCFA inhibition pathways

1.0 INTRODUCTION

1.1 Context

Many industrial effluents contain fats and oils in addition to proteins and

carbohydrates. The composition of these effluents is variable and is characteristic of a

particular indusu).. Some effluents contain only fats and oils while in others proteins and

carbohydrates are present. Effluents containing fats and oils originale from edible oil

manufacturing industries (Beccari et al.. 1 996: Harndi er al., 1 992). slaughterhouses

(Sayed el ai.. 1984). fried foods (Landine et al.. 1987). livestock fanns (Broughton er ai..

1998: Hobson er al.. 1981) and dairies (Perle er al.. 1995; Backrnan er al.. 1985).

Treating these effluents containing fats and oils is of concern for aesthetic reasons and

because of the high biochemical osygen demand (BOD) concentration. High BOD

emuents discharged to water bodies pose a threat to aquatic life and to the local

ecosystsm. These effluents can be treated at the site or they can be discharged to

rnunicipaI \\.astavater treatment systems.

Because of the high BOD loadings these wastes may impose on municipal treatment

sq-stems. on-site treatment is desirable to reduce the contaminant level. Biological

treatment. either aerobic or anaerobic. can be used to treat these effluents. Factors such

as BOD loading. foaming and impedance of oxygen transfer affect aerobic ireatment of

long chain fatty acids (LCFAs). However. higher BOD loadings c m be applied to

anaerobic systems and hence. they are more suitable to treat effluents containing fats and

oils. Additionallj.. anaerobic treatment offers advantages of no oxygen addition and

methane recovery.

Anaerobically. fats and oils are biodegraded to LCFAs and glycerol by

microorganisms (Hanaki et al.. 1981). In biological matment systems. LCFAs are

degraded ho~vever. LCFAs are inhibiton. to aceticlastic methanogens (Koster and

Cramer. 1987: Hanaki et al.. 198 1 ). Low degradation rates of LCFAs and the inhibitop

effects of LCFA byproducts as \vell. are major problems facing the development of

suitable anaerobic ueatment technology.

Several rssearchers have conducted LCFA degradation and inhibition studies between

35 to 5 5 OC. Ho~vever. research at louer temperatures is required to better understand the

degradation and inhibition of LCFAs in anaerobic systems. Johns (1 992) reported that

effluents from slaughterhouses in Europe are at approximately 20 OC while in Ausudia.

the? vary between 30 to 35 OC. Between 30 to 55 OC. Hwu (1997) reported that

inhibiton. effects. s lo~v degradation of oleic (Cis 1 ) acid and temperature are important

factors affëcting LCFA degradation. As temperatures were increased from 30 to 5 5 OC.

degradation rates and inhibitoc effects of oleic (Cis 1 ) acid incrmed (Hwu. 1997). For

some industrial effluents containing LCFAs. anaerobic treatment within the thermophilic

range ma? require raising the temperature of the incominp Stream. From an economical

point of vie\\.. adding rnergy ma>- not be a feasible. Hence. for reactors operating at

leu-sr than 30°C. it is important to understand LCFA degradation and inhibition caused

by these acids on anaerobic organisms.

This study. \\-hich focused on the anaerobic degradation of several common LCFAs

(linoleic ( C i s 2 ) . oleic (Cis ,) and stearic (C l s acids) found in fats and oils. therefore.

a a s conducted under room temperature conditions at 21 O C to answer a number of

questions. Would linoleic (Cl* 2). oleic (CI8 ,) and stearic (Cis acids be appropriate

substrates for anaerobic bacteria at 2 1 OC? If so, what are the rates and the pathways of

degradation of these LCFAs? Would these LCFAs and their degradation byproducts

inhibit acidogens. acetogens. aceticlastic methanogens and hydrogenotrophic

methanogens at 2 1 OC?

1.2 Research Objectives

The main objective of this research was to examine the degradation of linoleic (CI8 .).

oleic (Cis !) and stearic (C 18 acids and their inhibitoy effects on acidogens. acetogens

and methanogens at 2 1 i 1 O C .

To meet the main objective. the following specific objectives were developed:

1 . To confirrn the anaerobic degradation pafhrc.a~ and byprodwt distribution for linoleic

(Cls I). oleic (Cls 1) and srearic (Cl8 0) acids.

Degradation of LCFAs proceeds by P-osidation however. it is unclear whether

complete double bond saturation is necessi. for the pathway to proceed. This

objective \\.il1 imtstigate the degradation and byproduct distribution of linoleic

(C 18 .). oleic (C i s 1 ) and stearic (C 18 acids at several substrate concentrations.

2. To determine the inlzibiroc, eflects of linoleic fCls d , oleic (Cls 1) and siearic (Cl* a)

acids. inclitding mixrures of all three acids. on acidogenesis.

Glucose fennenting acidogens are part of the anaerobic microbial consortium and

LCFAs may inhibit glucose fermentation. This objective uill detemine the effects of

Iinoleic (C 18 ?). oleic (C I 8 1 ) and stearic (C s.o) acids and mixtures of al1 three acids at

several concentrations on acidogenesis.

3. Tu determine fhe inhibiroq~ effecrs of linoleic fCls.d. oleic (Cls 1) and srearic (Cls 0)

acids. ird.diilg ntixrures ofal2 rhree acids. on acerogenesis.

The conversion of butyrate. a glucose fermentation byprcduct. to acetate may be

inhibited in the presence of LCFAs. This objective will investigate the effects of

linoleic (Cis,2), oleic (ClS.!) and stearic (Cis:o) acids and mixtures of ail three acids at

sevcral concentrations on acetogenesis.

1. To determine the inhibitor-). effecrs of linoleic (Cls d . oleic (Cl8 1). and stearic (Cl8 O/

acids. and mixtures of ail rhree acids. on aceticlastic methanogenesis.

Acetate deri\-ed from butyrate fermentation and LCFA P-degradation is convened

to carbon dioside and methane by aceticlastic methanogens. However. in the

presence of LCFAs acetate conversion is inhibited. This objective will investigate the

effects of linoleic (Cig ,). oleic (Cls 1 ) and stearic (CIg O) acids and mixtures of al1

thres acids at ss\.eral concentrations on aceticlastic methanogenesis.

5 . To deîerritine the inhibirory effecrs of linoleic (C18.d. oleic (Ci8 and sreuric (Cl8 O)

acids irtclrrdiitg misrzcrrs of al1 rhree acids oit hydrogenorrophic merhanogenesis.

Hydrogen. a b>product of acetogenesis and P-osidation is combined with carbon

dioside to produce methane and water by hydrogenotrophic methanogens. This

objective w-iI1 investigate the effect of concentration dependence of linoleic (Cl8 ?)-

oIeic (Cls i ) and stearic (Cls O) acids and mixtures of al1 three acids on

h)-drogenotrophic rnethanogenesis.

1.3 Thesis Outline

Chapter 1 outlines various problems facing anaerobic wastewater treatment of LCFAs

and based on these issues several objectives are outlined. The Iiterature review presented

in Chapter 2 sections 2.2 and 2.3 briefly summarizes anaerobic reactions and an overview

of several treatrnent technologies is subsequently presented. Section 2.4. the main focus

of the Iiterature review. discusses sources. treatment, composition and structure of

LCFAs. Subsequent sections in Chapter 2 examine LCFA depadation, effects of

hydrogen and volatile farty acids on LCF A degradation. LCFA inhibitory effects and

factors affecting LCFA degradation and inhibition. Chapter 3 provides a description of

al1 materials and methods used in this study. Results for the operation of batch reactors

used to acclimate an anaerobic culture to glucose are discussed iri Chapter 4. Chapters 5.

6 and 7 discuss results for the degradation and methanogenic inhibitory effects of linoleic

(C 1s :). oleic (C 1s ) and stearic (C l 8 acids on methanogenesis. respectively.

Degradation of linoleic (C is .). oleic (Ci i) and stearic (C is acids are compared in

Chapter 8 and a mechanism is postulated. Inhibitop effects of individual and mixtures of

linoleic (Ci8 ?). oIeic (Cis i ) and stearic (Cis acids on acidogenesis. acetogenesis and

methanogenesis are discussed in Chapter 9. Inhibition data from Chapters 5.6, 7 and 9

are compared in Chapter 10 and possible inhibitory pathways are described. S w n m q

and conclusions are presented in Chapter 1 1. FinaiIy, Chapter 12 discusses the

engineering significance of this work and proposes future research that should be

conducted.

1.4 Publications

Several sections of this thesis have been published and subrnitted for publication in

con ference proceedings and refereed journals. Section 5.1.1 of C hapter 5 -0 bas been

publ is hed in Errvrrorrrnerrtal Engirreeririg 1999, Proceedirlgs of the ASCE-CSCE Natiorlal

Coriferetrce on Erw~rorrmenta/ Engiiieeririg (Lalrnan and Bagley, 1 999). In addition,

several sections of Chapter 5 are in press for publication in Water Research. Only

Section 5 . 1 -7.B.ii was escluded from the Warer Research subrnission. Section 6.1. I has

been publ ished in Em*irorlmenta/ E;rlgilleering 1999, Proceedings of the ASCE-CSCE

Il'atiot~al Corfererlce or1 E'wirorlmerttai Engineering (Lalman and Bagley, 1999).

2.0 LITERATURE REVIEW

To farniliarize the reader with anaerobic treaunent fundamentais. a brief introduction

to chemical reactions and treatment technologies is presented in Sections 2.2 and 2.3-

2.1 Overview

.4naerobic treatment is a proven technology and is widely used to treat effluents fiom

man' industries (Spsece. 1996: Wheatley. t 990). The technolog>. has been researched

estensively in order to understand the microbial degradation processes. implernentation

of proper operational strategies and the development of reactor technologies. However.

anaerobic treatment currently faces severaI problems relating to effluents containing fats

and oils. Al though this literature review emphasizes issues arising from the anaerobic

treatment of LCFAs. an oven-iew of anaerobic degradation reactions and a bnef

description of severaI treatment technologies is also discussed.

.A consortium of microorganisrns mediates anaerobic degradation of comples organic

substrates. During degradation. byproducts from one reaction s e n e as substrates for

other reactions in the sequence. Specific microbial populations essential for the process

to function efficiently mediate the reaction sequence. Ultimately. carbon dioside.

methane. \vater and biomass are major end products of anaerobic treatment.

Several reactor technologies currentIy used to treat effluents containing fats and oils

are esamined. A description is provided for selecred reactor configurations including

suspended grou-th. hybrid and attached growth. Finally. this review focuses on LCFA

sources. composition. biodegradation. metabolic byproducts. inhibitory effects and

factors affecting degradation and inhibition.

2.2 Fundamentals of Anaerobic Wastewater Treatment

2.2.1 Hydrolysis

Conversion of carbohydrates. proteins and lipids anaerobically to methane is linked by

several reactions. each mediated by a specific microbial population. The process, sho~-n

in Figure 2.1. is divided into hydrolysis. acidogenesis. acetogenesis and rnethanogenesis.

Hydroiysis. the first reaction step. is the degradation of complex organic pollmers into

monomers. Conversion of a polysaccharide (Figure 2.2) into glucose by hydrolsic

bacteria is catalyzed by arnalyse. an extra-cellular enzyme (Lehninger er al., 1999).

Cellulose- protein and lipid hydrol>-tic reactions are cataiyzed by cellulases. proteases and

lipases. respsctivel>-. Several esarnples of hydrolytic reactions and their negative free

energies reported in Table 2.1 show that organic polymers are hydrolyzed to monomeric

compounds.

Factors affecting enzymatic hydrolysis rates include substrate solubility. temperature.

pH. and the t>.pe of substrate (McInerney. 1988: Gujer and Zehnder. 1983). In contrast

to proteins. hemicellulose and lipids have higher hydrolysis rates. For example.

hydrol>-sis rate constants for hemicellulose. lipids and proteins are 0.54 (d-') (Ghosh et

al.. 1980). 0.4 - 0.6 (d-') (Heukelekian and Mueller. 1958). and 0.02 (d-') (Woods and

.Melina, 1965). respecti\.ely. Hemicelluiose hydrolysis occurs when cu(l-+ 6) o rp ( l+ 4)

ether linkages are esposed to hemicellulases.

Lipids and proteins uith hydrophobic components are less hydrophilic than

carboh>.drates and do not dissolve rsadily into the aqueous phase. During hydrolysis.

lipids and proteins are cleaved at ester and amide bonds to produce LCFAs and amino

acids byproducts. respectively (Figures 2.3 and 2.4). Protein hydrolysis rates are less

than hemicellulose because of the comples folding structure in complex proteins

(Lehninger er al.. 1999; Ghosh et al.. 1980: Woods and Melina. 1965). Complete

hydrolysis of peptide bonds is accomplished only after the quatemary or tertiary protein

structure unfolds to expose the primary amide bonding structure.

+ Lactate. propionate.

butyrate. ethanol, etc. -

-

Carbohydrates. proteins and lipids

v

Figure 2. I : .c\naerobic conversion processes (Gujer and Zehnder. 1983)

Long Chain Fatty Acids

(LCFAs)

Sugars

-

v Acetate

1 A

* CH& CO?

Amino Acids 5 -

-4 simple tripeptide protein \\ith its p r ï m q amide bonding structure is shown in

Figure 2.3. Under unfavorabie conditions' unfoidine the protein is not

Glucose monomer

O a ( 1 - 6 ) linkage

/

Figure 2.2: Structure of a polysaccharide (Lehninger ei al.. 1999)

Table 2.1 : Some hydrolytic reactions and free energies AG, (w-mole-' )

Sucrose +- H 2 0 - D-fnictose + 0-D-glucose -43 -6 ' GI>qfglycine -+ H 2 0 - 2 Glycine -9.2 ' Frtx rnergy values taken from Thauer et ai. (1977)' and Lehninger er al. (1 999)' Al1 free energ)- values reported in this table and other sections of this test are based on standard conditions at 25 OC and unit concentrations. AG,' is the reaction free energy adjusted to pH 7.

thermodynamically feasible because hydrophilic molecuIes on the outer protein structure

hide hydrophobic amino acid components of the structure within the complex tertiary and

quaternary cornplex (Lehninger et al., 1999). The rate determining step for protein

degradation is hydrolysis of the structure into free arnino acids (Heukelekian, 1958).

Lipids are hydrolyzed into LCFAs and glycerol by esterase enzymes. An example of

a typicaI lipid structure is shown in Figure 2.4. O'Rourke (1968) reported that lipid

hydrolysis was rate limiting during sludge digestion within the pH range fiom 6.7 to 7.4.

Carbosyi- terminal end 0

Alanine v

! O O CH3

I

bond amino acid

. CH-N I CH20H

Serine

residue

Amino- terminal end

Phenylalanine

Figure 2.3: Structure of a tripeptide (Lehninger et al.. 1999)

Figure 2.4: Structure of a triacylglycerol (Lehninger et al.. 1999)

Eastman and Ferguson (1 98 1 ) concluded that lipids are not degraded during sludge

digestion at pH 5.2. Using the first order rate constant as a rneasure of lipid hydrolysis.

OIRourke ( 1 968) reported increasing hydrolytic rates as the temperature increased.

The optimum pH for hydrolysis is variable and for carbohydrate degradation to

ducose. the masimum hydrolysis rate occurs at pH from 5.5 to 6.5 (Zoetemeyer er al.. C

1982). For proteins. the optimum pH is 7 and higher (Breure and van Andel. 1984) and

for lipids. the optimum pH has not been reported.

Several researchers have used kinetic models to fit data from hydrohsis studies. 3-oike

er al. (1 985) investigated fitting anaerobic degradation profiles for cellulose and starch to

kinetic models. Noike er al. (1 985) used the Contois-Chen mode1 to fit cellulose

degradation and the Monod equation to mode1 hydrolysis of starch into glucose. Based

on results from these models. cellulose hydrolysis was reported as the rate-limiting step.

The cause of the lo\v reaction rates may be related to the inability of cellulases to

hydrolyze cellulose into monomer units because of low cellulose solubility in solution.

2.2.2 Acidogenesis

Formation of acetate. propionate. n- and iso-butyrate. lactate. valerate and ethanol.

from carbohydrates. amino acids and LCFAs is the next step in the reaction sequence

follou.ing hydrolysis (Sahrn. 1983). Examples of several acidogenic reactions are shown

in Table 2.2. Hydrogen production accompanies the formation of acetate. propionate and

but>.rate from glucose. When hydrogen accumulates. formation of reduced VFAs

predominates to maintain low hydrogen partial pressures (pHz). Hydrogen removal by

sulfate reducing hydrogenotrophs or hydrogenotrophic methanogens influences

Table 2.2: Some acidogenic reactions and fiee enernies AG, (k~-mole-')

C6Hl2O6 + 4 H20 e 2 CH3COOe + 2 HC03- + 4 H-> + 4 H+ -206.0

CsHi206 + 5 H20 - CHjCHzCOO- + 3 HC03- + 5 Hz + 4 W -1 77.9

Cd4 1.06 -+ 2 CH;CH(OH)COO' + 2 IT -198.5

C3Hi206 + 2 HtO 3 CH;(CHc)2COO- + 2 HC0;- + 2 Hc + 3 Ht -253.8

C6Hi2O6 + 2 H20 + 2 CH;CH20H + 2 HCOf + 2 H -225.4

~ i e e ene rg values taken fiom Thauer er al. (1977)

fermentative bacterial product distribution patterns (Wolin and Miller. 1982). When

hydrogen is consumed by hydrogenotrophs, fermentative bacteria produce more oxidized

byproducts than at increased hydrogen levels. This synergy between hydrogenotrophs

and fermenters permits more energy per unit of substrate to be supplied to fermentative

bacteria (Wolin. 1976; Wolin. 1979: Mah. 1983).

2.2.3 Acetogenesis

During acetogenesis. acidogenic byproducts are converted to acetate. hydrogen and

carbon dioside by acetogens. Esamples of several acetogenic reactions are showm in

Table 2.3. Successful degradation of VFAs is only accomplished at low hydrogen partial

pressures. Syntrophic association by two or more species maintains low hydrogen partial

pressures (McInerney er oz.. 1979) between 10" to 1 o4 kPa (1 o4 to 10" atm) (Zehnder.

1978). The balance between hydrogen production and consurnption maintained by a

symbiotic relationship has been suggested to be not only biochemical, but also spatial

TabIe 2.3 : Some acetogenic reactions and free energies AG, (kl-mole-')

Free ensrgy values taken from Thauer et al. (1 977)

(Gujer and Zehnder. 1983). For exarnple, if hydrogenotrophs are inhibited, hydrogen

accumulation is prevented by acidogens through the formation of reduced VFAs

b>.products. This association behveen these organisms is termed syntropy and it is found

kvhen two metabolically different types of bacterial species depend on each other for

degradation of a certain substrate (Gujer and Zehnder, 1983).

32.4 Methanogenesis

The conversion of organic substrates to methane is achieved by methane producing

bac teria (MPB). Two types of methane-producing bacteria are hydrogenotrophic and

aceticlastic methanogens. Aceticlastic methanogens convert acetate to methane and

carbon dioside and hydrogenotrophic methanogens reduce carbon dioxide to methane.

Several methane-producing reactions are s h o w in Table 2.4 together with their free

energy values.

Table 2.4: Some methanoeenic reactions and free enereies AG,' (kJ-mo~e-')

CH;COO- + H- 4 CO? + C& -27.5

CO? t 4 H3 - CE6 + 2 H z 0 -139.1

4 CH;OH - 3 C h + COi + 2 HzO - 544.8

CHjOH + H1 - C& + H 2 0 - 149.8

3 CHOO- + 2H- e CHI + CO, + 2 HC03' - 302-6

CHOO- + 3 H2 + H' e C h + 2Hz0 - 178.3

3 CO -+ 2 H-O 4 C h + ;CO2 - 185.9

Free energy values taken from Thauer er ai. (1 977)

ApprosimateIy 70 % of the methane in anaerobic reactors is dcrived from

decarboxylation of acetate by aceticlastic methanogens. Therefore. acetate is the main

precursor for producing methane (Gujer and Zehnder, 1983; Boone, 1982;

Mah et al.. 1977: Smith and Mah. 1966; Jeris and McCarty, 1965). Carbon dioxide

reduction with hydrogen by hydrogenotrophic methanogens produces most of the

remaining methane (Table 2.4). Methane is also produced from other substrates such as

formic acid. methanol and methylamines but except for wastewaters containing rnethznol.

these substrates account for only minor quantities of methane (Koster. 1989).

Maintaining a balanced population of methanogens to sustain low hydrogen partial

pressures and preventing acetate accumulation is essential to the operation of a stable

reactor. Therefore. monitoring acetate is a proper means for controlling the performance

of a methane producing system. For example. accumulation of acetate is an early

\varning of a drop in pH and process instability (Ahring er al., 1995). Formation of

\.olatile fatty acids (VFAs) byproducts is variable and depends upon the hydrogen partial

pressure.

.An equilibriurn between the acid production rate (hydroiysis and acidogenesis) and

acid consumption rate (acetogenesis and methanogenesis) is n e c e s s q to maintain steady

state conditions. Additionallu. hydrogen production and consurnption must be balanced

to maintain low hydrogen levels. Under unsteady state conditions. VFAs and/or

hydrogen may accumulate to inhibitory levels.

Aceticlastic methanogens grow- about 5 to 10 times more slowly in comparison to

hydrogenotrophic methanogens because the free energy of reaction for acetate conversion

to methane and carbon dioxide is less than that for the reduction of carbon dioxide to

methane and \vater (van Leir. 1995). The biomass yields per unit of COD substrate

consumed is less in comparison to hydrogenotrophic methanogens. therefore. aceticlastic

methanogens are affected more by inhibitors.

2.3 Anaerobic Reactor Technologies Used to Treat EMuents Containing Long Chain Fat* Acids

2.3.1 Introduction

Various aerobic or anaerobic reactor technologies are avaiiable to treat effluents

containincg pollutants with a high chemical oxygen demand (COD). However, anaerobic

treatment is a more feasible means since it has the advantages of no oxygen addition and

can operate at higher COD loading rates. These advantages are the fundamental

operational differences between anaerobic and aerobic treatment. Figure 2.5 shows

hydraulic retention times (HRT) and COD operational ranges for aerobic and anaerobic

Chemical Osygen Demand mg-^-')

Figure 2.5: Operating HRT and COD ranges for aerobic and anaerobic bioIogica1 treatment technologies (Hall. 1992)

treatment systems. In comparison to aerobic treatment. high rate anaerobic systems are

operated u-ith less than Ida. hydraulic retention time and up to approsimately 30.000

rng.~- ' of COD (Hall. 1992). Because rhere are no osygen transfer limitations and no

thickening limitations +th proper biomass immobilization. anaerobic loading rates are

much higher in comparison to those applied for aerobic treatrnent.

Various anaerobic treatment processes shown in Figure 2.6 are ofien classified as low

or high rate systsms. Low rate treatrnent processes are designed to handle influent COD

concentrations from 1 .O00 to 12.000 r n g ~ " with long HRTs of up to 30 days (Hall.

1992). In contrast to the same influent COD concentrations fed to low rate systems, high

rate systems are operated with higher concentrations of volatile suspended solids (VSS)

and HRTs ranging fiom 0.1 to 3 days. The development of hi& rate systems has been

researched extensively over the past several years and many are instalied in new

industria1 wastewater treatment processes.

ANAEROBIC T R E A M N T TECHNOLOGIES

1 GROWTH

: COMPLETE i I I I MIX l

DIGESTER 1 1 l - - - - .. - - . - . -

1 SUSPENDED 1 HYBRiD 4

-- LAGOONS .

: ANAEROBIC 1 CONTACT : - - . - . - - - - . - . - . - . - . - . - . .

1 - - - - - - - - I -------- I

UPFLOW SLUDGE I 1

I BLANKET I I I L---,,,,-,--,,,,,d

r . - . - -.-.-.-.-.-.---.-. .

SUPPORTED GROWTH

r ' - . - . - ' L .'".'. r.-.-.-.-.- I -.- . - . - . - . - . -

1 FIXED : i EXPAhlDED / : 1 .

; BED i f FLUDIZED ; 1 . L . . . . . . . 1

I I BED

1

r--------L,,,---d, I UPFLOW SLUDGE I I BLANKET/ FIXED I I I L,,-,,-,,----,,,--

ANAEROBIC SEQUENCING : I

1

I BATCH REACTOR I

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Legend: LOH' rate ------ High rate -.------- LowMigh rate

(0. I - 2 kg COD -m".d-') ( 5 - 25 kg COD -~n'~-d- ' ) (0.1 - 25 kg COD -m-j-d")

Figure 2.6: Anaerobic treatment process classification (modified after Hall, 1992)

2.3.2 Low Rate Treatment

-4 commonly used low rate reactor configuration employed to treat effluents

containing LCFAs is anaerobic lagoons. Suspended growth low rate anaerobic lagoons

have been used to treat effluents from slaughterhouses (Dague et a(.. 1990). potato

processing (Landine et al.. 1987): dairies (Landine et al., 1988) and edible oils

(Southu-orth. 1979). Operating data for full-scale low rate lagoons provided in Table 2.5

show that these systems are loaded between 0.1 to 2 kg COD^"-^-'.

Table 2.5: Design parameters reported for anaerobic reactors treating eMuents containing fats and oi!s

Reactor Waste HigMow Scale Temp. COD CH4 Ref. Type rate m3 O C load - vield

kpem4.d-l

Lagoon Potato processing low 45000 27 0.13 0.3" 1 Lagoon Edible oil low 68000 45 0.6 - 0.9 - - 7 UASB (floc) Slaughter house high 10 30 5 3a 3 UASB S laughter house high 33 20 7 2.8" 4 (granulated) U.4SB Dairy high 400 30 7 - 5 A F S laughter house high 83 36 8 0.32~ 6 AC S lauphter house hi& 11120 35 3 0.24' 7

kg C H ~ - C O D - ~ ~ - ~ - ~ - ' ; kg CI& +kg COD" removed; m' biogas -kg COD" added; AF = anaerobic filter AC = anaerobic contact 1 Landine et al. (1 987); 2~outh\vorth (1 979); %ayed and de Zeeuw (1 988); 'saYed er al. (1 987): '~amson er al (1985); 6 ~ e t z n e r and Temper (1990); 'stebor er cd. (1 990)

2.3.3 High Rate Treatment

High rate systems treating effluents containing LCFAs include upflow anaerobic

sludge blanket (UASB) (suspended growth) (Sayed and Zeeuw, 1988), U.4SB

(granulated sludge) (Hwu. 1 997; Sayed et al., 1 987), anaerobic filter (Metzner and

Temper. 1990). anaerobic contact (Stebor er al.. 1990) and anaerobic sequencing batch

reactor (anSBR) (Dague and Pidaparti. 1992). Figures 2.7 to 2.9 show several reactor

Solid 1 Liquid Separator

Flocculator influent

Anaerobic reactor

7- Effluent

1

Sludge Recycle

Figure 2.7: The Anaerobic contact process flow schematic

Biogas

Biomass attached support material

J

Recycle

Figure 2.8: Down flow fixed film anaerobic filter schematic

configurations used to treat effluents containing LCFAs. Operational data for bench and

full scaIe installations are provided in Table 2.5.

High rate reactor configurations offer several advantages over the low rate lagoon

system. For exarnple. in the contact process (Figure 2.7), a flocculator is added following

the anaerobic reactor to assist in soiid / liquid separation (Schroepfer and Ziemke. 1959).

This reactor design configuration ailows for longer solid retention tirnes (SRTs).

-4naerobic sequencing batch reactors (anSBRs) (Dague, 1992) offer the advantage of

operational flesibility over continuous treatrnent systems. Because the anSBR reactor is

operated in batch mode. filling, reacting, settling and decanting are accomplished in a

sinzle vessel. The simplicity in design allows for no separate clarifier and no extemal

solids recycle. Anaerobic filters (Figure 2.8) present exceptional benefits by preventing

biomass \vashout through immobilization and a quiescent inlet region where large dense

biofilms are developed (Speece. 1983). The downflow configuration is normally used to

treat effluents containing linle or no suspended solids while operation in an upflow mode

is used to prevent plugging caused by emuents containing high suspended solids.

Howe~~er. lab scale studies have shown effluents containing high suspended solids (1 3

CL-') can be treated in the downflow configuration (Kennedy and Guiot. 1988). Another b

ds\vloped technolog!.. which involves no support for biomass immobilization. is UASB

reactors (Figure 2.9) (Lettinga and Hulshoff, 1992; Lettinga et al.. 1980). Dense granules

in UASBs avoid the added cost of packing material, which is necessary for biomass

retention. GranuIes with their high settling velocities and good settleability are

distinctive features of UASB reactors. Under some circurnstances, an additional

gas :' solid separator

I

Effluent

Recycle

Influent A 7

Figure 2.9: UASB flow schematic

clarification process is added ont0 the emuent to aid in soIid/liquid separation. Typical

loading rates for high rate reactors vary between 5 to 25 kg COD-^"-^-' (Hall. 1992).

2.4. Long Chain Fatty Acids

2.4.1 Sources and Treatment

Fats and oiIs are present in effluents from daines (Perle et al.. 1995)- slaughterhouses

(Sayed and Zeeuu.: 1988. Sayed et al.. 1987; Sayed et al.. 1984), [ivestock farms

(Broughton er al.. 1998; Hobson et al.. 198 l), and edible oil processing facilities (Becker

er al.. 1999: Beccari er al.. 1996; Hamdi et al.. 1992). As previously discussed. the

presence of these compounds in industrial effluents causes several problems for aerobic

treatment systems. The high COD in these emuents exceeds the allowable organic

loading to aerobic systems. In addition, LCFAs are surfactants and as such cause foam

and scum formation (Lemmer and Baumann. 1988). They also f o m an oily film around

microbial flocs. impeding osygen transfer and decreasing the efficiency of aerobic

organisms (Becker er al.. 1999).

Sekreral difficulties also arise during the treatment of fats and oils in high and low rate

anaerobic reactors. Stebor et al. (1 990) reported that effluents containine fats and oils

caused anaerobic reactors to operate inefficiently because of problems related to low

solubility. Io\- degradation rates and solids (fats and oils) flotation. Studies by Hnu

( 1997) reported sludge flotation during treatment of oleic (Cis !) acid in a UASB reactor.

In addition. fats and oils are inhibitory to anaerobic organisms (Broughton et al.. 1998:

Koster and Cramer. 1987. Hanaki er al.. 198 1 : Novak and Carlson. IWO).

During treatment. the first reactior, step is hydrolysis of fats and oils into free glycerol

and LCF.4s by lipases (Hanaki er al.. 1981). Under anaerobic conditions. glyceroI is

degraded to 1.3 propanedioi (Biebl et al., 1998) and subsequently to acetate (Qatibi el al.,

199 1 ). Hou.s\.er. LCFAs are not easily degradable and are inhibiton. to aceticlastic

methanogens (Rinzema et al.. 1994: Angelidaki and Ahring. 1992: Koster and Cramer.

1987: Hanaki et al., 198 1 ). Additionally. Angelidaki and Ahring (1 992) reported LCFAs

are inhibiton. to organisms consuming propionate and butyrate.

2.4.2 Composition and Structure

PhysicaI properties and chemical composition for several LCFAs used in this stud!.

are described in this section. Fat and oils are glycerol esters of fatty acids and the

predominant ester is the triglyceride which consists of a glycerol backbone with three

long chain fatty acids (Fonno. 1979). The molecular structure of a typical

triacylglyceride was previously shown in Figure 2.4. The less common di- and

monoglycerides consist of two fatty acids and one fatty acid molecule. respectively. I t is

difficult to distinguish between glycerides of fats and oils and according to Forrno

"Reversible changes in the state owing to variation in temperature may obliterate the common conception that fats are solids and oils are liquids. so today this distinction between the terms fat and oil is largely academic. The tems are still used comrnercially, but the? have on1 y limited significance".

Physical properties of linoleic (C 1 8:2). oleic (C 8: 1) and stearic (C 1 S:O) acids. three

LCF.4s used in this study, are shown in Table 2.6. Further data on physical and chernical

characteristics of other LCFAs is available in "Baileys' Industrial Oil and Far Producrs"

by A.E. Baile'. (1979). At room temperature. both linoleic (C18.z) and oleic (C18 !) acids

are liquid u-hile stearic (Cis acid is solid.

Tabie 2.6: Selected properties of linoleic (C18.1), oleic (C18:1) and stearic (CI8.0) acids LCFA Melting Point ( O C ) Boiling Point (OC) Densiîy

/ distillation messure (e-rn~-' ) 20 OC Linoleic (C 1 2 ) -5 229-230 / 16 mmHg 0.902 Oleic (Cr8 1 ) 13.4 228-229 / 15 mm Hg 0.893 Stearic (C 18 67-69 232 / 15 mm Hg 0.94 1 (Weast. R.C. and M.J. Astley (eds.) 1979).

The main edible oil group classification is shown in Table 2.7. Linoleic (C18 2) acid. a

LCFA ~vith 18 carbons and two cis double bonds located on carbons 9 and 12 is shown in

Figure 2.10. Free rotation about carbons 9 to 10 and carbons 12 to 13 is prevented and

causes the structure to be rigid. Linoleic (C18:,) acid, a common LCFA in many edible

oils. is found in safflower and tobacco seed oils with a composition of up to 75 % w/w

(Table 2.8).

H H H H H H H H O \ I I I I I I I I 4 Y' \ ,,C-C-C-C-C-C-C- C-C *\ ,G 1 1 1 1 1 1 I \ G H H H H H H H OH

-1 \

Figure 2.1 O: Linoleic (Ci ?) acid molecular structure

Oleic (Crs 1 ) - a LCFA with 18 carbons and one cis double bond located on carbon

9. is the rnost abundant acid and is found in almost every edible oil or animal fat

(Somtag. 1979). Thc cis double bond shown in Figure 2.1 1 prevents free rotation

between carbons 9 and 10 and aids in the rigidity of the structure. In teaseed and pecan

edibIe oils. oleic (Ci* j ) acid content can range up to 85% w/w and up to 60% w/w in

peanut oil ( S o ~ t a g . 1979). Typical edible oils containing oleic (Cis l ) acid are sho~bn in

Table 3.9.

Table 2.7: Main edible oil categories (Somtag. 1979) Principal fatty acid Oil Lauric (Ci 2 Coconut

Palm kemel Babassu

Palmitic (C Palm Oleic (C 18 1 ) Olive

Peanut Linoleic (C 1s :)(medium) Soybean

Cottonseed Sesame

Corn Linoleic (C 18 ?)(high) Sunflower

Safflower Eruic (CZi 1 ) Rapeseed

Table 2.8: Edible oils containing linoleic (C !3-)

acid (Somtag. 1979) Edible Oil Percent Linoleic Acid

(% %hT) Safflo~ver 75 Tobaccoseed 75 Poppyseed 65 Sunflo~ver 60 Soybean 50 Corn 50 Cottonseed 45

Table 2.9: Edible oils containing oleic (Cl8 l )

acid (Somtag. 1979) EdibIe Oil Percent Oleic Acid

Teaseed 85 Pecan 85 Olive 80 Peanut 60

H H H H H H H H 1 1 1 1 1 1 1 1 C-C-C-C-C-C-C-C-

1 1 1 1 1 1 1 H H H H H H H

Figure 2.11: Oleic (Cig 1 ) acid molecular structure

Stearic (C18:0) acid (Figure 2.12) is present in most fais and oils but at reduced levels

compared to oleic (C i8 . i ) and linoIeic (Cist) acids. Typically stearic (Cig.0) acid is found

in cocoa and tallow oils with an approsimate composition of less than 40% w/w (Table

2.1 O).

H H H H H H H H H H H H H H H H H O 1 1 1 1 1 1 1 1 1 1 1 1 1 l 1 1 1 4

H - C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C-C I I I I I I I I I I I I I I I I I \ H H H H H H H H H H H H H H H H H OH

Figure 2.12: Stearic (C is 0) acid moIecular structure

Table 2.1 0: Edible oils containincg srearic (C i8 .0)

acid (O'Brien, 1998'1 Edible Oil Percent Composition

(Y0 \v/w'1 Cocoa 31 Ta1 1 ou- 18.6

3.4.3 Biodegradation of LCFAs

Biodegradation of LCFAs proceeds via several steps including adsorption ont0 the

ce11 surface. movement across the ce11 surface and LCFA conversion to lower molecular

~veight components.

Adsorption onco the ce11 surface

The first step of the LCFA degradation process begins with adsorption ont0 the ce11

membrane. Adsorption is a physical or chernical process controlled by factors such as

temperature. pH. rnultivalent cations. agitation speed. agitation time and adsorbate

polarity (M>.ers. 1999: Daniels. 1980). The effect of temperature on adsorption is

variable and depends on the nature of the adsorbate and adsorbent. For example. as

temperature increases. the "stickiness" of surfaces decreases causing adsorbed ionic

surfactants to desorb (Myers. 1999). This type of adsorption can be modeled as a

physical process b>- considering surface forces to become weaker due to increased kinetic

energies as surface temperature increases (Adamson and Gast. 1997). In cornparison. in

some systems containincg nonionic surfactants. adsorption increases as temperature

increases (Tilberg and Malrnsten. 1993). The beha\ior of nonionic surfactant adsorption

is c o n t r e to ionic surfactants and can be modeled as a chemical process. As

temperature increases to an optimum value. chemical bonding between nonionic

surfactants and a surface increases and beyond an optimum temperature. desorption

begins to occur (Somasundaran er al., 1997).

pH strong1~- influences the adsorption of bacterial cells ont0 soils. clays. hydrous

metal osides and ion eschange resins (Daniels. 1980). Altering the pH reverses

adsorption once the adsorbate is attached to a surface. Adsorption of proteins ont0

surfaces is influenced by changes in pH. which affects the isoelectric point and modifies

the surface charge. Generally. stronger adsorption occurs between pH 3 to 6 (Daniels.

1980). The effect of pH on LCFAs adsorption ont0 microbial cultures has not been

in~eestigated.

Adsorption is affected b>- the binding of cations ont0 adsorbate or adsorbent.

Multi\.alent cations compete with adsorbates for active sites in solution and the addition

of inorganic salts promotes desorption (Daniels. 1980). Several studies have esamined

addition of CaC17 to decrease LCF.4 inhibition (Koster. 1989; Hanaki et al.. 198 1 ;

Galbraith er al.. 197 1). The role of ~ a - ' ions is unclear but binding of ~ a - ' ions with

LCFA carboxylic g o u p s is likely the mechanism preventing adsorption and hence.

inhibition.

.4n increase in agitation speed and reaction time enhances adsorption by increasing the

probability of surface contact. Generaliy, equiIibrium is reached within 15 minutes.

although in some cases, it may take a fe\v hours (Daniels, 1980). Afier equilibrium. if a

system is agitated violentl>. for long penods. the adsorbate may begin to desorb (Daniels.

1980).

Because LCFAs possess hydrophobic and hydrophilic components. their surface

orientation may be different. For esample. if the ionic component dominates at the

surface. the interaction wiil be such that the hydrophobic component is oriented towards

the solution. Han-ever. if the hydrophobic component prevails at the surface. the

h>.drophilic component is directed towards the solution (Ulrich and Stumrn. 1988). On

non-polar surfaces. adsorption of LCFAs is dominated by hydrophobic properties of the

nioleculs and the estent of the interaction increases with increasing hydrophobicity of the

carbon chain (Wrich and Stumm. 1988). Ho~vever. on polar surfaces. the ionic end of the

LCFA binds to the polar surface and the hydrophobic component is oriented towards the

solution.

Surfaces are classified as either as homogenous or heterogenous. Biological surfaces

are heterogenous in nature and made up of comples structures of carbohydrates. proteins

and lipids (Shinitzky. 1993). Adsorption of LCFAs ont0 these surfaces is complicated

and researchers have attempted to use homogenous surfaces to characterize the process.

On hornogenous surfaces such as rke hull, it is reported that addition of an isopropanol

cosolvent influenced the adsorption of LCFA emulsions (Proctor and Palaniappan. 1990).

Procror and Palaniappan (1 990) proposed that hydrogen bonding of the isopropanol

cosolvent to weak acidic sites on the adsorbent promoted LCFA binding by hydrophobic

interaction.

In contrast to rice hull surfaces. biological surfaces have variable adsorption sites and

characterization of the adsorption process is comples. Using aerobic and anaerobic

sludges as adsorbents. Hrudey (1 982) and Hanaki et al. ( 1 98 1 ) reported that LCFAs were

removed frorn the aqueous phase within 20 minutes and 24 hours. respectively. More

recent studies at pH 7.2 and 35 O C have shown that the adsorption of LCFAs ont0

eranulated anaerobic sludge follo\vs the Freundlich isotherrn model (r = 0.992) (Hwu. k

1997). H m (1 997) characterized the process and proposed a biosorption model

consisting of the follouing stages: 1. Adsorption. 2. Desorption. 3. Desorption and

degradation. and 4. Degradation.

Mo~wement across the ce11 surface

.4t the esterna1 surface of the outer membrane. LCFAs transverse the ce11 into the

periplasmic space via a trammembrane membrane protein (NuM, 1986). This

mechanism has been researched extensively by biochemists and ce11 physiologists

because LCFAs are important substrates for mammalian ceIl groWh (Mangroo et al..

1995). LCFAs are used for energy storage (triclyceride synthesis in mammalian cellsj.

lipid synthesis (in prokaryotic and eukaryotic cells) and energy production viap-

osidation (in prokaryotic and eukaryotic cells). The bacterium Escherichia coli has been

snidied as a model to investigate the LCFA uptake mechanism across ce11 membranes

(Mangoo er al., 1995).

Four steps have been identified in LCFA uptake by E. coli (Mangroo et al.. 1995). A

schematic membrane transport mechanism is showm in Figure 2.1 3. FadL, a transversal

outer membrane protein mediates LCFA movement across the membrane. Initiaily.

ionized LCFAs bind to FadL and are then transported across the peptidoglycan layer into

the periplasmic space. The transport mechanisrn across the peptidoglycan layer has not

been elucidated but it is possible a protein may be involved in mediating LCFA transfer

across the layer.

f -Medium-chain fatty

acids (C7-C 1 1)

FadL

acids (C 12-C 18)

OM

ACS Long chain fatîy acid ac y 1-CoA

COA' + ATP

cytoplasm

Figure 2. 13 : Proposed model of fany acid transport in Escherichia coli C7 10 C 1 fatty acids traverse the outer membrane via a membrane protein (FadL). C7 to CI I fany acids also enter the cell by diffusion. Fatty acid becomes activated by acyl-CoA synthetase (ACS) protein to f o m long chah fatty acid-CoA. OM = outer membrane; PG = peptidoglycan; PS = periplasmic space; IM = inner menbrane; FadL = membrane protein: ACS = acyl synthease Co.4. (Adapted fiom NUM, 1986)

-4 protein identified as Tsp has been proposed to facilitate binding and releasing of

LCF.4s across the periplasmic space (.4zizan and Black. 1994). Afier passing througb

the peptidoglycan layer. LCF.4s are protonated in the periplasmic space and diffuse to the

i ~ e r membrane. On the outer part of the inner membrane adjacent to the periplasmic

space. another proposed transversal inner membrane protein anchors protonated LCFA

molecules for activation by adenine triphosphate (ATP). Finally. on the inside of the

imer membrane. acyl-CoA synthetase activates fiee LCFAs into long chain acyl-CoA

cornpieses using ATP.

LCFA Siodegradation

Hydrogenation and osidation are two reaction stages identified during LCFA

degradation. It is unclear whether complete LCFA doubie bond saturation is necessary

before P-osidation. No\& and Carlson (1 970) postulated cornplete LCFA saturation is

required before P-osidation. Hom-ever. Canovas-Diaz et al. (1 99 1 ) reported unsaturated

LCFA byproducts during oleic (Ci s: ,) acid degradation. Assuming complete LCFA

saturation is the only pathway. then stearic (Crs:o) acid may enter into the P-oxidation

reaction. Ho\vever. in cornparison. for unsaturated LCF.4s. hydrogenation before P-

osidation will be required assuming the postulate b>- Novak and Carlson ( 1 970) is

correct.

Fujimoto et al. (1 993) reported linoleic (Ci8 ?) acid is first hydrogenated to form

several intermediate compounds. Several organisms listed in Table 2.1 1 are reported to

mediate hydrogenation of linoleic (Ci 8:z) acid. Fujirnoto et al. (1 993) also proposed

several possible pathways leading to the formation of stearic (Ci8:0) acid via the

formation of tram- 1 1 -0ctadecenoic acid. A mechanisrn postulated bp Harfoot (1 9 7 8 ) for

the formation of the trans intermediate is shown in Figure 2.14. In the active site. the

substrate is anchored to hydrogen bonds and n -electron interaction sites on the isornerase

enzyme. A series of proton transfer reactions is initiated by hydrogen bond formation at

the carbosyl end of the substrate molecule. Eventually. the conjugated diene product is

rt-leased ~vith the T-bond at carbon number 12 shifiing to carbon number 1 1.

Table 2.1 1 : Hydrogenation of linoleic (Cis -) acid b'v rumen bacteria (Fujimoto er al.. 1993)

Bacterium H ydrogenation Genus and species identification croup products

1 trans- 1 1 -C 18 1 Blrryrivibrio jibr isolvens I I trans- 1 1 -C 1 1 and Cls.o Unidentified II 1 trans-9 C i 8 1 B~cryri\:ibriojibrisolvens

Selenomonas ruminanhm

Se\.eral researchers (Kemp er al.. 1975: Rosenfeld and Tove. 1971 : Viviani. 1970)

have attempted to idsntifi hydrogen donors responsible for hydrogenation of unsaturated

LCF.4s. Viviani (1 970) tested several hydrogen donors and only pyruvate and formate

had a positi\-e effect on hydrogenation acti\.ity. Sirnilar experiments by Kemp er al.

( 1975) failed to demonstrate any effect of pyruvate. formate. succinate and a-

ketoglutarate on hydrogenation. In addition' using labeled substrates. Rosenfeld and

Tove ( 1 97 1 ) reported tritium was not the source of hydrogen for the formation of trans- 1 1

octadecenoic acid from linoleic (C is ?) acid.

The hydrogenation mechanism from oleic (Cl* ,) acid to stearic (Cis acid is not clear

and is based on the observation of the trans-1 1-octadecenoic acid byproduct. Although

Hydrogen bonding site

H-O

Enq-me site for interaction with x-electrons

H>.drogen donor site

Figure 2.14: Postulated mechanism of the interaction of linoleic (C 1s .) acid with active site of A '"cis. A ' ' -trans-isornerase and the conversion of substrate to cis-9.trans- 1 1 - octadecadienoic acid. (Harfoot. 1978; Kepler er al.. 197 1 )

no intermediates have been identified for oleic (Cis 1 ) acid hydrogenation. Kemp er al.

( 1975) suggested rhat the formation of 1 O-hydrox). stearic (Ci* acid may be a key

intermediate. More recently. several researchers ha\*e reponed the formation of 10-

hydrosy stearic (C acid from linoleic (C is ?) acid (Koritala and Bagby. 1992). It is

possible that hydrogenation or hydro1)-sis of unsaturated LCFAs may be possible routes

for detosification prior to metabolic osidation.

LCFA oxidation proceeds microbially via several reactions shown in Table 2.12

(Ratledge. 1991: Mackie er al.. 1991). Al1 five oxidative pathways have been observed in

pr0ka.q-otic and e u k q o t i c orpanisms but the predominant pathway in anaerobic cultures

is P-osidation. The first four pathways in Table 2.12 are discussed briefly and a detailed

analysis is presented for the P-oxidation reaction scheme since this is the major pathway

of concem in this study. In the first pathway, 2-methyl ketones are formed via terminal

carbos>.lic groups \\.hich are reduced with subsequent oxidation of the a-carbon ( i x . the

carbon in the &-position relative to the original carboxylic group). In eukaryotes such as

yeasts. carbon dioxide is produced as the terminal carboxylic group is cleaved during cu-

osidation. During w-osidation LCFAs are converted fiom mono-carboxylic acids to

hydres). carboxylic acids and in (w- 1 ) and in (w-2) oxidation- hydrosy fatty acids are

also fonned. Mid-chain oxidation of LCFAs such as oleic (Cis:i) acid leads to the

formation of 1 O-hydroxy stearic acid (Koritala and Bagby. 1992).

After complete or incomplete biohydrogenation. LCFAs enter into the P-osidation

reaction scheme. For e v e c P-oxidation reaction cycle sho\\n in Figure 2.15. N o carbons

Table 2.13: LCFA oxidation reactions Formation of 2-methvl ketones

CH;(CH:)"CHICH~COOH +- HzO + CH3(CH2),C(0)CH; + CO: * 2Hz

a-osidation

CH:(CHI),CH~CH:COOH + 2H10 + CH;(CH2),CH2COOH -+ COz + 3H2

w-osidation

CHj(CH2),COOH - H 2 0 + CH?(OH)(CHi),COOH + H2

[w-1) and (w-2) osidation

CHj(CH2jnCOOH + H-O -B CH3CH(OH)(CHr)n_lC00H + H,

CH;(CHi),COOH + HiO + CH3CHtCH(OH)(CH2)n-2COOH + Hz

P-osidation

CH j(CH2)nCOOH + 2Hi0 + CH3(CH2)n_2COOH + CH3COOH + 2H2

\ FAD / flavin

reduced flavin

NADH

Figure 2.15: P-Oxidation cycle showing individual enzyme reactions: 1 . Fatty acyl-CoA dehydrogenase (bacteria and mitochondrial, FAD-linked) or fatty acyl-CoA oxidase (perosisomal. flavo-enzyme): 2.2,3-enoyl-CoA hydratase; 3. 3-hydroxyacyl-CoA dehydrogenase (NAD' - dependent); 4. 3-oxoacyl-CoA thiolase) (Ratledge, 1994)

are removed from the LCFA molecule. The sequential removal of two carbons by

formation of acetate characterizes the f3-oxidation cycle. Although the reaction sites are

not the same for prokaryotes and eukaryotes. the reaction is essentially the same for al1

orpnisms (Ratledge. 1994). Several researchers investigating the degradation of C l e to

CI4 LCFAs have confinned the P-oxidation mechanism (Hanaki er al.. 198 1 : Weng and

Jeris. 1976: h ' o ~ ~ a k and Carlson. 1970: Jeris and McCarty. 1965).

The P-osidation cycle shown in Figure 2.15 consists of 4 steps. During the first

reaction in the c>.cle. LCFA molecules are oxidized by abstraction of two hydrogen atoms

from the a- and p carbons via fatty acyl-CoA dehydrogenase. Subsequently. the hydrogen

atoms are donated to FAD (flavin adenine dinucleotide). The resulting LCFA acyl-CoA

comples is then transfonned into a trans unsanirated isomer. Next. water is added across

the trans double bond b!. 2.3-enoyI-CoA hydratase forming a y-alcohol LCFA acyl-CoA

complss. The LCF.4 molecule becomes further osidized by 3-hydroq.acy1-CoA

drhydrogenase ~vhen nicotine adenine dinucleotide (NAD-) is reduced to NADH.

Acetate is released and 3-oxoacyl-CoA thiolase again activates the shortened LCFA

molecule using .4TP (adenine triphosphate). .4TP is hydrolyzed into -4DP (adenine

diphosphate) and a phosphate molecule. For stearic acid. the overall reaction is

summarized in reaction 2.1.

8 FAD i- 8 NAD- - 8 CoASH + 40 ATP + 38 H20 +- CH;(CHiCH2)7CH~CHzC(0)-SCOA

+ 9 CH;C(O)-SCoA + 8 FADH? -+- 8 NADH + 8H- + 40 ADP + 40 P, ( 2 - 1)

Roy et al. ( 1 986) and Kemp and Lander ( 1 984) have investigated degradation

pathways for unsaturated LCFAs. For linoleic (C 18.z cis-9,cis- 12) acid two degradation

pathways (Figure 2.16) are possible (Roy et al.. 1986). One pathway (the left one in

Figure 2.16) starts with three P-osidation cycles to f o m cisxis-3.9 dodecadienoic (C 2

Linoleic acid C 18:2 (cis-9. cis- 12)

Figure 2.16: Two possible pathways of linoleic (cis 9, cis I I ) ) acid P-oxidation. Enzymes: 1. A'-cis-A'-trans enoyl Co A isomerase: 2. A'-cis enoyl Co A hydratase: 3. 3-hydroxyacyl CoA epimerase; 4. A"-c~s-A' '-uans isomerase. (.4dapted from Roy ef al.. 1986)

(cis-3 . cis-9)) acid. Further 9-oxidation is prevented because the structure shoun in

Figure 2.1 6 is unable to undergo dehydrogenation by fatty acyi-CoA dehydrogenase.

This dehydrogenase enzyme is specific to removing two hydrogen atoms from the a and

carbons with subsequent formation of the trans isomer. Hydrogenation by a

hydrogenase or isomerizations by an isomerase are the two choices available for the

reaction to proceed. Isomerization takes place with the formation of trans.cis-2.6

dodecadienoic (C acid which enters the P-osidation cycle tsvice eventually forming

cis-2 octenoic (Cs 1 ) acid. Eventuall~.. cis-2 octenoic (CS 1 ) acid is hydrogenated and the

unsaturated octanoic (Cs:o) acid molecule is hydrolyzed to 3-D-hydroxy octanoic (Cs)

acid. An epirrierïzation reaction transforms 3-D-hydroxy octanoic (Cg) acid into 3-L-

hydrosy octanoic (Cs) acid which then undergoes P-osidation.

The alternate pathway (Figure 2.16) shows linoleic (C i8:2 cis-9. cis- 12) acid initiaily

undergoing isomerization to cis.trans-9.1 I octadecadienoic (Cis.2) acid instead of directly

entering the P-osidation. Subsequently. two hydrogenation reactions occur forming cis-

1 1 octadscenoic (C I)acid and then stearic (C 1s.o) (octadecanoic ) acid. The saturated

Cls 0 molecuIe mal. then enter into the P-osidation cycle. Although the rnechanism for

oleic (Cl8 ,cis-9) acid degradation has not been elucidated. based on the linoleic (Cis ,)

acid mechanism suggested by Roy et al. (1986). a similar mechanism is postulated and is

shonn in Figure 2.17.

Several isomerization and hyrogenation reactions take place during the degradation of

linoleic CC and oleic (C 18: ,) acids to acetate. A linoleic (Cl s,z) acid hydrogenation

mechanism proposed by Fujimoto et al. (1993) is shown in Figure 2.18. Two pathways

Oleic (C 1) acid cis -9 octadecenoic acid

Figure 2.17: T~vo possibk pathways of oIeic (Cl8 I (cis 9)) acid P-o'tidation. 7 9 9 Enz! mes: 1 . A -&-A -cis isomerase: 2. A -cis-4"-cis isomerase

Linoleic acid

cis-9. cis-12 C ->

Isomerizarion

/' cis-9. trans -1 1 C is 2

Reduction

f trans - 1 1 Cis 1

Reduction

f

C is 0 (Stearic acid)

Isomerization . trans-9- trans- 1 1 C s.2

Reduction

Figure 2.18: Possible hydrogenation pathway for linoleic CI^:^) acid by rumen bacteria ( Adapted from Fujimoto et al.. 1993)

in Figures 2.18 show that the saturated and unsaturated end products are stearic (C l 8 O)

and trans-octadecenoic acids. respectively. Canovas-Diaz er al. ( 199 1 ) and Novak and

Carlson (1 970) o b s e ~ e d palmitoleic (Cl6 1 cis-9) acid as a byproduct fiom LCFA

degradation. Formation of palmitoleic (Cl6 1) acid may take place via two possible routes

requiring isomerization of the single double bond in the parent oleic (Cl8 ,) acid

rnolecule. The detection of palmitoleic (Cl6 1) acid suggested that unsaturated LCFAs

ma? enter into the P-oxidation pathway. This observation is c o n t r q to the mechanism

proposed by Novak and Carlson (1 970).

SeveraI researchers have investigated the hydrogenation of a-linolenic (C 1 8 3 cis-9.cis-

12.cis- 1 5) acici in addition to linoleic (C 1 8 . ~ ) acid. Hydrogenation of a-iinolenic (C 1 j)

acid takes place by several rumen bacterial species (Kemp and Lander. 1983). A

proposed path~vay (Figure 2.19) shows that the acid is first isomerized to octadecatrienoic

(C l s 3 cis-9. trans-1 1 . cis- 15) acid and then hydrogenated to octadecadienoic (C 18 2 trans-

1 1. cis-1 5 ) acid. Subsequently. t~vo additional double bonds are hydrogenated to form

stearic (Cl acid via octadecenoic (C l s 1 trans- 1 1 ) acid. Both these products \vere also

reported by Kemp and Lander (1 984) during the hydrogenation of linoleic (Cr8 .) acid

(Figure 3.1 8).

The hydrogenation mechanism of a-iinolenic (Ci8 ;) acid is not coinpletely understood

since a \vide range of geometrical and positional isomers have been observed (Viviani.

1 970). But Fujimoto er al. (1 993) and Kemp and Lander (1 984) have proposed the

octadecenoic (CI 1 trans- 1 1 ) acid byproduct formed prior to formation of stearic (Ci*

acid as the most comrnon intermediate found in animal rumen studies. Hydrogenation of

a dienoic LCFA (ie. an unsaturated LCFA with N o double bonds) acid to stearic (C l 8 O)

acid has been suggested to involve two organisms (Harfoot. 1978). This postulate is

based on the accumu~ation of octadecenoic (C18.1 trans-1 1) acid pnor to the formation of

Linolenic (C 18 ;) acid

cis-9. cis- 1 2. cis- 1 5 octadecatrienoic acid (Cr s:3)

Isomerization

cis-9. trans- 1 1. cis- 1 5 octadecatrienoic acid (C l g ;)

trans-1 1. cis-15 octadecadienoic acid (Cl8 Z)

trans- 1 1 octadecaenoic acid (C l s . ,)

stearic (Ci g:o) acid

Figure 2.19: Proposed cr-Iinolenic (Cls ;) acid hydrogenation mechanism (-4dapted from Kemp and Lander. 1984)

stearic (Cr* acid. One bacteriurn is presurned to be responsible for the isomerization

step and another completes the hydrogenation reaction to stearic (Cl8 O ) acid.

Microor~anisms Mediatine LCFA Degradation

LCFAs are converted to shorter chah LCFAs. acetate and hydrogen by hydrogen

producing acetogens (Lorowitz et al.. 1989: Roy er al., 1986). A few LCFA degrading

organisms have been identified in pure cultures. Under thermophilic conditions. a LCFA

osidizing culture in association with kîethanobacrerium thermoautorrophicum AH and

.Lfefhanosavcina rhermophila TM-1 degraded Cls to Cio LCFAs and C8 to C2 VFAS

(Angeliddi and Ahring. 1995). Methane was the main byproduct from even carbon

LCFAs and odd carbon LCFAs ultimately yielded methane and propionate as final

byproducts.

S)-ntrophic removal of hydrogen by hydrogen utilizing microbes is an essential

componenr of the LCFA degradation process. Using a vanety of LCFA substrates.

se\.eral acetogenic hydrogen producing organisms have been identified together with

their s'mtrophic partners unàer mesophilic and thermophilic temperatures (Svetlitshnyi et

al.. 1996; Angelidaki and Ahring. 1995: Zhao. er al.. 1993: McInerney et al.. 198 1 ;

Lorowitz et al.. 1989: Roy et al.. 1986). Table 2.13 provides a list of several organisms

identified in LCFA degradation syntrophic reactions. In addition. several organisms

identified in hydrogenation of unsaturated LCFAs are also listed in Table 3.1 3. The key

role of these organisms is the formation of saturated or unsaturated substrates prior to p-

osidation.

2.4.4 Effects of Hydrogen and Volatile Fatty Acid

.4. Hydrogen

Hydrogenotrophic methanogens consume hydrogen. a byproduct of acidogenesis and

acetogenesis. M e n the hydrogen panid pressure is less than 1 O-' kPa (1 O" atm).

hydrogenotrophic methanogens are inhibited because the free energy for converting CO:

to CH4 becomes positive (Thauer er al. 1977). Thermodynamically. hydrogen levels

ereater than 1 oA kPa (1 od am) are unfavorable for the conversion of propionate to C

acetate by acetogens (Thauer et al.. 1977). If h>rdrogenotrophic methanogens cannot

consume hydrogen as the level rises. the system attempts to retieve the stressed condition

through the formation of more reduced VFAs.

B. T ola file Farg ,-l cids

Volatile fatty acids propionic and butyric. while serving as substrates for acetogens.

are inhibitory to methanogenssis. The undissociated forrns of these acids arc responsible

for organism tosicity (Huesemann and Papautsakis. 1986: Kashket. el al.. 1980). For

acetic acid. the ionized to unionized species ratio decreases 10.000 fold from a pH of 7 to

3. Herrero ( 1983) and Finean er al. (1 984) have suggested the movement of

undissociated acids into cells alters the pH and disrupts the proton pump. As a result.

membrane and intraceMar processes are disrupted.

Several researchers have investigated the effects of acetate on acetogenesis and

merhanogenesis. Using VFA concentrations between 500 to 35000 rng -~ - ' . Iannoni and

Fischer ( 1984) reported acetate to be the least toxic to aceticlastic methanogenesis at 37

OC in cornparison to propionate and butyrate. .4t 37 O C and pH 7, the degradation of

propionate decreased with increasing acetate concentration (Gorris et al.. 1989). Gorris

er al. (1 989) also reported butyrate degrading organisms were not influenced by the

presence of acetate. In cornparison. acetate at concentrations between 1 O00 to 2000

rng-~- ' . inhibited propionate degrading organisms at 37 O C and pH 7 (Mawson et al..

199 1 ). In thermophilic UASB reacton ( 55 O C and pH 7.0 - 7.7). acetate at 3000 mg-^-'

inhibited propionate degradation and no effect was observed when 3080 mg^“ of

butyrate was added (van Lier er al.. 1993). Variations observed in propionate inhibition

b>- acetate are most likely due to culture variabilit). and the conditions under which the

esperiments n-ere conducted. The activity of aceticlastic methanogens is also affected by

high acetate concentrations. At 6000 r n g - ~ - ' acetate. 37 O C and pH 7. aceticlastic

methanogenic inhibition was observed (Yang and Okos. 1987) while no inhibition was

obsened at 3000 r n g ~ - ' between 35 to 37 O C and pH between 7.0 to 7.1 (Yang and

Okos. 1 987: Huser er al.. 1982: Hobson and Shaw. 1 976). ln contrast. Van den Berg et

al. ( 1 976) reponed that acetate at 5900 r n g ~ - l did not affect aceticlastic methanogenesis

under similar temperature conditions.

2.4.5 Inhibito-. Effects

-4 . Eflecrs on Menzbrane and Macromolecules Funcrion

LCFAs have been reported to inhibit gram-positive bactena at low concentrations but

not gram-nsgative bacteria (Chemngton et al., 199 1 ). Gram-positive bacteria have ce11

\t-alls similar to methanogens (Ferq-, 1993) and hence. are expected to be susceptible to

LCFA inhibition. As discussed previously, NUM (1 986) exarnined the uptake of LCFAs

in E. coli. a gram-negative bacterium and proposed a mode1 for membrane transport.

LCFAs are able to traverse the cell membrane. become activated by acyl-CoA synthetase

and subsequently undergo P-ooxidation. Before entenng the cell. LCFAs may disnipt

several membrane components and inactivate many energy-linked reactions. For

esample. the)' interfere with H-. K-. Na- regulator proteins and other ce11 proteins

involved mith rnaintaining ce11 homeostasis (Chemngton er al.. 1991 ). LCFAs also act as

membrane disrupting agents causing leakage of proteins or ions fiom gram-positive

bacteria (Greenway and Dyke. 1979; Galbraith and Miller. 1973). Inhibition of nutrient

transport by LCFAs has been suggested to be caused by proton leakage across the ce11

membrane (Frsese er al.. 1973).

Upon entering the cytoplasm. carbosylic acids dissociate causing acidification of the

q-toptasm (Baird-Parker. 1980). The overall impact is a reduction in the ApH across the

membrane. reducing the electro-motive potential. At pH values iess than 7 to 8 within

the cell. most enzymes become inactivated and loose their activity (Lehninger et al..

1999). Tuttle and Dugan (1976) reported the iron-osidase system of Thiobaccilltrs

_ferrooxia'urïs \vas inhibited by l o ~ . pH caused by the presence of organic acids.

Synthesis of macromolecuIes such as DNA. RNA. proteins and lipids are also affected

by the presence of organic acids (Chemngton et al.. 199 1 ). The sensitivity of individual

biosynthetic pathu-ays depends upon the bacterium and the acid. No information is

available on the impact of LCFAs on macromolecule synthesis. However, shorter chain

fatty acids such as formic and propionic are h o w n to cause substantial disruption of

DNA, ELVA and lipid synthesis in E. coli (Chemngton et al.. 199 1 ).

B. Efects on Aizaerobic Organisms

in coId climat es, anaerobic reactors treating effluents containing LCFAs may operate

at psychrophilic (> O to 20 OC; Brock et al., 1994) temperatures (Johns et al., 1995). At

less than 30 OC1 the impact of LCFAs on anaerobic microorganisms has not been

esamined and only a limited amount of research has addressed the inhibitory effects on

methanogens and acetogens at psychrophilic temperatures. Several researchers have

reported the effects of LCF.L\s on aceticlastic methanogens at mesophilic (12 to 45 OC)

and thermophilic (42 to 70 O C ) temperatures (Brock et al., 1994). .4t 30 OC1 Koster and

Cramer (1987) found oleic (C~g:l), myristic and lauric (C17:0) acids to be more

inhibitory to aceticlastic methanogens than capric and caprylic (C8:o) acids. They

also reported aceticlastic methanogenic inhibition caused by oleic ( C I R . ~ ) , myristic (Cld:~),

lauric (CI-.O) and capnc (Clo:~) acids was concentration dependent. However, Koster and

Cramer ( 1 987) did not examine the effects of linoleic (ClsZ2) and stearic (C18:0) acids on

aceticlastic and hydrogenotrophic methanogens.

.At hisher temperatures of 55 OC, acetate degradation was disrupted by both oleic

(C1x-1) and steanc (Clg:~) acids (Angelidaki and Ahring, 1 993), with oleic acid

causing slightly more inhibition than stearic (C18:0) acid. However, no report of the effect

of these acids on hydrogenotrophic methanogens was provided. Angelidaki and Ahring

( 1992) also reported acetogens to be inhibited by oleic (Ci8:i) and stearic (C18:o) acids at

5 5 "C. Stearic (Clx,O) acid showed a threshold inhibitory concentration greater than 300

r ng -~ - ' for propionate and butyrate degradation. However, the inhibitory concentration

dependence of oleic ( C I S : ~ ) acid was not investigated. For aceticlastic methanogens,

oleic ( C I ~ : I ) acid at concentration between 100 to 200 rng-~- ' was reponed to cause

inhibition at 5 5 O C (Angeiidaki and Ahring, 1992).

Some evidence that LCFAs inhibit hydrogenotrophic methanogens has been reported

in the literature. Hydrogenotrophic methanogenic inhibition caused by a LCFA mixture

at 3 7 O C was reported by Hanaki et al. (1 98 1) but in cornparison the inhibition was less

than that observed for aceticlastic methanogens. Linolenic (Clg:3) acid was also observed

to cause inhibition of hydrogenotrophic methanogens at 39 O C (Demeyer and Hendrickx,

1967). AIthough a few studies have reponed the impact of LCFA on hydrogenotrophic

methanogens, no study has been conducted to investigate the effects of linoleic

oleic (C 18: 1) and stearic (C 1 8 : ~ ) acids at varying concentrations and at psychrophilic

temperatures.

Several reseruchers have examined the eflects of chemicai addition to moderate LCFA

inhibitory effects on methanogenic organisms. Calcium chloride has been reported to

reduce the inhibitory effects of LCFAs on aceticlastic methanogens (Angelidalci ef al.,

1990; Rinzema er al., 1989; Koster and Cramer, 1987; Roy et al., 1985; Hanaki er al.,

198 1 ; Galbraith et al., 197 1 ) . However, solids formation from calcium carbonate

precipitation is an operational problem and is an added treatment operating cost. Also.

addition of bentonite clay was reponed to moderate edible oit inhibition by binding and

removing slyceride tnoleate ester from the aqueous phase (Angelidaki et al., 1990).

.Addit ion of CaC12 and bentonite while effective in moderating LCFA inhibition,

however, cause significant operational problems due to inorganic solids removal.

2.46 Factors Affecting LCFA Degradation and Inhibitory Effects

-4. Srtbstrare Molecular Structure and Concentration

Inhibitor). effects of LCFAs on gram-positive and gram-negative bacteria have been

investigated at several concentrations to determine the minimum inhibitory

concentrations (MIC) (Galbraith et al.. 197 1 ). In general, gram-positive bacteria were

affected and no effect on gram-negative bacteria was observed. Inhibitory effects of

LCFAs w-ere also a function of molecular structure. Galbraith er al. (1 97 1 ) obsenred

low-er M C \.alues as the carbon chain length decreased and the degree of unsaturation

increased.

Seteeral researchers have investigated the effect of LCFA concentration on inhibition.

Increasing oleic (Cls 1) and stearic (Cls acid concentrations in mixed cultures incubated

at 55 OC demonstrated that low concentrations of the fiee LCFAs inhibited degradation of

acetatr. propicrate and bu?rate (Angelidalci and Ahring. 1992). They reported that oleic

(CI* ?) acid brtu-een 100 to 700 r n g ~ - ' and stearic (Cls acid at 500 mg^" were

inhibitoqr to \'FA degradation. Ho\ve\*er. at 30 OC. caprylic (Cg 0) acid kvas reported to

be less inhibitory than lauric acid and the inhibitory effects of oleic (Cis i) acid

were approsimatrly the same as for lauric (CI-, 0) acid (Koster and Cramer. 1987). Lauric

(C l i acid inhibited aceticlastic inhibition at 300 mg^-' and 50% of the methanogenic

activity was lost at about 800 r n g ~ " . Rinzema et al. (1989) observed that launc (Ci? 0)

acid initially caused inhibition of aceticlastic methanogens at a dose of 100 mg-^-' and 30

"C. At higher concentrations ( 1 150 to 1550 r n g ~ - ' ) . octanoic (Cg 0) acid inhibited

aceticlastic methanogens and only about 0.2% of the aceticlastic methanogens were able

to survive when the LCFA concentration exceeded this Iethal dose (Rinzema er al..

1 993).

B. Temperature Effecrs

Although several studies have been conducted to examine LCFA inhibition under

mesophilic or thennophilic temperatures. only one single comparative research work has

been conducted (Hwu. 1997) to examine LCFA inhibition as a function of temperame.

It is b o n - n that methanogens have higher thermophilic growth rates as compared to those

at mesophilic conditions (van Leir. 1995). Growth rates between 2 to 3 times higher are

generally found for thermophiles in comparison to mesophiles. In particular.

thermophilic hydrogenotrophic methanogens grow approximately 10 times faster than

mesophiles (\an Leir. 1995).

Oleic (Cis 2 ) acid tosicity was reported to decrease and methanogenic gronth rates to

increase as temperature increased ( H w u 1997). Hw-u (1 997) also concluded that

thermophilic suspended gro~kth sludge \vas more susceptible to inhibition in comparison

to granular mesophilic sludge. Since, the exposed surface areas of the two sludges are

different. it was postulated that the sludge \?th the greater surface area was more

susceptible to inhibition (Huu. 1997). Because of the physical differences between the

two sludges. a cornparison between their inhibitory properties is not suitable under

thermophilic and mesophilic conditions. Instead. it is more appropriate to compare

i n h i b i t o ~ properties of thennophilic or mesophilic cultures u?th similar physical

characteristics.

C. Solttbilir)! Effecrs

Solubility is one of several factors affecting the degradation of hydrophobie organic

substrates. For organic acids. the solubiiity decreases as the carbon chah length

increases (Ralston and Hoerr. 1 942). Hence. it is espected that the low mass trans fer of

these compounds to cells may reduce their degradation rates. From hexanoic (Cg 0) to

octanoic (Cs 0) acid. the solubility decreases significantly by 15 fold and reaches 2.9

r n g ~ - ' for stearic (Ci&o) acid (Table 2.14).

Even at their low aqueous solubility of approximately 3 r n g ~ - ' (Ralston and Hoerr.

1942). C 18 LCFAs adsorb ont0 membrane sites and initiate their degradation. Increasing

LCFA solubility (Bagby. 1993). decreasing surface adsorption (Myers, 1999)- and

increasing surface chernical reaction rates (Adamson. 1997) are a few of the many

interrelated factors affected as surface temperature increases. These factors are Iinked to

substrate conversion at a surface and the net effect is that increased reaction rates are

observed as surface temperature increases. Studies by Hwu (1 997) have s h o w higher

inhibiton. effects and increased oleic (C18 ?) degradation rates by a factor of

approsimately 1 as temperature increases from 30 to 55 OC. Although toxicity and

degradation are competing factors as temperature increases. Hwu (1997) concluded

greater methanogenesis was observed under thermophilic conditions in cornparison to +

lo~ver temperature conditions.

D. pH Efjects

The relative degree of dissociation of organic acids is pH dependent and a greater

proportion of undissociated molecules exist at low pH values (Freese et al.. 1973 ;

Hentges. 1967). For example, a decrease in 3 pH units causes a IO00 fold increase in the

undissociated form of the acid and lowering the pH enhances its inhibitory propenies

(Chemngton er al.. 199 1 ) . The external ce11 pH is normally between 5.5 to 9 and the

interna1 pH is conuolled to maintain homeostasis between 7.4 to 7.6 (Padan et al.. 198 1 :

Sloncze\vski et al.. 198 1 ) and 8.2 to 8.7 (Booth et al.. 1979; Largarde. 1 977). Thus.

organic acids become ionized to the same extent inside the ce11 assurning the dissociation

in aqueous solution is the same as inside the ce11 (Salmond et al.. 1984: Freese et al..

1973).

Table 2.13: Fatty acids aqueous solubilities and dissociation constants Acid Solubility ( m p - ~ ' l ) T pKa -4cetic (CI) very soluble 4.74' Propionic (C 3) very soluble 4.88' Buyric (CJ) very soluble 4-82' Valeric (Cs) 3 7.000 4.81' Hexanoic (C6) 9,680 4.85' Octanoic (CS) 680 - Decanoic (C 1 150 - Dodscanoic (Cl?) 55 - Tetradecanoic (C 1 4 ) 20 - Hesadecanoic (C 16) 7.2 - Octadecanoic (C Is) 2.9 - ~ a l s t o n and Hoerr. ( 1942): ' ~ a ~ b ~ . (1 993)

E. Coszibstrare and Thermodynarnic Effecrs

Addition of co-substrates. such as sugars. has been reported to enhance LCFA

degradation. The overall standard free energies. AG", for metabolic oxidative reactions

for Iinoleic (Ci8 ?). oleic (Ci* 1 ) and stearic (Cis.o) acids are shown in Table 2.15 and were

calculated based on the standard free energy values for LCFAs tabulated in Table

Beccari et al. (1995) successfully utilized 5700 r n g - ~ - ' of glucose to treat

approsimately 1000 mg^-' oleic (Cis ,) acid. Based on Becarri's (1 995) obsen-ations.

Huu ( 1997) suggested that coupling the free energies for glucose oxidation with oIeic

(C 1 8 1 ) acid degradation providsd a net overall negative free energy. It is possible a single

orgmism might be able to mediate both glucose fermentation and oleic (Cls ') acid

Table 2.1 5 : Free energy changes for C 18 LCFAs oxidation to acetate AGO' (k~-mole-')

(Calculations for LCFA free energy of formation values are tabulated in Appendis A)

Table 2.16: LCF.4s Gibbs free energy of formation values Compound -AG?

(H-mole-') CH;(CH2)sC00- 399.45 CH j(CHi)loCOO- 367.56 CH;(CH.)I iCOO' 35 1 .O8 CH;(CH2) 14C00- 332.50 CH3(CH~)_iCH=CH(CH2)7COO- 254.22 CH3(CHz)i6COO- 3 18.24 CH j(CHI)7CH=CH(CHZ)7COO- 239.62 CH;(CH? )~CH=CH(CH,)CH=CH(CHz)7COO- 161 .O4 (Calculations for LCFA free energy of formation values are tabulated in Appendis A)

degradation. The degradation reaction for glucose and oleic (C18:I) acid together with

free snergies are shown in Equations 2.2 and 2.3 and coupIing reactions together

providrs a net overall negative free energy of 254.9 kl-mole-'.

3 C6Hl106 + 6 -4DP + 6 PI + 9 CH;COO + 9 H- + 6 HzO + 6 ATP (2 .3) AG'' = - 3 moles * 21 5.25 kJ-rno~e-~

= - 645.75 kJ

LCF-4 P-osidation proceeds via a sequence of reactions to acetate and hydrogen

(Reaction 2.2). In the first step of the sequence. the P-ozridation of stearic (Crg O ) acid to

palmitic (Cl6 acid is show% in Equation 2.4.

Equation 2.3 is controlled thermodynarnically by the hydrogen partial pressure and

assuming 1 M concentration for the acids and a pH of 7. the hydrogen dependence is

derived from equation 2.5 (where AG" is the reaction free energy under standard

conditions and adjusted to pH 7, R is the gas constant. T is the temperature and Q is the

reaction quotient). The final espression is shown as Equation 2.6. Some fiee energy

expressions for several reactions considered for this study are shown in Table 2.17 and

plotteà in Figure 2.20.

AG' = AGO' - RT ln Q (2.5)

AG' = 50.82 + I 1 .l log [HZ] (LJ-mole-') (2.6)

The conversion of stearic (C18.0) acid to palmitic (C16-0) acid is possible over a wide

range of hydrogen partial pressure and the reaction becornes more thermodynamically

favorable as the hydrogen partial pressure decreases. In comparison, the conversion of

acetic acid to methane and carbon dioxide is independent of the hydrogen partial

pressure.

The reaction free energies s h o w in Table 2.17 suggest that P-oxidation of linoleic

(Cl8.?) and oleic (Ci8-*) acids into palmitic (Ci6.0) acid is themodynamically more

fa\*orable in comparison to stearic (Cl* acid. a saturated C 18 LCFA. The reason being

that hydrogen produced during P-oxidation is possibly used to hydrogenate the carbon

double bond hence. lowering the hydrogen partial pressure and favoring the formation of

palmitic (C i6 acid.

2.5 Summary

The development of suitable anaerobic treatment processes to remove LCFAs from

industrial effluents has progressed to a stage where issues relating to the degradation

mechanism and inhibiton. effects remain unresoived. A complete understanding of the

LCFA degradation reaction mechanism has still not been clearly established. Novak and

Carlson ( 1 970) proposed only saturated LCFAs initiate the P-oxidation degradation

mechanism. however. further studies by Canovas-Diaz et al. (1 99 1) have shown oleic

(C 18 1 ) acid may also enter into the reaction scheme. Hence, it is unclear whether or not

cornplete LCFA saturation is required to initiate the P-oxidation reaction. The impact of

LCFAs on anaerobic organisms has been investigated but the reported research has

focused mainly on the inhibition of aceticlastic rnethanogenesis. Additional work

Figure 2.20: Free energy vs hydrogen concentration for some P- osidation and rnethanogenic reactions. (Al1 reactions are numbered in accordance with Table 2.17. Curves for reactions 3 and 5 are approsimately the same)

is ho\ve\.er. required to identiQ the effects of individual and mixtures of LCFAs not only

on aceticlastic mcthanogens but also on acidogens. acetogens and h>.drogenotrohphic

methanogens.

Degradation and inhibition studies have been mainly focused at temperatures ranging

from 35 to 55 O C . In contrast. effluents at 20 OC or less will require additional energy to

reach a desirable reactor target temperature within the mesophilic or thermophilic range.

In some cases. increasing the reactor temperature within the mesophilic or thennophilic

range is not possible because of process or economic reasons, Therefore, developing

anaerobic technologies operating at lower than mesophilic temperature is required to treat

effluents containing LCFAs.

3.0 in4TERIALS AND METHODS

3.1 Experimental Pian

The esperimental pian was developed in accordance with the objectives of this

research. An experimental design matrix outlining the experiments conducted during this

work is shown in Table 3.1. Degradation experiments shown in Table 3.2 were designed

to confinn the LCF.4 P-oxidation mechanism and at the same time to determine LCFA

inhibition of the biomass. The effects of LCFA concentration on the f3-oxidation

mechanism were exarnined during the degradation studies.

Inhibition studies (acidogenesis, acetogenesis, aceticlastic methanogenesis and

hydrogenotrophic methanogenesis) were designed to investigate the effects of LCFA

concentration on the degradation of substrates specific to each population. Experimental

details for the three C l g LCFA studies are s h o w in Tables 3.3 to 3.7.

Note: O denotes experiment not perfonned, 1 denotes experirnent perfonned

Table 3.1 : Esperimentd design matrix

Details of interaction esperiments to determine the additive effects of individual and

mixtures containing Iinoleic acid, oleic (Cr8:I) acid and stearic (Cis:~) acids on

acidogens. acetogens and hydrogenotrophic methanogens are shown in Table 3.7. Al1

controls tvere prepared in duplicate and sarnples containing LCFAs were prepared in

triplicate. The culture used during the duration of this study was acclimated to glucose

LCFA Degradation Studies

Inhibition Studies Glucose Acetate Butyrate Hydrogen

Table 3.2: Linoleic (C 18.2)~ oleic (C 1 1) and stearic (C 1 g : ~ ) acids degradation studies

LCFA Conc., Glucose Diethyl ether rng-~- ' Conc.. r n g - ~ - '

O O No O O Yes O 1 O0 No 1 O O Yes 30 O Y es 5 0 O Y es 1 O 0 O Yes

Table 3 -3 : Acidogenic inhibition studies conducted with linoleic (C 1 8:2). oleic (C 18 1) and stearic (CI 8:o) acids

Glucose Diethyl ether LCFA Conc.. rng-L-' Conc.. r n g - ~ - '

O No O Yes No Yes Y es Y es Yes

1 O0 Yes 1 O 0

Table 3.4: Acetogenic inhibition studies conducted with linoleic (C 18:r). oleic (C18:l) and stearic (C18,0) acids

But>-ric -4cid Glucose Diethyl ether LCFA Conc.. r n g i ' l Conc.. rng-L-' Conc., mg^"

No Y es No Y es No Y es Yes Y es Yes

and over the duration of the study, duplicate glucose serum bottle controls were analyzed

for fermentation b~products during each experiment. Profiles of the fermentation

byproducts were used comparatively to determine if there were any changes w-ithin the

culture.

Table 3.5: Aceticlastic methanogenic inhibition studies conducted with linoleic (C1g.t). oleic (Cl8 1 ) and stearic (C WO) acids

Acetic Acid GIucose Diethyl ether LCFA Conc.. rng-~-' Conc., mg^-' Conc., r n g ~ - '

O O No O O O Yes O

1 O0 O No O 100 O Yes O

O 1 O0 No O 1 O0 O Yes IO 1 O0 O Yes 30 1 O0 O Y es 50 I O0 O Y es 1 O0

Table 3.6: Hydrogenotrophic methanogenic inhibition studies conducted with linoleic (C i 8 ?). oleic (C 18 1 ) and stearic (C 18 acids

Hydrogen. Glucose Diethyl ether LCF.4 kPa Conc.. mg^" Conc., r n p - ~ - '

O O No O O O Yes O

10.13 O No O 10.13 O Yes O

O 1 O0 No O 10.13 O Yes 10 10.13 O Yes 30 10.13 O Y es 50 10.13 O Y es 100

Table 3 -7: LNONSA interaction studies Individual LCFA Total LCFA Glucose. Butyrate. Hydrogen.

Conc.. r n p . ~ - ' Conc.. r n g ~ - ' mg^" rng-~- ' P a 33.3 ' 1 O0 100 1 O0 10.13 100 ' 300 1 O0 1 O0 10.13

' Contained equal concentrations of linoleic (C acid (LA), oleic (Ci8 ,) acid (OA) and stearic acid (SA).

3.2 Reagents

Linoleic (Ci 8:z) (99%). oleic (C 18.1) (>99%). stearic (C ig.0) (9g0/0). palmitoleic (C 16 *)

(98%). palmitic (CL6:0) (99Y0). myristic (CI4,o) (>99.5%). lauric (C12:0) (299.5%). capric

(C 1 0 (>99%)$ palmitoleic (C 16.1) (99%), caprylic (Cg:o) (>99.3%) and hexanoic (C6.0)

(99%) acids (Sigma Chernical Co.. St. Louis. MO) were used to calibrate the gas

chromatograph (Hewlett-Packard. HP 5890). The Dionex ion chromatograph (IC)

(equipped with an autosarnpler AS 401 a liquid chromatograph LC 20. a gradient pump

GP 30 and a conductivity detector CD 20) \vas calibrated with acetic (Ci) (99.8%).

propionic (C;) (99%). n-butyic (C3) (99%). i-butyïc (C4) (99.5%). n-valeric (Cs) (99%)

i-\?aleric (Cs) (99%) and hexanoic (99%) acids (BDH Chemicals. Toronto. ON). Acetic

and butyric acids for inhibition studies were also purchased from BDH Chernicals.

Toronto. ON. Hesane and methyl tertiary butyl ether (MTBE) were HPLC grade

(Caledon Laboratories, Ontario. Canada). Methane (99.995%). carbon dioxide

(99.999%) and hydrogen (99.999%) gases (BOC Gases, Toronto, ON) were used to

calibrate the gas chromatograph (GC). Carrier gases used were helium (99.999%) and

nitrogen (99.999%). Glucose (BDH Chemicals. Toronto. ON) degradation was

monitored with a glucose hesokinase kit (Sigma Chernical Co., St. Louis, MO). The kit

consisted of 0.75 mrnole NAD. 0.5 mmole ATP, 500 units glucose hexokinase (yeast),

500 units glucose-6-phosphate dehydrogenase. 1.05 mmole magnesium ions and 0.75 g

sodium azide.

3.3 Batcb Reactors

3-3.1 Inoculum Source

Seed culture to MO batch reactors was prepared using a 1 :6 mixture of anaerobic

digester sludge and granulated anaerobic biomass collected from the Toronto Main

Treatrnent Plant and a food processing plant in Cornwall. Ontario. respectively. A 4-L

somi-continuous reactor (Reactor A) mith a 3-L liquid volume was maintained at 2 1 OC

with 20.000 r n g - ~ - ' volatile suspended solids (VSS). Using basal medium at pH ranging

Table 3.8: Basal medium cl Parameter

K2HPOj (N&)~SOJ NaHCO; NHjHCO; h4gC12.4H20 KCl HjBOj FeC12.3H20 ZnCl? MnC 12.4H20 C U C ~ ~ . ~ H ~ O ( N H ~ ) ~ M O O ~ . ~ H ~ O C O C ~ ~ . ~ H ~ O NiC12.6Hz0 NazSeO; EDTA Resazurin Yeast extract

(adapted from Weigant and 1

racteristics Concentration. rnp L"

from 8.0 to 8.2 (Table 3.8), inoculum fiom Reactor A was diluted to 1500 r n g ~ - ' VSS

into a second 3-L semi-continuous reactor (Reactor B). Reactor B was also maintained

with a 3-L liquid volume. Biomass from Reactor A served as an inoculum source to

reactor B as needed and inocula for the serum bottles (1 60 mL) were collected from

Reactor B.

3 - 3 2 Operation of Inoculum Reactors

Rcactors A and B were operated in batch mode and fed with 1000 rng -~ - ' glucose

every 5 to 6 days (time when acetate and gas production measurements indicated that al1

glucose and byproducts were consumed). Glucose feed solution (30.000 mg^-') for both

reactors A and B \vas prepared in basal media. Operational stability for both reactors \vas

monitored using pH. alkalinity (as CaCO;) and VF.4 measurements. Prior to inoculation

of the 160 mL senun bonles. VFA concentrations. pH. alkaIinity (as CaCO;). total

suspended solids (TSS) and volatile suspended solids (VSS) were measured to

characterize the biomass in Reactor B. VFAs were measured to ensure no residual

remained in the reactor. The semi-batch reactor (Reactor B) was operated with a HRT of

approsimately 150 days and an organic loading rate of 0.21 g COD-L"-d-'. In

cornparison. Maillachemvu and Parkin (1996) reported a loading rate of 0.5

g COD-~ ' ' -d - ' to a culture in a batch reactor.

3.4 Hydrogen and Methane Measurement

Head space gis samples (20 PL) for hydrogen and methane were analyzed using a

He~vlett Packard 5890 gas chromatograph equipped with a thermal conductivity detector

(TCD) and a 30-m x 0.53-mm diameter CarboxenTM (Supelco) plot column. Analysis

u-as isothermal at 60 OC with nitrogen as the carrier gas at 5 ml-min" and the detector

and injector temperatures set at 250 OC and 200 O C , respectively. Hydrogen and methane

were detected at 1.19 and 1.78 minutes. respectively. The detection limit for hydrogen

\vas 0.063 3 kPa and for methane it was 0.484 kPa,

Calibration standards for the gas chromatograph were prepared in senun botrles (160

mL) that had been purged with nitrogen (99.998%) for 2 to 3 minutes. The bonles were

sealsd uith ~eflon' lined septa and capped with aluminum crimp seals. Knowm

quantities of hydrogen and methane were injected into the capped bottles. Triplicate

sarnples (20 PL) were prepared for each gas concentration measured. Calibration curves

for hydrogen and methane were prepared using gas samples ranging from 0.0633 to 3.17

kPa and 0.0483 to2.40 kPa. respectively.

Prior to analyzing headspace samples. a blank and two standards were prepared within

the calibration rangs and ana1)zed for carbon dioxide and methane. Gas standards were

also anal>zed after every 10 samples to ensure the instrument remain calibrated. During

the duration of the study, analyses of the standards were found to lie within less than 5%

of the calibration cunTe.

3.5 Volatile Fatty Acid (VFA) Measurement

During each experiment, 2 mL mixed Iiquor samples were periodically withdrawn

From the serurn bottles. The sample was split into two I mL aliquots, one for VFAs

measurement and the other for measurement of LCFAs. Deionized water (2 mL) was

used to dilute the VFA aliquots. Afier centrifugation at 1750 g for 5 minutes, the centrate

was removed. filtered through H-cartridges (Dionex Canada) and diluted with deionized

water to maintain a detector response within the range of the caiibration curves. The

filtered samples were analyzed using a Dionex Ion Chromatograph equipped with a 25

pL sarnple loop. a conductivity detector (CD 20). a 24-cm x 4-mm diameter AS 1 1

colurnn. an AMMSII micromembrane supressor and a lon~ac" ATC-1 cartridge (al1 fiom

Diones). The three eluents used were deionized water (eluent A), 5 mM NaOH (eluent

B) and 50 mM NaOH (eluent C). The eluent flows as a percent of the total flow of 2

ml-min" were as follows: O - 2 mins.. 93% A. 7% B: 2 - 6 mins.. A ramped from 93% to

0%. B from 7% to 100% 6 - 9 mins., B ramped from 100% to 50%. C ramped from 0%

to 50% and then held unti19.99 minsj and fiom 10 - 17 mins., 93% A, 7% B. This

rnethod provided detection of acetic (CI) acid. propionic (C;) acid. iso- and n-butyic (Cr)

acids. iso- and n-valeric (Cj) acid and n-hexanoic (C6) acid. The effective detection

limits (incorporating dilution) were 0.2 r n g ~ - ' for propionic (C3). i -butyric (Cr). n-

valeric (Cs) and i -valeric (C6) acid: 0.3 r n g ~ - ' for acetic (C2) and n-butyric (Cr) acid and

0.4 mg-l" for hesanoic (Cs) acid.

TripIicate standards for VFA analysis were prepared in basal medium using a 1500

mg^" VFAs stock solution. The stock solution was prepared with acetic (Cz) acid.

propionic (C ;) acid, i-butyric (Cj) acid, n-butyric (Cj) acid, i-valeric (Cs) acid. n-valeric

(Cs) acid and hesanoic (C6) acid in deionized water. Basal medium containing 0.5. 1. 2.

3.4 and 5 rngl - ' of each acid was filtered using Dionex H-cartridges and analyzed by

ion chromatography.

The quality control procedure used for VFA analysis was equivalent to that used for

gas analysis by GC. At the beginning of each ion chromatographic analysis, a deionized C

water blank together with VFA standards prepared in basal media were analyzed. A

standard followed by deionized water blank was placed in between every 10 to 15 sarnple

intenal. Sample c w over was initially investigated and never detected and eventually.

the blank was removed fiom the sample queue.

3.6. Development of a LCFA Delivery Strategy

Pure LCFA does not disperse very well in aqueous solution. Preliminary studies

showed aqueous solubility and adherence ont0 g l a s surfaces influenced microbial

contact with LCFAs. In studies conducted with the addition of pure LCFAs into senun

bottles and then inoculation with biomass, very little biodegradation was observed even

afier approsimately 50 days and most of the LCFA was visually observed adhering to the

glass surface.

Substrate insolubility is an important factor affecting microbial degradation of

hydrophobic cornpounds. Therefore, the bioavailability of these compounds will be

affected by their dissolution rates in solution. Work by Wodzinski and coworkers (1 972)

showed that microorganisms utilized only hydrocarbon molecules dissolved in the

aqueous phase.

One approach used by several researchers to increase LCFAs dispersion in aqueous

solution is to add them as sodium salts. LCFAs were reported to be melted a u bain-

marie and dissolved in hot NaOH (50 OC) (Rinzema et al., 1994; Angelidaki amd Ahring.

1992: Novak and Carlson, 1970). The advantage of using LCFAs prepared by this

approach is that an exact arnount of is added however, several problems may arise. Some

of the LCFAs may partition ont0 surfaces and the amount in solution becomes unknown

at the tirne of initial addition. If the amount partitioning is variable, then analyzing

triplicate samples becomes rneaningless. This becomes an important issue especially

when low LCFAs concentrations are added. Secondly, if the LCFAs adhere ont0

surfaces. they may be able to slowly undergo dissolution as the aqueous concentration

decreases via biodegradation. Again, the rate of dissolution may Vary within triplicate

bottles and different aqueous concentration would be measured in the samples. To avoid

these potential problems. an aitemative addition technique was investigated using a

several dispersing agents.

Several dispersing agents were evaiuated to determine their ability for delivering

LCFAs into solution. Researchers have reported using these chemicais to assist in

dispersing hydrophobie compounds in solution (Sikkema, er al., 1995). Ideally, the

dispersing agent must be miscible in aqueous and substrate phases, not influence

substrate degradation. be non-biodegradable and be nontoxic (Sikkema, er al., 1995).

Achieving al1 these characteristics is difficult for many dispersing agents: therefore.

evaluating their use must take into consideration the overall experimental objectives.

Several dispersing agents screened for LCFA delivery included ethanol, acetyl acetate.

diethyl ether and methyl tertiary butyl ether (MTBE). Aqueous solubilities in water for

these compounds are shown in Table 3.9.

Table 3.9: Dispersing agent solubilities 1 Chernical 1 Aaueous Solubilitv (w/wll. ' 1

Ethanol Ethyl acetate Diethyl ether MTBE

Miscible ,

7.7 l

6.9 4.3

'I~mallwood (1996); .'solubility at 25 OC

ï h e first criteria for choosing a dispersing agent were based on polarity and its ability

to solubilize LCFAs. Solubility data for LCFAs in ethanol is unavailable. However.

based on their solubility in acetone (a polar solvent) and n-heptane (a non-polar solvent)

(Bagby. 1993). some solubility is expected in ethanol. As a cosubstrate, the behavior of

ethanol was of concem because of its degradability and potential influence on LCFA

degradation. Ethanol could enhance LCFA degradation. Although LCFA solubilities in

acetyl acetate are unknown. it was considered as a dispersing agent because of its

polarity: however. concerns for its use were similar to those for ethano1 and it was

discarded as an option.

Ethers and esters are less polar in comparison to alcohols bearing the sarne number of

carbons (Smallwood, 1996). Ethers also have an advantage of being recalcitrant (Yeh

and Novak. 1994). Although the alcohol and ester were initially rejected as potential

dispersing agents. they were evaluated with the two ethers for their toxicity effect using

data from the literature. For the tosicity evaluation comparison. similar compounds were

esaminsd using data reported by Playne and Smith (1983) since comparative microbial

toxicity data for ethanol. ethyl acetate. diethyl ether and MTBE are unavailable. Playne

and Smith (1 983) reported toxicity. measured on a methane production basis. for isoamyl

alcohol. isoamyl ester and isoamyl ether (2.5 % (v/v)) to be approximately the same.

These researchers esamined the toxicity of several chernicals used for product extraction

from anaerobic process streams. Using data reported by Playne and Smith (1983),

diethyl ether and MTBE were identified as candidates for delivering LCFAs. In

comparison to diethyl ether, MTBE was more toxic (Lewis, f997) for use in the

laboratory and was eliminated as an option.

Two-mL of diethyl ether was the maximum amount found to be effective in dispersing

10 mg of C 1 8 LCFAs (1 0 mg per 100 mL of liquid). the maximum amount used in this

study. The concentration of LCFA stock solution used through out this study was 5000

r n g - ~ - ' . In control cultures, acetate was used as a measure of diethyl ether hydrolysis and

insignificant Ievels were detected. In cornparison to controls without diethyl ether. no

increase in methane levels were observed in control cultures receiving diethyl ether.

3.7 Long Chain Fatty .4cid (LCFA) Measurement

3 -7.1 LCFA Extraction- Method Development

LCFAs are relatively insoluble in aqueous solutions and an extraction protocol was

developed to ensure losses did not occur afier samples were removed frorn the serum

bottles. Solvent polarity. pH. ionic strength and extraction time were investigated to

determine their effects on the arnount of LCFAs recovered during liquid sarnple

extraction. Several organic extractives Lvere screened to compare solvent polarity and

extraction efficiency: chloroforrn. chloroform:methanol (1 : f ). hexane and hexane:MTBE

( 1 : l ) .

-411 samples for extraction studies were prepared in triplicate using 10 mL of an

anaerobic culture in 20 mL serum bottles. The bottles were sealed ~ ~ i t h TeflonB fined

septa. secured with alurninum caps and shaken using an orbital shaker (Lab Line

Instruments Inc. Mode1 No. 3520) for 10 minutes at 200 rpm. One-mL sarnples were

removed and transferred into 5 mL semm bottles with 2 mL of an organic extractive.

The extraction time was initially set at 5 minutes for a11 the extractives. However. for the

hexane:MTBE extractive. increased extraction effrciencies were observed when

comparing results for the 5 and 15 minute extraction times. Afier shaking. the samples

Lvere centrifuged for 5 minutes at 1750 g to separate the aqueous and organic layers.

One-pL samples of the organic phase were analyed by gas chromatography.

Chloroform. a polar solvent- \vas initially examined to determine the percent LCFAs

recovered afier extraction (Figure 3.1 ). Percent recoveries for dodecanoic (C 10) to

octadecanoic (C 18) LCFAs were greater than 90 percent for cultures receiving 56.25 and

1 2 . 5 m . However. l o w r LCFAs extraction efficiencies between 70 to 85 percent

nvre obtained for cultures receiving 5.625 r n p - ~ - ' . Also. less than 50 percent hesanoic

(C6) and octanoic (Cs) acids were recovered from cultures receiving 5.635 and 56.25

mg-L-'. To increase the amount of LCFAs recovered from cultures receiving

approsimate1)- 5 n q - ~ - ' . a 1 : I misture of rnethanol and chloroform was used and

recoveries increased to between 80 to 95 percent. Usine the methanoI:chloroforrn

mixture also improved percent recoveries for hesanoic (C6) and octanoic ( C g ) acids at ail

concentrations esamined (Figure 3.2). While an effective extractive. chlorofonn \vas

considered too tosic to be used in the laborator). on a long-term basis. As a result.

alternative sol\-ents were investigated based on data obtained from the

n~etl~ano1:chloroforrn study.

Hesane. a non-polar solvent. \vas the next extractive examined and LCFA extraction

efficiencies were found to be similar to chloroform (Figure 3.3). Percent recoveries were

comparable to chlorofonn for cultures receiving 56.25 and 1 12.5 r n g . ~ - I . However, at

5.625 mgLe ' . less LCFAs were extracted into the hexane phase in comparison to the 1 :1

methanol:chloroform misture.

Figure 3.1 : Percent LCFA extracted into chloroform (Averages for triplicate samples, error bars represent standard deviation for the samples)

Figure 3.2: Percent LCFA extracted into ch1oroform:methanol ( Averages for triplicate sarnples, error bars represent standard deviation for the samples)

Figure 3 . 3 . Percent LCFA extracted into hexane (.\verages for triplicate samples, error bars represent standard deviation for the sarnples)

I Herane:31TB E with SaCI and pH LCFA O Hcxanc O Heranc:MTBE

Figure 3.4: Percent recovery for 5.625 mg-^^' C6 to Ci* in hexane and hexane:MTBE: 5 minutes shaking at 200 rpm (Averages for triplicate samples, error bars represent standard deviation for the sampies)

IIIcrane: . \ ITBE with S ~ C I and pH U l iesane LCFA O Hexane:>lTB E

Figure 3.5: Percent recovery for 5.625 mg^-' C1 to Cis in hexane and he'rane:MTBE: 15 minutes shaking at 200 rpm (Averages for triplicate samples, error bars represent standard deviation for the samples)

Several parameters were investigated to increase the amount of LCFA extracted into

the hexane phase. Longer extraction times fiom 5 to 15 minutes assisted in increasing the

amount recovered for cultures receiving 5 -625 mg-^-' LCFAs (Figures 3.4 and 3.5).

Adding a 1 : 1 mixture of MTBE and hexane, lowering the pH (2 drops of 1 : 1 H2S04) and

adding NaC1 (0.05 g) were examined to fùrther increase percent recoveries (Figure 3.5).

Greater than 95 percent CIO to Ci* LCFAs were extracted into the hexane:MTBE phase at

lower pH values and increased ionic strength.

For octanoic (Cg) acid. the incremental amount recovered at longer extraction times

increased by approximately 25 percent and minor increases were observed for hexanoic

(CG) acid. The final extraction protocol developed for the remainder of this study

consisted of adding 0.05 g NaCl. 2 drops of 50 % H2S04. 2.0 mL of a 1 : 1 mixture of

hesane and MTBE to ImL aqueous samples in 5 mL serum bottles. The bottles m-ere

shaken using an orbital shaker at 200 rpm for 15 minutes and centrihged for 5 minutes at

1750 g to separate the organic and aqueous phases. One-pL samples were removed from

the organic phase and analyzed by gas chromatography.

This method quantified saturated LCFAs (dodecanoic (Clo to stearic (Cl8 acids

and unsaturated C 18 LCFAs (linoleic (Ci8 ,) and oleic (Cl* 1 ) ) with a 85 to 90 percent

removal efficiency. Octanoic (Cs acid was characterized with a 75 percent removal

efficiency.

3 -7.2 LCFA Extraction- Phase Partitionhg Studies

LCFAs are biodegraded only when they adhere ont0 LCFA degrading organisms.

Because Cis LCFAs have only limited aqueous solubility (2.9 mg^-'). it was n e c e s s e

to examine if the LCF.4.s partitioned ont0 the glass surface or with the biomass. Serurn

bottles (20 mL) were inoculated \vith 10 mL of 1500 m g ~ ~ ~ - ~ - l biomass and kno\r-n

quantities of linoleic (Cls,z) acid \vas added from a 5000 r n g - ~ - ' diethyl ether stock

solution. Afier feeding the cultures with 100 r n g ~ - ' linoleic ( C [ S . ~ ) acid, 3-3 mL samples

Lvere removed from the serum bottles and split into 1 mL (sarnple A) and 2 mL (sample

B) portions. Sarnple B was centrifuged and 1 mL of the centrate removed and placed into

a 5 mL via1 (Sample C). The amount of LCFAs in samples A (culture + basal medium)

and C (basal medium) was determined using the extraction procedure outlined in section

3.7.1 . The average linoleic (C 8 2 ) acid concentrations distributed within the microbial

culture (sample A) and centrate (sample C) were 100 + 5 r n g * ~ - l and 5.8 + 0.6 mg^-'.

respective1 y (Figure 3 -6) . The slightly higher linoleic CI^:^) acid concentration (5.8 i 0.6

Figure 3.6: Linoleic acid extraction partitioning studies

mg^-') greater than the solubility of 3 r n g ~ - ' measured in the aqueous phase (Ralston

and Hoerr, 1942) was probably due to the presence of diethyl ether.

3 . 7 . 3 LCFA Anal ysis

Extracted samples (1 -PL) were analyzed by gas chromatography using a Hewlett

Psckard 5890 chromatograph equipped with a flarne ionization detector (Fm) at 250 OC,

injector at 250 O C , a 30-m x 0.53-mm diameter Nukol (Supelco) column and, helium

carrier gas flow at 5 ml-min? The oven temperature programme was as follows: 0.5

minutes at 90 OC, a 20 OC-min" ramp to 180 OC and a final hold at 180 OC for 9 minutes.

The effective detection limits ranged from 1 rng .~ - ' (in the bottle) for oaanoic (Cs:()) to

hesadecanoic (C 1 and 2 r n g - ~ - ' for stearic (Ci 8:o), oleic (C !) and linoleic (C 18.z)

acids.

Triplkate calibration standards for LCFA analysis of 1, 2. 3.4, 5, 10, 3Ot 30.40. 50

and 100 r n g - ~ - ' were prepared in a 1 : 1 mixture of hexane:MTBE usinp a 1500 mg^"

LCFAs stock solution. The stock solution was prepared with caprylic (Cs) acid. capric

(CIO) acid. lauric (Ci2:o) acid, myristic (Ci4:o) acid, palmitic (C16.0) acid, palmitoleic

( C l S I ) acid. stearic acid, oleic (Cis.i) acid and linoleic acid in diethyl ether (Sigma

Chernical Co.. St. Louis, MO).

At the beginning and end of entracted sarnple analysis. a solvent blank followed by a

series of standards was placed in the queue to ven. the calibration. To check for sample

c w o v e r . a calibration standard of differing concentrations followed by a solvent blank

was placed between eveq 6 to 7 sarnples.

During esperiments with the 160 mL serum bottles. known quantitics of LCFAs were

added to a culture blank to determine the extraction efficiencies. Sodium chloride (0.05

g). concentrated H2S04 (2 drops) and 2 mL of a 1: 1 ratio of hexane:MTBE were added to

a I mL sample and estracted for LCFAs.

3.8 Serum Bottle Preparation

Degradation and inhibition studies were conducted in 160 mL serum bottles using a

liquid \volume of 100 mL and a 70% N, - 130% CO, b atmosphere headspace. Bottles were

inoculated under a 70% N2 /30% CO, - atmosphere with 96,98 or 100 mL of culture from

Reactor B. Using the 5000 r n g . ~ ' ' LCFA stock solution. varying amounts of diethyl

ether of up to 2 mL were added to culture bottles to provide concentrations of 0, 10, 30,

50 and 100 r n g ~ " . In triplicate bottles receiving less than 2 mL of the LCFA stock

solution. additional diethyl ether was added to maintain a constant total amount of 2 mL.

Triplicate bonles inoculated with 96 mL of culture received in addition to the LCFA

stock solu~ion and diethyl ether. 2 mL of stock solutions (5000 mg-^-') containing

glucose. acetic acid or butyric acid. Triplicate bottles for hydrogen studies contained a

total culture \.olume of 98 mL, 2 mL of LCFA stock solution and 6 mL of hydrogen at

10 1 -3 kPa and I l O C . Control bonles in duplicate contained 98 mL of inoculum and 2

mL of stock solution (5000 mg-L-' containing glucose. acetic acid or but>-ric acid).

Duplicate control bottles for hydrogen studies with and without diethyl ether contained

culture volumes of 98 mL and 100 mL. respectively plus 6 mL of hydrogen at 10 1.3 kPa

and 71 OC. For degradation and inhibition studies. control cultures were prepared \%<th

and nithout diethyl ether.

After inoculation. bottles were sealed with ~eflonB-lined silicone rubber septa and

aluminum crimp caps and pressurized with 20 mL of 70% N, - /;O% CO, - to avoid the

formation of a negative pressure in the headspace during sampling. To prevent

photos~mthetic activiv and LCFA photolytic reactions. the bottles were covered with

alun~inum foi1 and agitated in an orbital shaker (Lab Line Instruments) at 200 rpm and

2 1 O C * 1 "C for the duration of each study. Using a 2.5 mL syringe, liquid and headspace

samples \vere periodically withdrawn to mesure selected pararneters. At the completion

of each esperiment. culture bonles were sacrificed and liquid samples removed for pH,

a1 ka1 ini ty (as CaC03). TSS and VSS measurements.

Anaerobic conditions in serurn bottles were rnaintained with 50 r n g ~ - ' ferrous

chloride and 50 r n g ~ - ' sodium sulphide and determined using resazurin (al1

concentrations are reported as in bottle). A11 Iiquids used to prepare the semm bottles

were maintained under anaerobic conditions except diethyl ether solutions. Based on a

study conducted uith 2 mL diethyl ether, 98 mL of culture plus sodium sulphide and

ferrous chlofide at 10,25.50 and 100 mg-^-', it was determined that anaerobic conditions

were best maintained using 50 mg^-' of each compound. Trace amounts of oxygen in the

diethyI ether soIutions caused aerobic conditions in cultures receiving less than 50

mg- L" .

3.9 Glucose Measuremeat

Samples (1 -mL) removed fiom 160 mL serum bottles for glucose analysis were

Jiluted with 2 mL of deionized water and centrifbged for 5 minutes at 1750 g. The

centrate was removed and filtered with Diones H-cartridges.

Triplicate standards for glucose analysis ranging from 1 mg-^-' to 20 r n g ~ - ' glucose

were prepared with basal media in I O rnL vials. A 5000 r n g ~ - ' glucose stock solution

used for al2 standard solutions was prepared with deionized water. If necessq . samples

were hrther diluted with deionized water so as to maintain the glucose concentration

ivithin the calibration range. Next. 2 mL of a glucose hexokinase reagent at 50 % of the

recomrnended dilution (Glucose HK reagent, Sigma Chemical Co., St. Louis, MO) was

added to 2 mL of the sample. The mixture was left standing at room temperature for 5

minutes and analyzed at 340 n m using a Spectronic 21 D spectrophotometer (Milton Roy,

Rochester. NY). This method established a detection limit of 0.5 mg-^" (in the semm

bottle). The procedure was modified from Methods of Enzymaric Analysis (Kunst et al..

1983).

Blanks and standards for glucose analysis were placed between every 12 samples for

analysis. Standards were prepared using the same matris and dilution as for the samples.

3.10 Total Suspended Solids (TSS), Volatile Suspeaded Solids (VSS), Alkalinity and pH Measurements

Duplicate and triplicate senim bottle samples were analyzed for total suspended solids

(TSS) (5 mL). volatile suspended solids (VSS) (5 mL), alkalinity (as CaC03) (5 mL) and

pH ( I O mL). At the end of each esperiment. samples kvere rernoved afier the botties were

shaken and crimp seals removed. Duplicate samples from Reactor B were also analyzed

for TSS (5 mL). VSS (5 mL). a l k a h i h (5 mL) and pH (10 mL). Ali analyses were

conducted in accordance with Standard -44ethods (APHA. 1992).

4.0 BATCH REACTORS

4.1 Eaperimental Results

Batch reactors A and B were fed with approximately 3 g of glucose every 5 to 6 days

and produced on average 2.0L of gas. A schematic of Reactors A and B is showvn in

Figure 4.1. The average alkalinity was 4800 mg-L-' t 300 r n g - ~ - ' (as CaC03)

Figure 4.1 : Reactor schematic (sarne for A and B)

Ninogen Gas Bio gas I L

and the pH range \vas 7.4 to 7.6 in both reactors. The VSS contents in Reactors A and B

wzre rnaintained at approximately 20,000 and 1500 r n g - ~ " , respectively. Glucose was

not monitored: however, VFA and methane byproduct profiles are shown in Figures 4.2

and 4.3, respectiveiy. for a representative sarnple fiom Reactor B. Acetate and iso-

butyrate mêxirnurn concentrations of about 300 r n g - ~ " were reached within

approsimately 7 hours while propionate reached a maximum concentration of 270 r n g - ~ "

within 23 hours. Al1 the VFA byproducts were removed within approximately less than 6

f r--L,

Water Level

Reactor -W 4L

Outlet Pon \ ,O J

Magnetic Stirrer f late

0

O 1 3 5 6

Time, days

Figure 4.2: Reactor B &cose byproduct degradation profiles. (Average for duplicate samples shown.)

I Theoretical

Figure 4.3: Reactor B gas production profiles. (Average for duplicate samples shown.)

days. During each experiment. cultures removed from Reactor B and placed into 160-mL

serum bottles were inoculated with 100 r n g - ~ - ' glucose. The bottles were monitored for

byproducts for approximately 24 hours. Acetate concentration profiles are shown in

Figure 4.4 for representative culture samples removed fkom Reactor B at different

sampling periods.

ïi me, days

Figure 3.4: Acetate profiles for glucose degradation in semm bottles. (Average for duplicate sarnples shown.)

4.2 Discussion of Results

Initial operation of reactors .A and B with 1000 r n g ~ - ' glucose and using between

1500 to 2000 r n g ~ - ' alkalinity (as CaCOi) was unsuccessful. On several occasions

before the serum bottle (160 mL) experiments were conducted, the pH in both reactors

decreased to less than 4. Subsequently, both reactors operated successfùlly for the

rernainder of the study at an increased alkalinity of approximately 4800 r n g * ~ - ' (as

CaC03).

VF.4 profiles for Reactor B show glucose degraded to acetate, propionate and butyrate

\vas consumed to methane and carbon dioxide within 5 to 6 days. Conversion of glucose

to \TAS and then to methane in Reactor B and semm bottle studies was consistent

throughout the study. Before each experiment, duplicate samples from Reactor B were

analyzed for W A s by ion chrornatography. The culture was used to conduct the LCFA

esperiments only when the M.4 concentration was less than 5 mg^?

5.0 DEGRADATION OF LINOLEIC ACID AND ITS LNHIBITORY EFFECTS ON METE4NOGENESIS

5.1 Experimental Results

5 . 1 . 1 Linoleic (C 1 8 : ~ ) Acid Degradation

Under anaerobic conditions at 21 OC and using 1600 + 60 r n g - ~ - ' VSS, linoleic (CIP:Z)

acid was degraded to below detection limits within 25 days (Figure S. 1). Saturated fi-

oxidized LCFA byproducts, palmitic (C16:0) and myristic (C14:o) acids were observed at

al1 linoleic (C18:2) acid concentrations examined. Maximum concentrations of major

saturated LCFA byproducts shown in Table 5.1 were a hinction of the initial linoleic

(C ,g,-) acid concentration. Trace amounts of lauric (C12;0) acid (data not showii) were

obseneed only in cultures receiving 100 mg^" linoleic acid. In addition, hexanoic

O 5 1 O 15 20 25 30

Time, &y

Figure S. 1 : Linoleic (C18:2) acid (LA) degradation profiles. (Average for triplicate samples.)

Table 5.1 : Maximum concentration of byproducts formed during linoleic (Ci8:t) acid degradation '

I I 1 Product Formed Initial Linoleic (C 2 ) Acid Concentration. r n g ~ - ' (PM)

1 I

Stearic (C 18 acid O!eic (Ci8 1 ) acid Paimitoleic (C 16 1 ) acid Palmitic (Cl6 0) acid Myristic (Cl4 0) acid

1 O (36)

t Lauric (C12.0) acid ND'

Hexanoic (C6) acid i-Valeric (C j) acid n-Valeric (Cs ) acid

I ~ v e r a ~ e * standard deviation for triplicate bottles shown. 7

-ND = not detected.

50(179) 1 lOO(327)

ND ND ND

Capric (C 1 0 acid Caprylic (Cg 0) acid

(C6) acid \vas first observed in cultures fed with 100 r n g ~ - l linoleic (Cls:?) acid afier da.

50 (Figure 5 .2 ) and reached a maximum concentration of approximately 37 m g ~ - l (321

pmoles.~'l) on day 80. Similady, palmitoleic (CI6:,) acid was first observed on day 10

and reached a maximum concentration of 10 mg^" (41 pmoles-L-') on day 13 in cultures

receiving 100 r n g ~ - ' linoleic (C1a.z) acid.

Palmitic (C16.0) acid reached maximum concentrations of 29 and 46 r n g - ~ - ' (1 13 and

1 78pmoles-~-') at day 30 and was completely removed within approximately 90 days in

cultures fed wiîh 100 and 50 rng-L-' linoleic CI^:^) acid (Figure 5.3). In cultures

receiving 10 r n g - ~ - ' linoleic (Ci8:?) acid, palmitic (C16:0) acid reached a maximum

< 5 ND ND

ND ND ND < 20 < 10

i-Butyric (CJ) acid n-Butyric (Cj) acid Propionic (C ;) acid

p m o l e s - ~ ~ '

c 20 < 50 < 100

< 20 < 50 < 100

ND 1312 ND

12* 1 21 13

ND c 20

-

< 20 < 50 < 100

-

321 1 113 c 20

3292 2 115 Acetic ( C d acid

ND 3 0 1 1

ND 113 I 12 3914

c 10 1 < 10

483 I 32 / 2567 I 50

ND 1912

41 I 18 17819 127 I 14

concentration of 3 r n g * ~ - ' ( 1 2 pmoles-~-l) at day 10 and was undetected &er day 50

(Figure 5.3).

Figure 5.2: LCFA concentration profiles in cultures receiving 100 r n g ~ - ' Iinoleic (Clsiz) acid (LA). (&Average for triplkate samples.)

Time, & ~ s

Figure 5 .3 : Palmitic (C16) acid profiles in cultures receiving linoieic (C18:2) acid (LA). (Average for triplicate samples.)

In cultures fed with 100 and 50 r n g * ~ - ' linoleic acid, profiles for palmitic (C16:0)

acid were sirnilar to those observed for myristic acid (Figure 5.4). Myristic

(Cli,o) acid reached maximum concentrations of 9 and 29 mg-^" (39 and 127

pmoles-~-l) ar days 30 and 60, respectively and was undetected within approximately 80

to 95 days in cultures receiving 50 and 100 r n g - ~ - ' linoleic (C1g:2) acid (Figure 5 -4).

Removal times were similar for palmitic (C16:0) and myristic (C14:o) acids in cultures

receiving 100 and 50 mg^-' linoleic (Cis,~) acid. Myristic (Ci4:o) acid was removed

within 30 days in cornparison to 50 days for paimitic (C16:o) acid (Figures 5.3 and 5.4) in

cultures fed with 1 O r n g - ~ - ' linoleic ( C l 4 acid.

OIeic ( C ~ S , ~ ) acid was observed at ail linoieic acid concentrations examined

(Figure 5 . 5 ) . Both oleic ( C i x : i ) and palmitoleic (C16:1) acids were found in cultures

receiving 100 rngL-' linoleic (CIsLr) acid however, only oieic (C18:I) acid was detected in

cultures receiking 10 r n g ~ - ' and 50 mg^" linoleic (Cig:?) acid. In cultures receiving 100

r n g ~ - ' linoleic (Gia:-) acid, oleic (Cln,l) and palmitoleic (C16,1) acids reached maximum

concentrations of 5 mg L-' (1 9 pmoles-~*') and 10 rng-L" (4 1 pmoles-~- ') after

approsimately 1 I and 15 days, respectively.

Xcetic acid was produced in ali cultures receiving linoleic (C18:2) acid. Acetate

concentration initially increased up to approximately 60 mg^" between day 20 to 25. the

time during which linoleic (C18:t) acid was degraded to below detection levels- Between

day 20 to day 60, acetate levels did not increase significantty (Figure 5.6) while palmitic

(C 16.0) and myristic (Ci4:o) acids accumulated to maximum concentrations. However,

afier day 60, concentrations of both paimitic (&:O) and myristic (C14:0) acids began to

0 10 20 30 4 0 5 0 60 7 0 80 90 100

Time. days

Figure 5.4: Myristic (Ci4:o) acid profiles in cultures receiving linoleic (Ci*::) acid (LA). (Average for tnplicate samples.)

O 5 10 15 20 2s Timc, àavs

Figure 5 . 5 : Oleic (CIg:l) acid (0.4) profiles in cultures receiving linoleic (C18-2) acid (LA). (Average for triplkate samples.)

decrease (Figures 5.3 and 5.1) and large quantities of acetate of up to 200 r n g - ~ - '

accumulated in cultures receiving 50 and 100 r n g - ~ - ' linoleic (CIg:2) acid. At I O mg-L-' - linoleic (Cig:-) acid, most of the acetate was removed by day 70. Trace quantities of

propionate (C3), iso- and n-butyrate (C4) and iso- and n-valerate (CS) were detected at dl

linoleic (C :) concentrations examined (data not shown).

-

O 20 4 0 60 80 100 120 1 40

Time, days

Figure 5.6: Effect of linoleic (CIg:2) acid (LA) concentration on acetate production. (Average for triplicate samples.)

\,lethane \vas produced at al1 linoleic (C18.~) acid concentrations esarnined (data not

shown). Mass balances shown in Figure 5.7 were calculated by convening al1 the

byproducts (escludin_g methane) to both a linoleic (CIR:~) acid basis and a carbon basis for

cultures receivine - 10, 50 and 100 r n g - ~ - ' linoleic ( C i g : ~ ) acid (see example calculations in

Xppendis B). AIthough fluctuations were noted, in panicular for cultures with 100

r n g - ~ ' ' linoleic (CIO:2) acid, the mass balances indicate that the primary products of

linoIeic acid degradation were detected and measured.

Figure 5.7: Linoleic (C18.2) acid degradation study mass balance. (Averages for triplicate samples. error bars represent standard deviation for the samples)

5.1.2 Inhibiton Effects of Linoleic (CI8:?) Acid on Methanogenesis

-4. -4 cerare Degradation

Acetate degradation was examined using 1600 i 100 m g L-' VSS in the presence and

absence of diethyl ether for duplicate control cultures (Figure 5.8). Within approximately

2 da!.s. cultures containing neither linoleic (CI8.?) acid nor diethyl ether consumed the

added 100 mg^-' acetic acid. Acetate degradation was inhibited and complete

consumption was accompIished within approximately 14 days in the presence of diethyl

ether. although acetic acid was 80 to 90 % removed within 2.5 days (Figure 5.8).

Acetate degradation profiles for cultures receiving Iinoleic (Cl8 -) acid are shown in

Figure 5.9. No inhibition was observed when comparing cultures receiving only diethyl

ether uith those receiving 10 mg^" linoleic (Cigi2) acid. However, at concentrations of

30 rngL-' or greater (Figure 5.9), linoleic acid inhibited acetic acid consumption.

Up to 10 days after inoculation, none of the initially added acetate was removed in one

culture receiving 100 rng.~- ' linoleic (Clg:Z) acid (Figure 5.9, test :! 1). In a later study

( 100 mg^" linoleic acid test R), aceticlastic methanogenesis was inhibited up to

day 15 in comparison to the control cultures. M e r day 10, however, the inhibition was

relieved in one culture (test ff2) but remained in the other culture even at day 25 (test g1).

Initial acetate degradation rates for control cultures and those receiving 10 mg^'

linoieic (Clw -) acid are sho\\-n in Table 5.2. In comparison to control cultures, diethyl

ether reduced the acetate degradation rate by approximately 77%. The presence of I O

r n g - ~ " linoleic acid decreased the initial acetate degradation rate by approximately

30°% in cornparison to the diethyI ether control.

O 0.5 1 tS 2 25

-w Figure 5.8: Acetic acid removal in the absence of linoleic acid (LA).(DE = diethyl ether, average for duplicate results)

+ 10 mglL LA test 2

-30 mglL L A test 1

- 5 0 mglL LA test 1

e l 0 0 mg/L LA test 1 - I I - + 100 mg/L LA test 2

O 5 10 15 20 25 30 35 Time, days

Figure 5.9: Acetic acid removal in the presence of linoleic (C18:2) acid (Average for tnplicate samples shown)

1 Average of duplicates, ' ~ v e r a ~ e and standard deviation of triplicate samples. DE = diethyl ether, LA = linoleic (CIK:t) acid, ND = Not degraded

Table 5.2: lnltial acetate degradation rates for varying iinoleic (CIR:?) acid concentrations

B. m*droger Comwnpiiotl

i. Hydrogen uptake 1 day afier linoleic (Clg:~) acid addition

Hydrogen uptake profiles shown in Figure 5.10 are for duplicate control cultures in

the presence and absence of diethyl ether. Al1 experiments were conducted using 1550 k

90 r n g - ~ " VSS. The differences in rate constants observed among control cultures are

Condition Esamined

Control Control + DE 1 O mg^" LA 30 r n g - ~ " LA 50 rng.~" LA

Initial Acetate Deeradation Rates ( mg~-'-d-') 2

85 1 9'

13.6 k 0.3' 2

-

ND

ND A

ND

negligible indicating that diethyl ether caused insignificant inhibition of

hydrogenotrophic methanogens. At the four concentrations examined, tinoleic (CIR:P)

acid did not significantly inhibit hydrogenotrophic rnethanogenesis and hydrogen was

consumed within 12 hours (Figure 5.1 1).

The shape of the hydrogen uptake profiles was non-linear and followed a first order

kinetic expression (equation 5.2, where Hz is the hydrogen concentration (pmoles /

bonle) and k is the first order rate constant, d-'). The first order expression was

used to compare the data sets using least-squares regression.

The hydrogen first order rate constants were detennined for the data in Figure 5.1 1

and are shown in Table 5.3 . NI data sets provided rZ > 0.97 for the least-squares

Fisure 5 . IO: Hydrogen removal in the absence of linoleic (Ci rr:2)

acid (LA). (DE = diethyI ether, Average for duplicate results shown)

Figure 5.1 1 : Hydrogen removal in the presence of linoleic (Cis:2) acid (LA). Experirnent performed 1 day after L.4 inoculation. (Averages for tri plicate samples, error bars represent standard deviation for the samples)

regession. Based on the Tukey's paired cornparison procedure (Box et al.. 1978) at a 95

"io confidence level, the rate constants for cultures receiving O (without diethyl ether), O

(with diethyl ether) and 10 r n g - ~ - ' linoleic (Cig,z) acid were not significantly different

from each other but were significantly different from those for cuitures receiving 30, 50

and 100 rne .~ ' ' linoleic (Cin,2) acid. No significant differences were found between rate

constants for cultures receiving 30, 50 and 100 r n g ~ - ' linoleic acid.

i i . Hydrogen uptake 18 days and 35 days afier linoleic ( C l g , ~ ) acid addition

Cultures. inoculated with linoleic (Clg,2) acid and receiving hydrogen one day after

preparation of the serum bottles, were injected with hydrogen again on day 18 and day

35. Data for hydrogen profiles shown in Figures 5.12 and 5.13 for day 18 and day 35,

respectively were used to caiculate first order constants for hydrogen uptake rates shown

in Tables 5.4 and 5.5. Statistical differences between rate constants for dôy 18 and day

3 5 were evaluated using the Tukey's paired cornparison procedure (Box et al., 1978).

Table 5 - 3 : First order rate constants for hydrogen removal 1 day afier adding LA

' LA Concentration, rng-L1 First Order Rate Constant, d"

Average andstandGd deviation for triplicate samples shown DE = diethyl ether, LA = linoleic (Cie:2) Acid

O (without DE) O (with DE)

10

6 l ime , hrs

0.3871 0.006 0.3791 0.006 0.374 I 0.006

Figure 5.12: Hydrogen removal in the presence of linoleic (Clg2) acid (LA) 18 days afler LA addition. (Control duplicates; Average for triplicate samples and the error bars are standard deviation for the samples)

Significant differences were found between the rate constants s h o w in Tables 5.4 for

cultures receiving 10 and 30 r n g - ~ " linoleic (C1gZ2) acid in cornparison to those cultures

receivine 50 and 1 00 mg^" linoleic (C s:z) acid. Uptake rate constants for cultures

Figure 5.13 : Hydrogen removal in the presence of linoleic (Cls:~) acid (LA) 35 days afier LA addition. (Control duplicates; Averages for triplicate samples, error bars represent standard deviation for the samples)

Table 5.4: First order rate constants for hydrogen removal 18 days afier adding LA

1 Duplicates, ' ~ v e r a ~ e and standard deviation for tnplicate samples L.4 = linoleic (C1g2) k i d .

LA Concentration, rng -~~ '

O (without DE) O (with DE)

10 30 50 1 O0

First Order Rate Constant, d"

1

0.4 19' 0.396~ ,

0.374 t 0.007~ 0.359 i 0.001~ 0.323 t 0.004~ 0.324 + 0.009' .L

Table 5.5 : First order rate constants for hydrogen removal 3 5 days after adding LA

receiving 50 and 100 mg-^-' linoleic (Cis:z) acid were significantly different from those

L.4 Concentration. r n g - ~ - '

O (without DE) O (with DE)

1 O 30 50 1 O0

receiving 30 mg^" linoleic CI^,^) acid. In comparison, no significant difference was

First Order Rate Constant, d"

0.384~ 0.364'

0.337 I 0.006~ 0.332 I 0.0003~ 0.3 18 A 0.003' 0.330 I 0.006~

obsenxd for cultures receivine 50 and 100 r n g ~ - ' linoleic (Cin:2) acid At day 35.

' ~ u ~ l i c a t e s ~ ' ~ v e r a ~ e and standard deviation for triplicate samples L.4 = Iinoleic (C18:2) Acid

significant differences between the uptake rate constants were not observed at any of the

linoleic (C18:') acid concentrations examined. Duplicate cultures with and without

diethyl ether Lvere not compared because the Tukey's paired comparison procedure

requires the same sample size for ail data sets.

5.2 Discussion of Results

5 -2.1 Linoleic (C 1 ~ : ~ ) Acid Degradation

Oleic (Clx:i), palmitoleic (CK,:~), palmitic (Cic;~), myristic (Cid:~), lauric CE:^) and

acetic acids were byproducts of linoleic acid degradation. Canovas-Diaz et al.

( 1 99 1 ) and Novak and Carlson (1970) also observed these byproducts. Although,

octanoic (Cg) acid was not detected, hexanoic (C6) acid was detected for the first time as

a byproduct of linoleic acid degradation, providing fiirther support that the

degradation mechanism proceeds via P-oxidation (Jeris and McCarty, 1965; Novak and

Carlson, 1970; Weng and Jeris, 1976). The distribution of byproducts was a function of

the initiai linoleic (Cln:;) acid concentration with palmitoleic (C16:1) and hexanoic (Cs)

acids detected only in cultures receiving 100 r n g - ~ - ' linoleic (C18:Z) acid.

ln this study, stearic (CIR:~) acid was not detected, however, palmitoleic I CI^:^) acid, an

unsaturated P-oxidized LCFA byproduct, was obsewed. The conversion of linoleic

(C18:2) acid to palmitoleic (C16:l) acid (Table 5.1) is contrary to the mechanism proposed

by Mackie el al. (1991) and Novak and Carlson (1970) that complete saturation of al1

double bonds occurs pnor to P-oxidation. Fujimoto et al. (1 993) proposed that linoleic

CI^:^) acid is first hydrogenated to stearic (CI8:o) acid by several bacteria and

subsequently P-oxidized to acetate. In contras, Kontala and Bagby (1 992) observed the

formation of 1 O-hydroxy-octadecanoic acid (ie. a hydroxylated stearic (Cls:~) acid

compound) during the anaerobic conversion of linoleic (CIR:~) acid. They did not report

the formation of any P-osidized byproducts.

.A mechanism for the formation of palmitoleic (C16:l) acid has not been previously

reported. A proposed pathway for the formation of palmitoleic (CIG:~) acid from linoieic

(CIR:t) acid is shown in Figure 5-14. In the first pathway (A), linoleic CI^:^) acid is

hydrogenated at carbon 12 to produce oleic (Cls:~) acid. SubsequentIy, oleic (C1s:J acid

undergoes P-oxidation and a double bond isomerization to form palmitoleic (C16:l) acid.

The second pathway (B) involves hydrogenation of linoleic ( C l 4 acid at carbon

number 9 followed by P-oxidation to form palmitoleic (C16:l) acid. In both pathways, the

formation of palmitoleic ( c16 :~ ) acid takes place via isomerization of carbon-carbon

double bonds (Canovas-Diaz et a/., 199 1). Experimental support for the first pathway is

provided by studies conducted by Canovas-Diaz el a/. (1 99 1) in which palmitoleic (C 16: 1)

acid was observed during the degradation o f oleic (C18-i) acid. Results fiom this study

coupled with the work by Canons-Diaz et al. ( 1 99 1 ) strongly suggest that hydrogenation

takes place prior to the P-oxidation during the formation of palmitoleic (Cl6 [ ) acid.

Oieic (Cl8 I ) Acid

CH j(CHz)jCH:CH2CH:CH=CH(CH2)7~~:~

Isomerization

Palmitoieic (Cl6 ,) Acid

CH j(CH2)aCH2CH2=CHiCH2CH2(CHL)5C02H

Figure 5.14: Proposed pathway for formation of palmitoleic (Cl6-*) acid fiom linoleic (Ci8 2 ) acid

In cultures receiving 50 and 100 mg-L-'linoleic CI*,^) acid, both palmitic (Ci6,a) and

myristic acids accumulated simultaneously and remained constant for

approximately 60 days. Sirnilarly, Canovas-Diaz et al. (1 991) observed that myristic

(CiJIo) acid was completely degraded only afier approximately 80 days. In contrast,

HeukeIekian and hIueIler ( 1958) reported that 80% palmitic (C16:O) acid was found to be

removed fiom a raw sewage sludge feed containing mixed LCFAs within a shorter time

of 25 days.

5.2.2 Linoleic (C i s : t ) Acid-Methanogenic Inhibition Studies

The influence of LCFAs on methanogens has been previously reported by a number of

researchers (Beccari rr al., 1996; Hwu , 1997; Angelidaki and Ahring, 1992; Canovas-

Diaz er al., 199 1 ; Koster and Cramer, 1987; Hanaki, et al., 198 1 ; Demeyer and

Hendricks, 1967). However, no research has esamined the effect of linoleic (C is:2) acid

on aceticlastic and hydrogenotrophic methanogens.

In this study, the effect of diethyl ether on aceticlastic methanogens was investigated

and it was found to inhibit acetate consumption in cornparison to cultures receiving only

acetate. Nevertheless, acetate was steadily consümed in cultures fed with acetate and

diethyl ether. Aceticlastic methanogenesis was also inhibited under al1 linoleic (CIX:~)

acid conditions esamined. Linoleic (Cig:z) acid was degraded without any lag period

during test # 1 and the acetate concentration remained greater than the amount initially

added. In two cultures receiving 100 mg-^" linoleic (C18:2) acid (test # 1 and test #t),

acetate concentration increased (> 100 mg-^-') for test 3 1 while the acetate concentration

decreased afier a lag of about 10 days for test #2. Linoleic (C18:2) acid was removed

within the same tirne period for both cultures (data not shown). Hence. variation in the

acetate profiles suggests that LCFA byproducts may be more inhibitory to cultures during

test 81 compared to test #2.

No evidence of synergistic interaction benueen LCFAs and diethyl ether was obsenred

in cultures receiving acetate. In the presence of 14.2 g - ~ - l of diethyl ether and a change

in linoleic (Cl* z) concentration of 20 rng.~- ' (from 10 to 30 rng-~' ')? a s ipifkant

~rariation in acetate consurnption \vas shown (Figure 5.9). When comparing cultures

receiving dieth)-1 ether to those receiving 10 r n g - ~ " linoleic acid. not much

difference in acetate consurnption was observed.

The presence of diethyl ether was found to cause minimal inhibition of

hydrogenotrophic methanogenesis in comparison to controls without diethyl ether.

Houxxer? in cultures receiving greater than 10 mg-L-I linoleic (Cisz) acid.

hydrogenotrophic rnethanogens were slightty inhibited in comparison to the controls.

AIthough statisticall>r significant, the inhibition is likely not severe enough to cause

hydrogen accumulation in anaerobic reactors. In comparison to controi cultures, Hanaki

et ai. (1 98 I ) reported that a mixture containing LCFAs ranging fiorn CIO to Cls inhibited

acet ic lastic niethanogens. However, they did not identify the LCFA or concentration

responsible for causing the inhibition. Additionally, based on manometric measurements.

Deymeyer and Henderickv (1967) reported that approximately 70 r n g ~ - ' of linolenic

(C 1 *.;) acid inhi bi ted hydrogenotrophic methanogens.

The first order rate constants at day 1, 18 and day 35 in Tables 5.3, 5.4 and 5.5.

respectively show that hydrogenotrophic methanogens were not significantly affected by

long esposure to linoleic (C 18:?) acid and its LCFA f3-osidation byproducts.

5.3 Summaq

Linoleic (C18:2) acid was degraded to shoner chain LCFAs and ultimately to acetic

acid at 21 OC. The initial concentration of linoleic ( C l 4 acid affected byproduct

distribution and removal from solution. Saturated and unsaturated LCFAs were observed

0-osidized byproducts at al1 concentrations examined. Oleic (CI 8; I), palmitic (C 16~0) and

rnyristic (C l4,o) acids were observed in cultures with 10, 50 and 100 r n g - ~ - ' linoleic

( C 4 acid. Additionally, palmitoleic (C16:l), lauric (Clz:o) and hexanoic (CG) acids were

observed only in cultures receivins 100 mg^" linoleic (C 18,t) acid. Hydrogenation of

unsaturated LCFAs is not necessary for substrates to enter the P-oxidation reaction since

both palmitic (C,G:O) and palmitoleic (Cl6:1) acids were obseiwd simulatneously.

Aceticlastic methanogens were inhibited by diethyl ether but hydrogenotrophic

methanogens were unaffected. Linoleic (C18:t) acid was found to inhibit aceticlastic

methanogens but only slightIy inhibited hydrogenotrophic methanogens. Above a given

linoleic (C 1 8 , ~ ) acid threshold concentration of 10 m&, aceticlastic methanogens were

severely inhibited. Although, some differences were observed when comparing cultures

recei\ing linoleic (Cix.2) acid, rnost of the hydrogen was removed within 12 hours.

M e r prolong esposure to linoleic (Cl*,?) acid and its 0-oxidation byproducts,

hydrogenotrophic methanogens were not significantly affected.

6.0 DEGRADATION OF OLEIC ACID AND ITS INHIBITORY EFFECTS ON METHANOGENESIS

6.1 Experimental resuits

6.1 . 1 Oleic (C 1 8 : ~ ) Acid Degradation

Degradation profiles for oleic (Cis:~) acid and its P-oxidized byproducts at 21 OC are

shown in Figures 6.1 to 6.4. Ail experiments were conducted using 1600 + 100 r n g - ~ - '

VSS. Oleic (Cix:l) acid was consumed within about 10 days in cultures receiving less

than 30 r n g ~ * ' . In cornparison. oleic (Cls,i) acid was undetected in less than 30 days

(Figure 6.1) when oleic (Clgl) acid greater than 50 mg-L1 was added.

No LCFA byproducts were observed when 10 rng-~- ' oleic (Cl8,,) acid was added.

However. palmitic (Ci6 O), myristic (C14:a) and acetic acids were detected in cultures fed

lvith 30, 50 and 100 mg^-' oleic (Clsi) acid. Palmitic (CIs,o) acid accumulated to

between 70 and 80 mg*^-' within days 10 to 20 and was undetected within 30 days

Figure 6.1 : Oleic CI^:^) acid (OA) profiles in cultures receiving oleic (Crs:i) acid. (Average for triplicate samples.)

in cultures receiving 100 rng-~ ' ' oleic (Cls:~) acid (Figure 6.2). Palmitic (Cls) acid

accumulation was limited and was degraded to undetectable ieveis within approximateiy

12 and 28 days in cultures receiving 30 r n g - ~ - l and 50 rng-L-' oleic (Ci8:i) acid,

respectively. Similady, myristic (Clr:~) acid (Figure 6.3) accumulated to 10 mg-^-' in

cultures receiving 50 r n g l - ' and 100 mg-L-' oleic (Cls,i) acid. No stearic (Cls:o),

paimitoIeic ( C I ~ J ) , lauric (C12:0) nor hexanoic (C6) acids were detected at any oleic (CM:~)

acid concentration examined.

In cultures receiving 10, 30 and 50 r n g - ~ " oleic (Clarl) acid (Figure 6.4), less than 10

r n g ~ - ' of acetate was detected. However, acetate accumulated between 20 to 30 days but

was undetected within 45 days in cultures receiving 100 r n g - ~ - ' oleic acid.

Mass balances are shown in Figure 6.5 for al1 oleic (Cie,l) acid concentrations

esamined. The baIance is based on a sum of oleic (Cis:r) acid, LCFA fi-oxidized

O 5 10 15 20 25 30 TI me, days

Figure 6.2: Palmitic (&:O) acid profiles in cultures receiving oleic (C1n:i) acid (OA). (Average for triplicate samples.)

Figure 6.3: Myristic (Cl4:o) acid profiles in cultures receiving oleic (Ci ,) acid (OA). (Average for triplicate samp les.)

O 10 20 30 40 50

Time, days

Figure 6.4: Effect o f oleic (Ci8:1) acid (OA) concentration on acetate production. (Average for triplicate samples.)

byproducts plus acetate and is reponed on an oleic (C18:1) acid and carbon basis (see

exarnple calculations in Appendix B). Although fluctuations are seen in Figure 6.5, the

profiles account for the initially added oleic acid and al1 the byproducts of P-

osidation.

6.1.3 Inhibitoq Effects of Oleic (Cis:r) Acid on Methanogenesis

In cornparison to controls without diethyl ether, acetate degradation was inhibited in

the presence of diethyl ether (Figure 6.6). Al1 experiments were conducted using 1 500 rir

70 r n g - ~ - ' VSS. Profiles in Figure 6.7 show acetate degradation was also inhibited by

oieic (C,SJ) acid at al1 concentrations examined. Although inhibition was observed at 10

Time, days

Figure 6.5: Oleic (CIR:I) acid degradation study mass balance. (Averages are for triplicate samples, error bars represent standard deviation for the samples)

O 5 10 15 20 25

liffi*~ Figure 6.6: Acetic acid removal in the absence o f oleic ( C l g : ~ ) acid. (OA = oieic (CIR:I) acid, DE = diethyl ether, average for duplicate results)

Figure 6.7: Acetic acid removal in the presence o f oleic (Clg:~)

acid (OA). (DE = diethyl ether; Duplicate control; Average for triplkate samples)

rn=-~-' oleic (Cip;I) acid, acetate was undetected between day 25 and day 30. In

comparison. acetate accumulated and was not degraded in cultures receiving 100 rng~-l

oleic (C 18: 1) acid up to day 25.

Initial acetate degradation rates are s h o w in Table 6.1. In the presence of diethyl

ether, the acetate degradation rate was reduced by approximately 85% in comparison to

control cdtures. When comparing cultures receiving diethyl ether, oleic (C18:1) acid also

influenced aceticIasric methanogens at 10 r n g - ~ - ' as a 60% rate reduction was observed.

Table 6.1 : Initial acetate degradation rates for varying oieic (C is : , ) acid

B. kiydmger~ Cotmcmpriorr

Profiles for control cultures without the presence of oleic (Clg:~) acid in Figure 6.8

sholv that hydrogen removal was not affected by the presence of diethyl ether. Al1

esperiments were conducted using 1600 i 80 r n g - ~ " VSS. In the presence of oleic

(C acid, small differences were observed between profiles for cultures receiving 10,

30. 50 and 100 mg^-' oleic (Cig,l) acid. Hydrogen uptake rate constants shown in Table

6.2 were calculated assuming first order kinetics. Using the Tukey's paired comparison

procedure (Bos er al., 1978). the first order rate constants for al1 the oleic (C18:[) acid

concentrations h

Condition Examined

Control Control A DE 10 r n g ~ - ' 0.4 3 0 mg^-' 0.4 50 mg-^" OA 1 00 mg L-' O A

Initial Acetate De radation Rates S ( mg-L- *d-') 54' 8'

3 -C 1' ND ND ND

Duplicates, 'r2verage and standard deviation for triplicate sarnples. ND = Not degraded, DE = diethyl ether, OA = oleic (Clgl) acid.

concentrations examined were compared. The constants for cultures receiving 30, 50 and

100 r n g - ~ - ' were not signiticantly different fiom each other but were aatistically

Figure 6.8: Hydrogen removal in the absence of oleic (Cis:~) acid (0.4). (DE = diethyl ether, Average for duplicate results shown)

Figure 6.9: Hydrogen rernoval in the presence of oleic (Clg: , ) acid (OA). (Averages for triplicate samples, error bars represent standard deviation for the samples)

different (95 % confidence) frorn those receiving IO mg^"

Table 6 2: First order rate constants for hydrogen removal in the presence of oleic (C,g:l) acid

6.2 Discussion of Results

6.2.1 Oleic (C 18: 1 ) Acid Degradation

By combining the results presented in Chapter 5 with those presented in this chapter

the question of whether hydrogenation of unsaturated Ci* LCFAs takes place prior to P-

osidation can be addressed. During linoleic (Clg12) acid degradation, oieic (CI 8. l) acid

[vas formed as a trace product (Figure 5 . 9 , indicating that hydrogenation could occur

prior to P-osidation. However. the results in Figure 6.1 show that oleic (Cls:~) acid was

degraded within the same time period as linoleic acid. This indicates that

hydrogenation of iinoleic (CIR:2) acid was not required prior to P-oxidation. If

hydrogenation of linoleic (CIsz2) acid was a necessary step prior to P-oxidation, oleic

(Cis 1 ) acid should have accumdated to rnuch higher concentrations, to the order of the

initial linoleic acid concentrations, assuming the rate of conversion is sfow. Based on the

results presented in Chapter 5 and those obtained frorn studies with oleic (Cl*:,) acid, it

Oleic (Clg:~) Acid Concentration, mg L-'

O (without DE) O (with DE)

10 30 50

1 O0

appears that complete saturation was not required before P-oxidation since no stearic

First Order Rate Constant, hf'

0.300' 0.295'

0.262 I 0.004' 0.222 i 0.008' 0.209 k 0.001' 0.212 ,+ 0.003'

' ~u~ l i ca tes ' ' ~ v e r a ~ e and standard deviation for tnplicate samples DE = diethyl ether

(Clg) acid \:as detected as a by-product of either linoleic (Cl&?) or oleic CI^:^) acid. This

observation fûnher suggests that complete saturation was not required. Funher support

for this hypothesis is provided by observation of palmitoleic (Cl6:I) acid (Figure 5 .3 )

fiom linoleic acid experiments.

Although complete hydrogenation does not appear to be a necessary step prior to P-

osidation. the data from linoleic (C18:2) and oleic (C18:1) acid degradation support the

hypothesis that hydrogenation and P-oxidation may occur concurrently. The formation of

palmitic (Cl6.0) and palmitoleic (Cl6.1) s i r n u l t a n e ~ ~ ~ l y (Figure 5.3) supports this point of

view .

The primary byproduct of oleic (Clgri) acid degradation was the h l l y saturated

palmitic (C16:O) acid (Figures 6.2). If P-osidation occurred before hydrogenation,

unsaturated Clo LCF.4s should have been observed. The possibility exists that

unsaturated C16:1 LCFAs were produced but then were rapidly converted to the relatively

slou.ly degrading palmitic (C16:o) and myristic acids. Studies with saturated C16:0

and unsaturated C16;1 LCFAS would be required to test this hypothesis. In addition to

palmitic (C 16:0) and myristic (CIJ:~) acids, Canovas-Diaz el al. (199 I ) also observed the

presence of palmitoleic (C16.1) acid during anaerobic conversion of oleic (Ci8 ,) acid.

In previous studies by Novak and Carlson ( 1 970), only trace amounts of Ciz:~ to Cg:O

acids and no acids were observed during degradation of linoleic (Cle:~), oleic (C18:1),

stearic (C 1 pdmitic (C i6:0) and rnyristic (C i4:o) acids. However, in cornparison,

significant concentrations o f C16:o and Cr+o acids were detected fiom oleic ( C I R : ~ ) acid

depadation. No Cg and C I O acids were observed at any concentration o f the oleic (Cigl)

acid examined. These observations support the hypothesis that f3-oxidation and not a- or

w- oxidation is the prirnary mechanism for Cl8 LCFA degradation in anaerobic systems.

Accumulation of palmitic (&:O) and myristic (C1.4 acids during the degradation of

50 r n g - ~ - ' and 1 00 rng-~- ' oleic (C 18:~) acid (Figures 6.2 and 6.3) suggested that these

byproducts may be inhibitory to the microbial cornmunity. However, inhibitory effects

of organisms consuming palmitic (&:O) and myristic ( C I J : ~ ) acids were eventually

relieved as these acids were degraded and hence. removed fiom solution. In cornparison.

linoleic (CIK:f) acid caused a greater residual inhibition of its byproducts than did oleic

(Cix:l) acid as the inhibition was relieved sooner, afier approxirnately 5 to 25 days in

oleic CI^:^) acid fed cultures.

The mass balance accounted for al1 the initially added oleic (Cig:~) acid and its B-

osidized byproducts up to day 15. The decrease observed after day 15 is due to the

consumption of acetate.

6 .2 .2 Oleic ( C I X : ~ ) acid-Methanosenic Inhibition Studies

Similar to studies conducted with linoleic ( C I R : ~ ) acid, no evidence was found for

acetate inhibition caused by synergistic effects between diethyl ether and oleic (CI*: 1)

acid. Inhibition of acetate consumption was observed in cultures receiving greater than

I O r n g ~ - ' oleic (Cla:l) acid. In cornparison, Angelidaki and Ahring (1992) and Koster

and Cramer (1987) reponed greater than 300 rng-~-' oleic (Clg:~) acid inhibited

consumption of acetate. Differences in concentration at which inhibition is initiated in

this work and that reported by Angelidaki and Ahring (1 992) and Koster and Cramer

( 1 987) are probably due to culture adaptation as welI as the high temperature conditions

of 55 OC and 30 OC respectively, under which the latter studies were conducted.

Additional differences may be due to the VSS concentration used in this study in

comparison to those reported by Angelidaki and Ahring (1992) and Koster and Cramer

(1987) in their studies. Angelidaki and Ahring (1992) reported al1 experiments were

conducted with 5% v/v cattIe manure instead of VSS concentrations. Koster and Cramer

(1 987) did not report VSS concentrations in their study.

Methanogenic inhibition by milk fat containing oleic (Cis;l) acid was also reported by

Perle el al. ( 1995). Although acetictastic or hydrogenotrophic methanogenic inhibition

was not specifically investigated, they reported significant methanogenic activity losses

for cultures inoculated with milk fat or oleic ( C I * : ~ ) acid.

6.3 Summary

Oleic (Cl*:,) acid was degraded to shorter chain LCFAs and ultimately to acetic acid at

2 1 "C. During oleic (Cisyi) acid degradation, intermediate length Cio to Cg fatty acids

were not obsewed as P-osidation degradation byproducts at any concentrations

examined. In comparison to studies with iinoleic ( C I R : ~ ) acid, palmitoleic (Cl6 1). lauric

( C 4 and hexanoic (C6) acids were not detected dunng the degradation of 100 r n g ~ - '

oleic (C l x . 1) acid. Oleic (Clx:~) acid was degraded to pairnitic (C ic:o), myristic (C14:0) and

acetic acids at 2 1 "C. LCFA byproduct distribution profiles were a function of the oleic

(Clx:~) acid concentration. Acetic acid did not accumulate in cultures receiving less than

100 mg-L" oleic (Cia:l) acid during the degradation studies.

Diethyl ether inhibited aceticlastic methanogens but not hydrogenotrophic

methanogens. Aceticlastic methanogens were affected by oieic (C1s:1) acid at al1

concentrations examined. At greater than 10 rng-~-' oleic (C 18:~) acid, aceticlastic

methanogens were initially inhibited but the inhibition was relieved after approximately

10 days. Although hydrogenotrophic methanogenesis was inhibited, the inhibition was

minimal.

7.0 DEGRADATION OF STEARIC (Ciri:o) ACID AND ITS lNHIBITORY EFFECTS ON METHANOGENESIS

7.1 Experimental Results

7.1 - 1 Stearic (Cl s:o j Acid Degradation

Steanc (C~~NI) acid degradation profiles at 2 1 O C are shown in Figure 7.1. Al1

esperiments were conducted using 1600 i 80 rng -~ ' ' \%S. .Mer 55 days, approsimately

50 to 60 % of the stearic acid remained undegraded in cultures receiving 10, 30.

50 and 1 00 rng-~-'. No LCFA byproduas were observed, however, acetate was detened

at al1 stearic (&O) acid concentrations examined (Figure 7.2). Acetate accumulated to

between 40 to 50 rng~-l and was degraded after day 40 only in cultures dosed with 10

rng~-l. However, in cultures receiving 30, 50 and 100 r n g - ~ " stearic (C1s:o) acid, acetate

was not degraded. Degradation rates calculated from slopes of the curves in Figure 7.1

are shown in Table 7.1 for al1 stearic acid concentrations examined. The rates are

s h o w to increase with the stearic acid concentration from 10 to 100 rng~'~.

A mass balance for al1 the stearic (CIx:o) acid concentrations examined is shown in

F ip re 7 .3 . The balance is based on convening the acetate byproduct to a stearic (Cla:o)

acid basis and adding it to the amount of measured stearic (Cig,o) acid. .A carbon balance

is also presented in Figure 7.3 (see example calculations in Appendix B).

Table 7.1 : Stearic (C ,*:O) acid degradation rates S tearic (C 18:0) Acid

Concentration, mg- L - ~ 10 30 50 1 O0

Degradation Rate, pg LCFA-mg VSS-~-~" ,

0.040 f 0.005 0.141 k0.017 . 0.250 t, 0.007 0.471 + 0.064

O 10 2 O 3 0 4 0 50 6 O Time, d a y

Figure 7.1 : Stearic (C18.0) acid (SA) degradation profiles in cultures with stearic (Ci* acid. (Average for tnplicate samples.)

O 10 20 30 40 50 60 lime, &ys

Figure 7.2: Acetate production profiles for cultures fed with stearic (C 1 *:O) acid (SA). (Average for triplicate samples.)

Figure 7.3 : Stearic (C s:o) acid degradation mass balance. (Averages for triplicate samples, error bars represent standard deviation for the sampIes)

7 1.2 Inhibiton. Effects of Stearic (Cig:o) Acid on Methanogenesis

A. A cerate Degradation

Diethyl ether and stearic ( C i s ; o ) acid inhibited methanogenesis at al1 concentrations

examined (Figures 7.4 and 7.5). In comparison to controls, diethyl ether reduced the

acetate degradation rate by approximately 87%. Unlike linoleic (Cia,2) and oleic (C18:l)

acids. however, increasing concentrations of stearic (C1r:o) acid provided no additional

inhibition. Initial acetate degradation rates for control cultures and for those receiving

stearic (Cis:o) acid are shown in Table 7.2. The acetic acid degradation rate for al1 stearic

(CIX.O) acid concentrations was approximately 45 % of the degradation rate in the diethyl

ether controls (Table 7.2). During the duration of the experiment, stearic (Cis:o) acid was

not completely degraded and remained in the system. Based on the Tukey's procedure,

Table 7.2: Acetate degradation rates for varying stearic acid

Figure 7.4: Acetic acid removal in the absence of stearic ( C i s : ~ ) acid (SA). (S.4 = stearic ( C i s : ~ ) acid, DE = diethyl ether, average for duplicate results)

concentrations

there are no significant (95 % confidence) differences observed between the rates at any

of the concentrations examined. Al1 experiments were conducted using 1500 + 100

Condition Examined

Control Control + DE 10 r n g ~ - ' SA 30 r n g ~ - ' SA 50 r n g ~ - ' SA

3

Acetate Degradation Rates ( mg^" -d-')

1

61 -8' d

7.90' 3.66 î 0.23~ ,

3-50 I 0.30' 3.32 1 0.13'

1 00 r n g ~ - ' SA w

3.78 I 0.33' A

T~uplicates, '.Average and standard deviation for uiplicate samples DE = diethyl ether. SA = stearic (&:O) acid

Figure 7.5: Acetic acid removal in the presence of stearic (CI8:O) acid (S.4). (DE = diethyl ether; Duplicate control; -4verage for tnplicate samples.)

Comparing control cultures, the presence of diethyl ether had very little effect on

hydrogenotrophic methanogens and most of the hydrogen was removed within 12 hours

(Figure 7 . 6 ) . Similarly, profiles for cultures dosed with 10, 30, 50 and 100 r n g ~ - ' stearic

(CIR:O) acid show most of the hydrogen was removed within 12 hours (Figure 7.7). Rate

constants for hydrogen uptake were calculated assuming first order kinetics and are

shown in Table 7.3. Using the Tukey's procedure to compare the stearic (Cls:o) acid first

order data indicated that there were significant (95 % confidence) differences observed

between the rate constant for cultures receiving 10 rng-~- ' stearic (Clsa) acid in

cornparison to those receiving 30, 50 md 100 rng-~ - ' stearic (Cis:o) acid. No significant

A no SA. no DE

r m SA. with DE

Figure 7.6: Hydrogen removal in the absence o f stearic (CIK:O) acid (SA). (DE = diethyl ether, Average for duplicate resuits shown)

O I> 7 4 6 8 10 12

Time, hrs

Figure 7.7: Hydrogen removal in the presence o f stearic (Cig:o) acid (SA). (Averages for triplicate samples, error bars represent standard deviation for the samples)

differences were observed for cultures receiving 30, 50 and 100 rng-~-' stearic (Cls:a)

acid. hl1 experiments were conducted using 1500 + 90 mg^" VSS.

I Duplicates, '!iverage and standard deviation for tnplicate samples

DE = diethyi ether

Table 7.3 : First order rate constants for hydrogen removal in the presence of stearic (C I~MI) acid

7.2 Discussion of Results

7.2.1 Stearic (Cix.o) Acid Degradation

hlackie er al. ( 199 1 ) and Novak m d Carlson (1 970) proposed that complete double

bond saturation is necessary to initiate the P-oxidation. However, our results show

stearic (Cls:~) acid to be less degradable than linoieic (CIR:~) and oleic (CIR,~) acids.

Therefore. the hypothesis that stearic (Clx:") acid is the only Cl8 acid to initiate P-

Stearic (C l*:~) Acid Concentration, r n g ~ - '

O (without DE) O (with DE)

10 30 50 1 O0

i

osidation is unlikely. Support for P-oxidation of unsaturated LCFAs is evident by work

conducted during this study and that by Canovas-Diaz et al. (199 1) with the formation of

palmitoleic acid from linoleic CI^:^) acid (chapter 5 of this work) and from oleic

(C acid, respectively.

Degradation - rates shown in Table 7.1 increased with stearic (Clg:~) acid concentration.

Lower degradation rates observed for steuic (C18:0) acid in cornparison to linoleic

and oleic (C lg:~) acids may be due to slow uptake of stearic (C1a:o) acid into the cell. The

First Order Rate Constant, d-'

m

0.385' 0.38 1 '

0.378 + 0.012~ ,

0.362 I 0.017' 0.341 k 0.008' 0.342 +, 0.004'

biochemicai reason for observing low stearic (Cls:~) acid degradation rates is unclear, but,

it is possible that the formation of a stearic (Clg:~) acid acetyl CoA complex to initiate the

P-oxidation pathway is the rate determining step.

Palmitic (CIG:O) and myristic (C14:o) acids. detected during the degradation of linoleic

(C ls:z) and oleic CCix:i) acids. were not obsen-ed at any of the stearic (Cls:o) acid

concentrations examined. Angelidaki and Ahring (1 995) reponed the same obsemation

.Accordin~ to the mass balance (Figure 7.3), acetate and stearic ( C I R : ~ ) acid accounted for

al1 the initially added stearic (Cis.o) acid up to day 20. Afier day 20, the decrease in the

rnass balance profile is Iikely due to degradation of acetate produced fiom P-oxidation of

stearic ( C i g : ~ ) acid.

7.2.2 Stearic (CIR:O) Acid-Methanogenic Inhibition Studies

.Ut hough aceticlastic methanogens were inhibited at al1 stearic (Ci x:o) acid

concentrations examined at 2 1 OC, almost al1 of the acetate disappeared within 25 to 30

days. No evidence Lias found for acetate inhibition caused by synerçjstic effects between

diethyl ether and stearic (C,e:o) acid. Inhibition of acetate degradation at threshold

concentrations greater than 300 mg^“ stearic (Cis:u) acid was reponed at 5 5 'C

(-Angelidaici and Ahring, 1992). Disagreements between aceticlastic n~ethanogenic

inhibition data reported in this work and that obtained at thermophilic temperatures by

Angelidaki and -4hring (1 992), are likely due to differeiices in temperature and VSS

concentration at which both studies were conducted. Although acetate accumulated in

cultures receiving gea te r than 1 O r n g - ~ - ' of stearic (Cla,o) acid during the degradation

studies. no acetate accumulation was observed when acetate was fed together with stearic

(Cig:~) acid to examine aceticlastic methanogenic inhibition. It is possible that the

accumulation of acetate during the degradation studies may be due to an initial contact

time for stearic (Cis:~) acid to disrupt aceticlastic methanogens. During this time, stearic

(C 18:0) acid has a greater opportunity to inhibit aceticlastic methanogens even before

acetate is produced from P-oxidation. In comparison, when acetate and stearic (C1g:o)

acid are added together, there is no contact time with stearic (Cig:~) acid in the absence of

acetic acid. Stearic acid does disrupt the aceticlastic methanogenic organisrns but

to a lesser degree in comparison to that observed during the degradation studies and

cornplete acetate consumption is accomplished.

The effects of varying stearic (Ci8:o) acid concentration on hydrogenotrophic

methanogenic activity have not been previously reported. No significant differences in

the first order rate constants were obsewed for cultures receiving stearic (Clx:o) acid.

Although hydrogenotrophic methanogenic inhibition \vas observed, the eîTect was

minimal as most of the hydrogen was removed within the same time period as the control

cultures.

7.3 Summary

No LCFA P-oxidized byproducts were observed during the degradation of stearic

(Clx:o) acid. Acetate accumulated and remained undegraded up to day 55. Aceticlastic

methanogens were inhibited by diethyl ether and al1 stearic (Ci8:o) acid concentrations

examined. The inhibition was independent of the stearic (Clm) acid concentration. In

corn parison to control cultures, hydrogenotrophic methanogens were unaffected by

diethyl ether. In cultures receiving 10 r n g . ~ - ' stearic (C18:o) acid, hydrogenotrophic

methanogens were less affected than cultures receiving 30, 50 and 100 r n g ~ - ' stearic

(CIS:O) acid

8.0 COMPARISON OF LCFA DEGRADATION STUDIES

8.1. Products of Linoleic (Cls:~), Oleic (Cle:1) and Stearic (Clrr:o) Acid Degradation

Linoleic CI*:^) and oleic CI*:^) acids were degraded within 25 to 30 days at al1

concentrations examined. In cornparison, after 55 days, 50 to 60 % stearic (Clgo) acid

remained in cultures receiving fiom 10 to 100 r n g - ~ - ' . LCFA and M A byproducts

observed in cultures receiving 100 mg-L" LCFA are s h o w in Table 8.1. Byproducts

fiom Clrs to CIO were detected in cultures dosed with linoleic (Cl&?) acid but not in

cultures fed with oleic (Clg:l) and stearic (Cl&()) acids. The byproducts observed confirm

the biodegradation pathway and suggest that unsaturated LCFAs are able to undergo P-

oxidation. Product distribution variations between C 18 fatty acids in Table 8. l are Iikely

Lauric (C 1 z : ~ ) 1 D 1 ND 1 ND I

Table 8.1 : Byproduas detected in cultures receiving 100 mgL-' LCFA t

Byproduct Oleic (Cl*:-,) Stearic ( C l s . ~ ) Palmitoleic (C 16:1)

Palmitic (C 16:0)

hlyristic (C1j:a) ,

due to different degradation pathways but it may also be due to the inhibitory effects of

Capric (CIW) Octanoic (Cg) Hexano i c (C6) Butyric (C4) Acetic (Cz)

the parent LCFA compound.

LCFA

Large quantities of acetate (1 50 to 200 mg-L-') were produced and accumulated until

D = detected, h D = not detected

D ND D

ND D

day 90 in cultures receiving 50 and 100 rng-L-' linoleic (Clg:l) acid However, for oleic

Stearic (Clg:~) acid - -

M> ND N D

Linoleic (Cis:?) acid D

ND D D

D

Oleic (Cig:~) acid -

ND ND D D -

ND ND ND ND D

ND ND ND ND D

(C [a, ,) acid, acetate did not accumulate in cultures dosed with less than 100 r n g - ~ " oleic

acid. A< 100 rng-~-' oleic CI*,^) acid, acetate accurnulated to approximately 30

r n g ~ - l by day 30. but, less than 5 mg-l-' was observed by day 40. In comparison, for

cultures receiving greater than 10 rng*~- ' stearic (Cig:~) acid, acetate accumulated at day

50 to between 40 and 50 r n g * ~ - ' .

The different byproducts that were observed during degradation of C 18 LCFA may be

due to the hydroeenation mechanism taking place prior to P-oxidation. For esample, it is

possible that oleic (Clg:~) acid is first activated by acetyl CoA to form oleate acetyl CoA

with subsequent hydrogenation to stearate acetyl CoA. Eventually, stearate acetyl Co.4

enters into the 13-osidation pathway to form palmitic and myristic (C14:a) acids. In

contrast, linoleic acid may be hydrogenated to oIeic ( C I R : ~ ) acid or to cis- 12-

octadeceneoic acid. Finally, both acids are P-oxidized and isomerized into palmitoleic

(C r 6 : i ) acid. .Alternatively, linoleic (C1X:2) acid may become activated by acetyl CoA with

subsequent hydrogenation to form stearate acetyl Co.4. Stearate acetyl CoA is, then, j3-

osidized to palmitic (C16:O) and myristic (C14:0) acids and successively oxidized to lauric

( C I Z : ~ ) and capric ( C I O : ~ ) acids.

Rernoval times are summarized in Tables 8.2 and 8.3 for linoleic (Ci8::) and oleic

(Clg:~) acids, respectively. No data are shown for stearic (Citpo) acid because LCFA

byproducts were not observed during its degradation. In general, the time for rernoving

P-oxidized byproducts from solution was less for oleic (Cl8:i) than for linoleic CI^;^)

acid. No firm conclusions can be made for variation in LCFA byproduct removal times

observed for oleic (C18:l) and linoleic (C1g:2) acids based on the maximum LCFA

Table 8.2: Byproducts detected in cultures receiving linoieic (Clg:~) acid I

B yproduct

# - .. .

D = trace moun t detected. ND = not detected, NR = not removed

Linoleic (C 18:~) Acid Concentration, mg L-' (pM)

10 (36) 1 50 (179) 1 100 (357) Removal Time (davs) / Maximum Concentration (uM)

Stearic (Clg:0) Oleic (C1gl) Palmitoleic (Cia:~) Palmitic (C16.0)

-

Ml~is t i c (C 14 0)

Lauric (C 12

Capric (C 1o:o) Octanoic (Cs) Hexanoic (CG) Butyric (Cj) Acetic (C2)

ND 10/30+,1

ND 85/113112

ND 1 5 / 1 3 f 2

ND 42/ 13 kO.4

22/21 F3 N D ND ND ND ND

105 1483 ': 32

71 / 4 0 i 3 ND ND ND N D M)

NR / 2567 t 50

Table 8 .3 : Byproducts detected in cultures receiving oleic (Clg:~) acid

ND 15 / 19k2

70/41 i 18 85 / 178 k 9

- - -

84 / 127 i 14 D D

ND 43 / 3 2 2 1 113

M)

NR 1 3292 k 115

B yproduct

Stearic (CIK:O) Palmitoleic (Cl‘: 1 )

Palmitic (C 16:0) Myristic (C 140)

. -., 1

Hesanoic (CG) ND ND ND Butvric (Ca) ND ND

Oleic ( C ~ H : ~ ) Acid Concentration, rng*~" (pM) 30 ( 1 08) 1 50 (179) 1 100 (357) Removal Time (davs) 1 Maximum Concentration (uM)

D = trace amount detected, ND = not detected, 'less than 5 rng.L" detected

.. ,

Lauric (C 1 2 : ~ ) 1 ND

byproduct concentration o b s e ~ e d . However, it is possible that linoieic acid

inhibited organisms mediating the degradation of palmitic (Cl6:($ and myristic (C14:0)

acids significantly more than oleic (Clg:i) acid. For example, up to 178 pM palmitic

h?> ND

I 0 / 2 4 5 10 10182 5

ND ND ND

Capric (Clo:~) Octanoic (Cd

ND ND ND

M)

hD

h?) ND

2 6 / 1 1 5 I 8 2 5 / 4 2 + 1

ND ND

26 /289& 21 2 5 / 4 6 4 1

(CL6 0 ) acid was removed within approximately 85 days in cultures receiving 100 mg^"

linoleic (CL8:2) acid but up to 289 pM palmitic (C 16:o) acid was removed within 26 days in

cultures receiving 100 mg.^-' oleic CI^:^) acid.

Initial degradation rates for linoleic CI^:^). oleic (C18.1) and stearic CI^.^) acids are

surnmarized in Table 8.4. In general, degradation rates for stearic (Cia:o) acid are

approximately 10 times lower than those for linoleic (Ci8:2) acid and between 4 to 14

times Iower than those for oleic (C 18: 1) acid.

1 O0 1 3.53 10.12 1 2.35 ,+ 0.1 5 1 0.47 I 0.06 1 Average and standard deviation for triplicate samples

Table 8.4: Initial LCFA degradation rates

The unsaturated LCFAs. linoleic (C18:?) and oleic ( C i s . ~ ) acid, were more easily

degraded than stearic (C acid, a saturated LCFA. Lower degradation rates observed

for stearic (Cl8 O) acid in cornparison to Iinoleic (CI8.2) and oleic (Cig:l) acids are possible

due to the high activation energy required for the formation of LCFA-CoA, the activated

f o m of LCFAs which initiates the P-oxidation reaction. The formation of the linoieate

(C 1 ?)-C oA. oieate (C 8 , ,)-CoA and stearate (C 18 .0 ) -C~A complexes are shown in

reactions 8.1. 8.2 and 8.3, respectively. After the LCFA-CoA cornplex is formed, the P-

osidation reaction proceeds with the formation of a shorter LCFA moIecule, acetate and

hydrogen. A second reason why unsatwated LCFAs are more easily P-oxidized is due to

the free energy of reaction in which the production of palmitic (C16:o) acid may or may

Concentration. mg^-'

1 O 30 50

Initial LC FA Degradation Rate. pg LCFA ~ ~ v s s - ' -d" Linoieic (C 18.2) Acid

0.49 I O. 12 1.22 k 0.16 1.91 I 0.18

Oleic (C 8: *) Acid 0.56 t 0.03 1.42 i 0.09 0.88 I 0.05

Stearic (C 1 go) Acid 0.040 i 0.005 I

O. 14 & 0.02 1

0.25 + 0.01

not be favourable. Free enersy values provided in Table 8.5 are for standard conditions

at 25 'C' 1 atm and unit concentrations. These free energy values suppon our findings

that linoleic (Cls:Z) and oleic (CIR:~) acids were more easily converted to pafmitic (C16.")

acid in comparison to stearic (C IR:^) acid. Under reaction conditions, the conversion

stearic (Clx:o) acid + CoA + stearate (CIR:O)-CoA + Hz0 (8.1 )

oleic (CIR ,) acid - CoA + oleate (CIR:I)-COA + HtO (8.2)

linoleic (CIY:O) acid + Co.4 -, 1inoIeic (CIX.~)-COA + H'sO (8.3)

Table 8.5: Free energy values for P-oxidation of iinoleate (CIX:~), oieate (CIS:~)

of stearic (CiXU) acid to paImitic (Cl6:~) acid becomes more feasible but in cornparison.

and stearate (C1g:o) to palmitate (CIG:O)

the conversion of linoleic (CIX:~) and oleic (Clg.1) acids remains more favourable

Reaction Stearate - 2 H - 0 + .4c' - Palmitate + 2H2 +- H- OIeate +- 2 H 2 0 + AC- -i Palmitate - + H

assumins the same LCFA concentrations.

AG": kl-mole-' 50.82 -27.8

8.2 Possible Pathways for Linoleic Acid Degradation

Several researchers proposed LCFAs to be P-oxidized by hydrogen producing

acetogens to acetate and hydrogen (Jeris and McCarty, 1965; Novak and Carlson, 1970;

Weng and feris, 1976). Hydrogenation of unsaturated LCFAs to stearic (Clg:~) acid prior

to 0-osidation has been proposed by Mackie et al. (1 991) and Novak and Carlson (1 970).

Linoleate - 2 H 2 0 -, Ac' + Pairnitate + H- I -106.38

Throughout this work, stearic (Cl*:-,) acid was not detected as an intermediate when

linoieic (Clg:2) and oleic (C18:l) acids were used as substrates. Several proposed

degradation pathways shown in Figure S. 1 for the conversion of C 18 LCFAs to palmitic

(C16:O) acid are based on data from this study. It is clear that complete LCFA

hydrogenation prior to B-oxidation is not necessary to produce the products observed.

In cultures fed stearic (C18:o) acid, a large amount of substrate remained undegraded

and no LCFA byproducts were observed suggesting that stearic (Clg:~) acid cannot

readil y undergo P-oxidation. Pathway 1 - 1 a- 1 b shows the conversion of Iinoleic

acid to stearic (CI8:O) acid is thermodynamically feasible however, the degradation of

stearic (CIS:O) to palmitic (&:O) acid is not possible under standard conditions. Based on

the calculated free energies, pathway 1-2-2a is not possible under standard conditions

since the conversion of oleic (Cls:~) acid to palmitoleic (C16:1) is thermodynarnically not

feasible. Because palmitoleic (C16:l) and paimitic (C16:0) acids were observed byproducts

from Iinoleic (Cig:~) acid, a Iikely route is 4-2a. To account for the oleic CI^:,) acid

observed during the conversion of linoleic (C1gZ2) acid, another possible pathway is 1-3.

No palmitoleic (Cl6:,) acid was observed when oleic (Clg:l) acid was used as a

substrate and a possibte route for its conversion to palmitic (&:O) acid is pathway 3 . The

overail free energy of conversion from iinoleic acid to palmitic (&:O) acid is

approximately -106.38 U-mole'' (Pathway 5 ) . It is unlikely a single enzyme can

accomplish this reaction in a single step. Pathway 5 is essentially composed of

hydrogenation, isomerization and P-oxidation steps and it is a combination of reactions in

pathways 1, 2, 3 or 4. Based on al1 the LCFA byproducts observed during the P-

osidation of linoleic (C18:2) acid, nvo pathways, 1-3 and 4-2a, likely mediated the

production of oleic (Clg,~), palmitoleic (C16:I) and palmitic (&:O) acids fiom linoleic

(CIR." acids.

At this stage, it is clear that the degradation mechanism is cornplex. The available

information in the literature does not elucidate the mechanism completeIy. Additional

work using radiolabeled tracers and pure cultures is required to thoroughly understand the

cornplete rnechanism.

palrnitoleic (C i6: 1) acid t

Pathway 1

Pathway 3

-78.57

palmitic (&O) acid

4 J Figure 8.1 : Proposed C 18 LCFAs degradation pathways. (Free energy values shown are in kJ-mole-' and were calculated assuming standard conditions. These complete reaction and fiee energy values are shown in Chapter 2)

9.0 INHIBITORY EFFECTS OF LLNOLEIC (Ci#& OLEIC (Cis:,) AND STEARiC (Cis:o) ACIDS ON ACIDOGENIC, ACETOGENIC AND METELANOGENIC ACTiVITY

9.1 Experimental Results

9.1.1 Acidogenic Inhibitory Effects - Glucose Degradation

Glucose degradation profiles are shown in Figures 9.1 and 9.2 for controls and

cultures receiving individual and mixed LCFAs. The culture concentration used in this

evperiment was 1500 k 70 rng-~' ' VSS. In cornparison to control cultures. diethyl ether

did not affect glucose degradation and undetectable levels of glucose were reached within

50 to 60 minutes. In the presence of 100 rng.~' ' of stearic (Cig:~) acid or 100 rng-~- ' oleic

(C 18: 1 ) acid, complete gIucose degradation was accomplished within 60 to 70 minutes.

However, in the presence of 100 mg-^" of linoleic (Clgr:) acid or mixtures of linoleic

(Ciw::). oleic (C'a: 1) and stearic (C18:o) acids ranging £Yom 100 to 300 rng-~' ' total LCFAs,

(Conditions 5, 6 and 7 in Table 9 . 1 ) longer removal times of up to 130 minutes were

observed.

Glucose degradation rates shown in Table 9.1 were calculated assuming zero order

kinetics. The degradation rates (Table 9.1) show a 3.7% difference between controls with

and without diethyl ether. However, in cultures receiving LCFAs, glucose degradation is

inhibited. At 100 r n g - ~ - ' , the addition of oleic (Clg:~) and stearic (Cis:o) acids reduced

the glucose degradation rate by 20 % versus diethyl ether alone while a 60 % rate

reduction was observed for cultures receiving linoleic ( C I R : ~ ) acid* Cultures receiving

total LCFA concentration of 100 r n g - ~ ' ' (Condition 6) inhibited glucose consumption

more than those receiving only 100 r n g - ~ - ' stearic (Cig,~) acid (Condition 3) or oleic

(CIR:~) acid (Condition 4).

eSo LCFA. S o DE

S o K F A . S o DE

ASO LCFA. with DE

0x0 KFA. with DE

Figure 9.1 : Glucose degradation profiles for cultures with and without diethyl ether. &CFA = long chain fatty acid, DE = diethyl et her)

O 20 40 60 80 100 1 20 140 'üm, mins

Figure 9.2: Glucose degradation profiles for cultures receiving individual and mixed LCFA substrates. (SA = stearic (Clg:~) acid, OA = oteic (C 8: 1 ) acid, LA = Iinoleic (Ci s:2) acid, average for triplicate samples shown.)

In cultures receiving 300 mg-^-' of LCFA (100 r n g - ~ " iinoleic (C18:~) acid plus 100

mg^" oleic (C 18, 1)acid plus 100 r n g - ~ ' ' stearic (C ls,o) acid), the degradation rate was

approsimately the same as for cultures receiving only 100 mg-^-' linoleic (Claz) acid.

Comparing cultures receiving LCFA mixtures (Conditions 6 and 7), the additional rate

reduction observed at the higher LCFA concentrations was likely due to the presence of

higher linoleic (C18:t) acid concentration.

Table 9.1 : Glucose degradation rates for individual and mixed LCFA substrates

Glucose degradation rates were analyzed statisticaily using the Tukey's paired

cornparison procedure. Significant differences in glucose consumption were observed in

cultures receiving stearic (C18:o) acid and oleic (Clg:~) acid versus those receiving linoleic

CI^,?) acid and LCFAs mixtures. When comparing glucose consumption for cultures

receiving LCFA mixtures, si_enificant differences were observed in cultures fed with 100

and 300 mg^“ total LCFAs.

Condition Examined

I

1. Control 2. Control + DE 1 100 mg-L-' SA 4 100 mg-L-' OA 5. 100 rng.L-' LA 6. 33.3 r n g ~ - ' LNOAISA 7. 100 rngL-' LNONSA

9.1.2 Acetoçenic lnhibitory Effects - Butyrate Degradation

The effects of diethyl ether, linoleic oleic CI^:^) and stearic (CIU:O) acids on

butyrate consumption are shown in Figures 9.3 and 9.4. The culture concentration used

Glucose Degradation Rates ( p p n g ~ ~ s"*rnin'l)

I

6-19' 5-96'

4.88 i 0.04' 4.81 *0.1l2 2.22 i 0.0 1'

3.367 i 0.001'~ 2.27 I 0.01

1 Duplicates. ' ~ v e r a ~ e and standard deviation for tnplicate samples? '~o ta l

LCFA concentration = 100 rng-~", '~ota l LCFA concentration = 300 rng-~- '

in this esperiment had 1600 + 50 r n g ~ - ' VSS. In the absence and presence of diethyl

ether in control cultures, butyrate was completely degraded within 0.5 days and 2 days,

respectively. In cultures receiving 100 r n g - ~ - ' stearic (Cis:a) acid or 100 r n g - ~ - ' oleic

acid, butyrate degradation was inhibited reaching undetectable levels on day 2 and

day 7. respectively. In cornparison at day 10, less butyrate removal was observed in

cultures receiving 100 mg-^" Iinoleic acid (condition 5 in Table 9.2), 100 mg-^-'

total LCFA (condition 6 in Table 9.2) and 300 mg-^-' total LCFA (condition 7 in Table

9.2).

Degradation rates for butyrate s h o w in Table 9.2 were calculated assuming zero

order kinet ics. Diet hyl ether inhibited butyrate consumption by approximatel y 60% in

comparison to control cultures. Addition of linoleic (Cigr2), oleic (Cig:i) and stearic

(Clgo) acids fûrther reduced the butyrate degradation rate. In comparison to control

Figure 9.3: Butyrate degradation profiles for cultures with and without diethyl ether. (LCFA = long chain fatty acid, DE = diethyl ether)

Figure 9.4: Butyrate degradation profiles for cultures receiving individual and mixed LCFA substrates. (SA = stearic (Cls:~) acid, 0.4 = oleic (CI8:,) acid, LA = linoleic (CisI2) acid, average for triplicate samples)

cultures receiving diethyl ether, cultures receiving linoleic (Cls::), oleic (C1s:l) and stearic

(C18:0) acids showed rate decreases of 92%, 78% and 2S0A, respectively

The addition of 100 r n g ~ - l total LCFA (33.3 r n g . ~ " linoleic CI^, oleic (Clxl) and

stearic (Clx:~) acids, condition 6 in Table 9.2) lowered the butyrate degradation rate in

comparison to cultures receiving a single LCFA. A fùrther rate reduction was also

observed in cultures receiving 300 rng.~- ' total LCFA (100 rng-~- ' linoleic

oleic(C1~ 1) and stearic (Ci8:o) acids) in comparison to conditions 3 to 6. In contrast to

cultures receiving diethyl ether alone, almost complete inhibition (99% butyrate

degradation rate reduction) was observed in cultures receiving a total of 300 mg-^"

LCFA.

Table 9.2: Butyrate degradation rates for individual and mixed LCFA substrates

C- I

' ~ u ~ l i c a t e s . '~verage and standard deviation for triplkate samples, ?oral LCFA concentration = 100 rng-~-'. ' ~o ta l LCFA concentration = 300 r n g - ~ - '

Condition Exarnined

1. Control

Based on the Tukey's paired comparison procedure. the butyrate degradation rates for

Butyrate Degradation Rates ( p n - m n ~ ~ ~ - ' -da?.')

119.3'

each case esarnined \vas significantly different fiom every other case.

9.1 -3 Methanogenic Inhibiton Effects - Hydrogen Consurnption

DiethyI ether had no significant effect on hydrogenotrophic methanogens (Figure 9.5).

The action of individual and mixed LCFA substrates on hydrogen uptake is shown in

Figure 9.6. The culture concentration used in this experiment had 1500 + 80 r n g - ~ - ' VSS.

Assuming first order kinetics (which showed r2 > 0.97 for al1 cases), the average

hydrogen uptake rate constants deterrnined for single and mixed LCFAs concentrations

are shown in Table 9.3. Based on the Tukey's paired comparison procedure (Box et al..

1978) at a 95 % confidence level. the differences in average rate constants for cultures

receiving 100 r n g - ~ - ' stearic (Clg,l) acid and those fed with linoleic (CIg:2) acid, oleic

Tinie, hrs Figure 9.5: Duplicate conuol cultures hydrogen profiles with and without diethyl ether. (DE = diethyl ether, Average for duplicate results shown)

Figure 9.6: Hydrogen profiles for cultures receiving individuai and mixed LCFAs. (SA = steanc ( C l g : ~ ) acid, OA = oleic (Cls:!) acid, LA = linoleic acid; Duplicate controls; Averages for triplicate samples, error bars represent standard deviation for the samples)

(C 1 g ,) acid and mixtures of three LCFAs (Condition 6 and 7 in Table 9.3) were

statistically significant. There was no evidence of any synergestic interaction due to

mixtures of LCFAs on hydrogen consumption.

9.2 Discussion of Results

9.2.1 Glucose degradation

Table 9.3: First order rate constants for hydrogen rernoval in the presence of individual and mixed LCFAs

Linoleic (ClS.:) acid was more inhibitory to glucose degradation in comparison to

oleic (Cis i ) and stearic (Cig:o) acids. Based on the degradation rates, inhibition caused by

stearic ( C i ô , ~ ) and oleic (CIg:~) acids was approximately the same. No comparison has

been made in previous research of the effects of single saturated and unsaturated LCFAs

on glucose degradation. In comparison to the rate observed for stearic ( C I R : ~ ) acid, the

Condition Examined

1 . Control 2. Control -+ DE 3. 1 O0 mg^" S A

presence of a single double bond had no major effect on glucose degradation.

addition of a second double bond (Iinoleic (C'S:~) acid) decreased the rate sign

J

First Order Rate Constant, d-'

0.409' 0.388'

0.374 0.006'

However,

ificantly.

4. 100 r n g - ~ " OA 5. 100 mg-^" LA 6. 33.3 r n g - ~ " LAIOAISA~ 7 100 mg^" LA/O.WSA"

0.260 I 0.006~ 0.263 I 0.013' 0.245 ,+ 0.01 1' 0.255 +. 0.003~

'~u~l ica tes , 'AveraSe and standard deviation for triplicate samples ' ~ o t a l LCFA = 100 r n g ~ ' ' . " ~ o t a l LCFA = 300 rng.L-', LA = linoleic (C 1s.:) acid, 0-4 = oleic (C1g:I) acid, SA = stearic (Cis:~) acid.

One possible explmation may be related to the effects of LCFAs on ce11 membrane

receptors that are responsible for glucose uptake. Linoleic (C18:2) acid may have a higher

binding affinity for these receptors in comparison to oieic (C18:l) and stearic (C18:0) acids

hence causing a greater inhibitory effect on glucose uptake.

The combined effect of mixed LCFAs (Conditions 7 in Table 9.1) on glucose

degradation shows that there is no synergistic interaction to hnher lower the rates below

those observed for linoleic (Cis:?) acid (Condition 5 in Table 9.1). However, the

concentration dependence of linoleic (Ci8:2) acid inhibition on glucose degradation is

clearly shown.

The effect of mixed LCFAs on glucose degradation was examined by Hanaki et al.

( 1 98 1 ) using 2500 mg^" VSS at 37 OC. They reponed glucose fermentation was

uninhibited by increasing concentrations (O to 2000 mg-^-' as oleate) of a fatty acids

mixture containing saturated Cie to C18 LCFAs and mono-unsaturated LCFAs fiom Ci? to

CIg. The difference between Our work and that reported by Hanaki et al. (198 1) is that

the parameters used to rneasure glucose degradation are not the same. Our work reported

direct measurement of glucose using an enzyme assay while Hanaki el al. ( 1 98 1) reported

acetate (Cz ) , a byproduct of glucose degradation as their measure of inhibition. The error

reported in their work is that in addition to acetate (C2) decived fiom glucose degradation,

they also measured acetate (C2) arising from LCFA P-oxidation. As a result, the data

reported by Hanaki er al. (198 1 ) is an inaccurate measure of the inhibitory effects of the

fatty acid mixture on glucose fermentation. Additionally, in comparison to the effect of

LCFAs mixtures reported by Hanaki et al. (1 98 l), Our work examined the effect of

individual and mixed C 18 LCFAs on glucose degradation.

9.3.2 Butyrate Fermentation

Diethyl ether inhibited butyrate fermentation in comparison to cultures without diethyl

ether. Single and mixed C 18 LCFAs affected butyrate degradation at al1 the conditions

examined. At 100 mg-^" LCFA, butyrate degradation rates decreased as the number of

doubIe bonds increased in the Cl8 homologous series from stearic (Clx:o) acid to linoleic

( C l 4 acid..

Based on the degradation rates, cultures receiving mixed LCFAs were inhibited more

in comparison to those receiving individual C 18 LCFAs. This synergistic relationship

was more obvious as the LCFA mixture concentration increased. This synergism may

have been caused by inactivation of receptors responsible for transpon of butyrate into

cells. If a specific LCFA such as linoleic (CIR:~) acid is able to bind to a butyrate

receptor. its action may enhance the binding of oleic acid and / or s t eak (Clg:~)

acid to other butyrate receptors. Synergistic effects of LCFAs on anaerobic organisms

have been previously reported by Canovas-Diaz et al. (1 99 1 ) and Koster and Cramer

( 1 987). Canovas-Diaz et al. ( 199 1) reported that decreased degradation rates were

observed when oleic and myristic (Ci+o) acids were added together as a mixture,

in comparison to cultures degrading the individual acids. In the presence of capric (Clo.o)

and myristic (Cl4:o) acids, Koster and Cramer (1987) reported lauric (Ci2:o) acid to have a

synergistic inhibitory efîect on rnethanogenesis.

Angelidaki and Ahring (1992) reported that higher than 500 rng*~-' oleic (Cltt,i) acid

in hibited but yrate and propionate fermentation. Although a threshoid value causing

inhibition was not determined, these studies showed that 100 mg^" oleic (CI~: l ) acid

inhibited butyrate fermentation. Differences between our resuIts at 21 OC and those

reponed by Angelidaici and Ahring (1 992) at 55 OC may be a reflection of temperature

variation but also to culture adaptation. It should be noted that in their report (Angelidaki

and Ahring, 2993) the VSS concentration was not available and this may also contribute

to the difference between the two studies,

9.2.3 Hydrogen Consumption

Cunently no research has reported on inhibition of hydrogenotrophic methanogenic

by direct measurement of hydrogen for single LCFA compounds and LCFA mixtures at

several concentrations. Research by Demeyer and Henderickx (1967) has, however,

reported that linolenic (C18:3) acid inhibits methane production fiom H2 and COz and

Hanaki rf al. (198 1 ) reponed inhibition of hydrogenotrophic methanogens by LCFAs.

In the presence of a LCFA mixture at 3 7 O C , Hanaki et al. (1 98 1 ) found

methanogenesis to be inhibited although al1 o f the hydrogen added was readily utilized.

Similarly, in our studies with LCFA mixtures, even though hydrogenotrophic

methanogenesis was slightly inhibited, the hydrogen was consumed to undetectable

levels within approximately 8 hours, similar to control cultures (Figure 9.5).

Stearic (Cix:a) acid did not affect hydrogen consumption in comparison to control

cultures with diethyl ether. The lower first order kinetic constants observed for linoleic

(C18:2) and oleic (Cii:]) acids, in comparison to stearic (Cis:o) acid, illustrate the inhibitory

effect caused by the presence of double bonds on hydrogen consumption. Although

lower first order constants were obsemed for oleic (Cis:]) and linoleic (Clgz) acids, the

hydrogen consumption was unaffected by addition of a second double bond.

First order rate constants, determined for cultures receiving 100 and 300 mg-L" mixed

LCFAs and unsaturôted LCFAs, were not significantly different based on the Tukey's

paired corn parison procedure. These results suggest that there is no inhibitory synergy

between LCFA mixtures on hydrogen consumption by hydrogenotrophic methanogens.

Mixed LCF.4s ranging fiom 100 to 300 mg-^-' did not inhibit hydrogen consumption

slightly. Assuming negligible effects of stearic (C18:0) acid on the first order rate

constant. the inhibitos. effects of linoleic ( C I S : ~ ) and oleic acids in LCFAs

mixtures are approximately the sarne as cultures receiving only linoleic ( C 1 4 acid or

oleic (Clg:~) acid.

9.3 Surnmary

Glucose fermenters were affected under al1 the conditions examined. The effect of

LCF.4s on glucose consumption is as follows: 100 mg-^-' linoleic (CIsc2) acid z 300

r n g - ~ " total LCFA > 100 mg-L" total LCFA > 100 mg-L-' oleic (C18,i) z 100 rng-L-'

stearic ( C , S . ~ ) acid. For butyrate consumption the inhibitory effect of 300 r n g - ~ - ' total

LCFA > 100 rng.L-' total LCFA > 100 mg^-' iinoleic (ClsZ2) > 100 mg^-' oleic ( C l s , ~ ) >

100 rng-L-' stearic (Cip:i) acid Although inhibition of hydrogenotrophic methanogenesis

was statistically sigificant between cultures receiving stearic (C18:o) acid versus those

receiving linoleic (Ci8:2) acid, oleic (C18:l) acid and LCFA mixtures, the inhibition was

minimal as most of the hydrogen was consumed within less than 10 hours.

10.0 DISCUSSION OF METHANOGENIC STUDIES AND LCFA LNHIBITION MECaANISM

10.1 Methanogenic Studies

10.1.1 Aceticlastic Methanogens

Under anaerobic conditions and in the absence of inhibitors, acetate conversion to

methane and carbon dioxide by aceticlastic methanogens is unaffected. However, in the

presence of LCFAs, aceticlastic methanogens are inhibited. During the degradations

studies, Iinoleic ( C 4 , oleic (CIS:I) and stearic (C 1g:o) acids at dl concentrations

exarnined, inhibited aceticlastic methanogens.

Variation in acetate profiles during the degradation studies for the three C 18 LCFAs

esarnined rnay be due to diflerent concentrations of LCFA byproducts produced. For

esample, larger amounts of palmitic (CtG:O) and myristic (Cta:o) acids were produced

during P-oxidation of 100 mg^-' linoleic acid compared to the amounts produced

during degradation of 100 m g - ~ - I oleic acid. These compounds may inhibit

aceticlast ic methanogens and affect acetate consumption.

The binding of C 18 LCF.4s to ceII receptors responsible for transport of acetate into

the rnethanogenic cell may play a role in inhibition. This possibility may explain why

acetate accumulated during LCFA degradation studies but was often consumed during

the inhibition studies. Assume that LCFAs and acetate molecuIes are able to bind to

these receptors sirnultaneously. Aiso assume that acetate when bound is transponed into

the ce11 while LCFAs when bound cause irreversible damage. During the LCFA

degradation studies, no acetate was initially available to compete with LCFAs for acetate

receptor sites. Therefore, most of the receptors may have been damaged so that when

acetate was produced, it accumulated in solution. As time progressed, more receptors

became available and the amount of acetate decreased as those aceticlastic methanogens

that were able to survive increased their population size.

During the inhibition studies, acetate was added simultaneously with linoleic (C18.z),

oleic ( C l 8 , ) or stearic CI*:^) acids. If binding is competitive, at low LCFA

concentrations, acetate concentration may have been sufficient to bind to most of the

receptors. This allowed acetate consumption to proceed. However, as LCFA

concentrations increased, LCFA molecules out-cornpeted acetate for receptor sites. The

higher the LCFA concentration, the less competitive acetate became and the acetate

consumption rate decreased.

In cornparison to cultures receiving Iinoleic or oleic (Cig:l) acids, acetate was

consumed initially and no lag was observed for cultures receiving stearic (C18:o) acid.

Stearic (Clgo) acid may behave differently to linoIeic (C 18~2) or oleic CI^:^) acids by

binding reversibly to the ce11 receptors without causing any damage. Hence, acetate

binds and is transported into the ce11 when stearic (Clg:~) acid is released from the

receptor.

That less aceticlastic methanogenic inhibition is caused by saturated LCFAs is

supported by Demeyer and Hendrickx (1967) who reported that LCFA inhibition

increased with increasing the number of double bonds. They aIso demonstrated that

LCF.4 inhibition increased as the LCFA carbon chain increased.

10.1.2 Hydrogenotrophic Methanogens

ln this study, hydrogenotrophic methanogens were inhibited to a much lesser extent

t han acet iclastic rnethanogens. Hanaki et al., (1 98 1 ) reported hydrogenotrophic

methanogens were less sensitive to a LCFA mixture in comparison to aceticlastic

methanogens. Although some inhibition was observed, no clear trends can be

determined. Under al1 the LCFA conditions examined, inhibition of hydrogenotrophic

methanogens is not expected to impact anaerobic degradation to the extent that inhibition

of aceticlastic methanogens will.

10.2 LCFA Inhibition Mechanism

The inhibitory effects of LCFAs on anaerobic fermentation have been exarnined

previously (Rinzema et al., 1994; Angelidaki and Ahring, 1992; Koster and Cramer,

1987; Hanaki et al., 198 1 ; Demeyer and Henderickx, 1967). However, the molecular

basis of LCFA inhibitory effects on anaerobic organisms or more specifically, on

aceticlastic and hydrogenotrophic methanogens has not been examined. Figure 10.1

shows several possible pathways for LCFAs ceIlular uptake. These pathways are based

on LCFA literature research in anaerobic degradation, food presewation and uptake into

eukaryotic cells. It is possible LCFAs may follow either of three routes: activate the

outer membrane sensory proteins and be transported into the cell, inactivate the outer

membrane enzyme or integrate into the cellular membrane.

Researchers have proposed a glucose uptake mechanism for eukaryotic cells (Van

Winkle, 1995; MueckIer et al., 1985) and ~ising the glucose model, a similar approach

can be developed for LCFA uptake into bacterial cells. LCFAs may first activate a

sensory membrane protein which causes the ce11 to initiate the uptake process. Mangroo

er al. ( 1 995) and Nunn (1986) have examined the transport mechanism of LCFAs into

adipocytes and Escherichia coli, respectively. Both researchers suggest a transponer

L-_-* Disrupt proton cliemical potential

IXFAs b Activaie .-+ I'ransportcd b B-oxidation - no inhibition but

Inactivate outer Disnipiion o f membrane function for membrane example, transporter protein enzyriies

out er irito the ceIl iiicnibraiie enzy riies

L lntegation into the cellular Phospholipid formation meiiibraiie

nossible bvproduct inliibiiiori.

b Iiiact haie enzymes

t- Disniption o f membrane function for example, transporter protein

hisertion into the membrane causes cell lysis

Figure 10.1 : Possible IdCFA inhibition pathwiiy s

protein, fadL. is responsible for the transmembrane movement of LCFAs into cells. But

the mechanism is not completely understood at the molecular level. It is possible that the

fadL protein is synthesized and inserted across the ce11 membrane afier outer membrane

sensory proteins detect the presence on LFCAs on the ce11 membrane. Subsequently, the

fadL protein is inserted into the ce11 membrane to initiate the LCFA transport process.

When transported into cells, LCFAs may undergo P-oxidation (Weng and Jeris, 1976;

Jeris and McCarty, l96j), bind to cellular enzymes (Ferdinandus and Clark, 1969) or

disrupt the proton chemical potential (Sikkema et al., 1995). During P-oxidation,

hydrogen producing acetogens degrade LCFAs to shorter chain LCFAs, acetate and

hydrozen. LCFAs can also bind and inactivate metabolic enzymes. Octanoic (CR) acid

was reported to cause inhibition of several enzymes responsible for the synthesis of new

LCFAs from precursors such as pyruvate, in lipogenesis and intermediates of the

tricarbosylic acid cycle (Ferdinandus and Clark, 1969). LCFAs may disrupt the proton

chemical potential across cellular membranes and inactivate reactions such as ATP

synthesis. Obstructing membrane transport tunction such as interaction with ATPase,

LCFAs are also able to uncouple the proton potential used for oxidative phosphorylation

(Sikkema er al., 1995). Rottenberg and Kashimoto (1986) and Fay and Farias (1977)

reported simulating proton and potassium ions leakage across various membranes upon

addition of LCFAs.

Another cellular pathway for LCFAs is integration into membranes (Figure 10.1 ).

When present in membranes, LCFAs may undergo transformation to become

phospholipids, disrupt membrane fùnctions or cause cell lysis. Phospholipids are a major

component that is essential to the function of cellular membranes. Greenway and Dyke

( 1 979) repoxted approximately 0.9 % linoleic (C 1 8 : ~ ) acid was incorporated into the

p hospholipid component of the outer membrane of Staphylococcus aureus and free

linoleic (Clg::) acid seemed to be the growth inhibitory substance and not the linoleic

( C 1 8 . 2 ) acid component present in the membrane lipid bilayer.

One role of proteins in cells is for transpon of molecuies into or out of cells.

Membrane proteins responsible for this function are called transporter proteins. Possible

damage caused to these proteins by C 18 LCFA was discussed previously.

LCFAs are able to insert into cellular membranes and cause ce11 lysis. Some research

has been conducted on the effect of LCFAs on ce11 lysis. Using Staphylococclts airrerts

Greenway and Dyke (1 979) reponed the release of a 260-nm material was dependent on

the linoleic acid concentration. They aiso used stearic (Ci8:o) acid at an equivalent

linoleic ( C ~ S . ~ ) concentration greater than 50 r n g ~ - ' and reponed that the 260-nm

substance was not released.

Linoleic (Cis::) acid is reported to behave as a surfactant by altering the interfacial

tension between the bacterial membrane and the bulk aqueous phase of the growth

medium (Greenway and Dyke, 1979). The ability for a compound such as linoleic ( C i 4

acid to migrate to cellular surfaces and lower the interfacial tension (Le. increase the

wettability of the surface) is related to its surface tension and sohbility properties. For

esample, in comparison to linoleic (C18:2) acid, stearic (C18:o) acid was reported not to

inhibit gro~kth at 30 "C since it is a much poorer surfactant (Greenway and Dyke, 1979).

Thus. LCFAs such as linoleic acid may be more easily degraded and yet cause

more inhibition in comparison to stearic (Crg:~) acid.

Stearic (Clg:~) acid was less inhibitory to aceticlastic methaogens in cornparison to

linoleic (Clrc:?) and oleic (C 18~1) acids. The soap solubilities of the Ci* LCFAs

homologous series for linoleic (Clg:~) and oleic (Cls:~) acids are approximately equal

(Irani and Callis, 1960) and the same solubility is assumed for stearic (Clg:~) acid.

Therefore, a factor other than the solubility that is likely affecting the inhibition, is the

chemical structure. Stearic ( C l s : ~ ) acid has no double bonds and the molecule is less rigid

cornpared to oleic ( C I R . ~ ) and linoleic (C18:?) acids. It is possible that this structural

difference may cause ce11 membrane receptors to have a low binding affrnity for stearic

(&:O) acid and thus, affect its degradation and inhibitory characteristics.

11.0 SLMMARY AND CONCLUSIONS

11.1 Summary

1 1 .l. 1 LCF A Degradation

Linoleic (CI*:-) and oleic (CIg:l) acids were degradable, however, in cornparison,

stearic acid was not completely B-oxidized at al1 the concentrations exarnined (O to

100 mg-~- l ) . Palmitoleic (C16:1) (less than 10.5 r n g ~ - ' ) , hexanoic (Cs) (less than 37.0

rng-~") and trace arnounts of lauric (Ciz:0) acids were observed in cultures receiving 100

r n g - ~ ' ' linoleic (Cis.:) acid. Oleic (CI~ : l ) , palmitic (C16:0), myristic (Cli:o) and acetic acids

were detected in al1 cultures receiving linoleic ( C 1 4 acid. Within less than 35 days,

linoleic (Cis:z) acid was degraded at al1 concentrations examined. The length of time to

comp~etely remove palmitic (Cl6:*) and myristic (Cld,~) acids from the system was

concentration dependent with long rernoval times observed at high dosages of linoleic

(Cin:2) acid. In cultures receiving higher than 10 r n g - ~ - l linoleic (Clg,2) acid, acetate (Cz)

accumulated from P-oxidation of LCFA byproducts.

Palmitic (Cll,o). myristic and acetic (Cz) acids were observed at al1 oleic (Cla:,)

acid concentrations examined. In cornparison to linoleic (C1s:2) acid, palmitic (C16:0)

and myristic (C li,o) acids were removed within shorter time periods in cultures receiving

oleic (C1s,l) acid. This suggests that oleic (Cia:l) acid might be less inhibitory than

linoleic (Ci8:2) acid to organisms responsible for B-oxidation of palmitic (C16:0) and

myristic (Cli:o) acids. Although acetate (C:) was consumed at al1 oleic (Cig:l) acid

concentrations examined, inhibition of aceticlastic methanogenic was observed in

cultures receiving higher than 50 r n g ~ ~ l .

No LCFA P-oxidation byproducts were observed in cuItures fed with stearic

acid. However, acetate (C2) was produced at ail concentrations examined. About 50 to

60 % of the stearic (C18:o) acid added remained undegraded after day 5 5 . Acetate ( C l )

accumulated to between 30 to 50 mg^" in cultures receiving greater than 30 r n g ~ . '

stearic (Cis:o) acid. It is unclear why stearic ( C i 8 : ~ ) acid is not easily degradable in

comparison to linoleic (C18:2) and oleic (Cig:l) acids. However, as previously discussed

in section 8.1, it is Iikely that the conversion of linoleic ( C I ~ ? ) and okic (Cig:~) acids is

favored thermodynamicaIIy. Based on reaction free energies, the most feasible

degradation pathway for linoleic acid is conversion to paimitoieic (C16:1) acid and

then hydrogenation to palmitic C CI^:^) acid. For oleic (Crs:~) acid, direct conversion to

palmitic (Clb .* ) acid is thermodynamically the most likely pathway.

1 1.1.2 LCFA Inhibition

-4. -4 cidoger~s

Glucose degradation by acidogens was affected at al1 individual and mixed LCFA

concentrations esamined in comparison to control cultures. Linoleic (CIS:~ ) acid by itself

caused the greatest inhibition in comparison to cultures receiving oleic (Cis:~) or stearic

(Ci *,O) acids. Linoleic (C is,z) acid at 100 mg-^-' inhibited glucose fermentation

approximately 34 % more in cornparison to a mixture containing equal amounts of the

three acids with a total LCFA concentration of 100 r n g - ~ - ' . Cultures receiving linoleic

acid slone at 100 mg^" caused approximately the same degree of inhibition as

those receiving a LCFA mixture containing equal arnounts of the three acids at a total

concentration 3 00 mg^-'.

B. A cetogem

-4t al1 individual and mixed LCFA concentrations examined, butyrate fermentation

was affected. The effect was strongly dependent on the degree of LCFA unsaturation

with the greatest inhibition occurring in the presence of linoleic (Cis:=) acid. Combining

linoleic (C18:2), oleic (Cls:l) and stearic (Ctg.O) acids in equal quantities for a total LCFA

concentration of 100 rng-~- ' was more inhibitory than either of the three acids alone at

100 r n g - ~ - ' The increased inhibitory effect due to the LCFA mixtures on butyrate

fermentation was caused by a synergistic effect of al1 three acids acting together and the

increased inhibition observed at greater concentrations of LCFA mixtures was due to the

presence of larger quantities of al1 three acids.

C. Me~haizogerw

i. Aceticlastic methanogens

-4ceticlastic methanogens were inhibited by linoleic oleic (Cig:l) and stearic

(Cl s :~ ) acids at al1 concentrations exarnined. The highest aceticlastic methanogenic

inhibition caused by linoleic and oleic (Cls:l) acids was in cultures receiving more

than 10 r n S - ~ - l of each LCFA. At greater than 1 O mg-^" linoleic (CItt2) and oleic (CI*:,)

acids, acetate (C-) consumption was not observed within 25 days.

i i . Hydrogenotrophic methanogens

In cultures receiving single LCFA substrates, hydrogenotrophic methanogens were

slieht ly inhib ited under al1 the concentrations examined. Steanc (C 18:~)) acid caused the

least inhibition when comparing cultures receiving individual LCFA substrates. The

inhibitory effects of al1 the LCFA mixtures were approximately the same as those

experienced by cultures receiving linoieic CI^:^) or oleic (Cls:~) acids. Under the

experimental conditions examined, hydrogenotrophic inhibition was independent of

LCFA concentration and also independent of the presence of individual or mixed C 18

LCFAs.

1 1.2 Conclusions

1 . Lilroleic (Cls,y arld oieic (Cls,r) acids ulere artaerobicall): degraded rtrlder the

corldiciotls emntirled. S~enric (CIPrl%) acid rcSas more d~flicltit to degrade t h 1 lirloleic

(CM:~ m d oieic (ClPtl) acid. LCFAs B-oxidized byproducts were observed oniy in

cultures fed with linoleic (C and oleic (C 1 g : ~ ) acids.

2. Complete b iohydroger~aliott of rrttsaiccrated LCFAs is r1ot necessas. 10 ir~itiate rhe P-

oxidarion ntechmtisni. Both saturated and unsaturated C 16 byproducts were observed

in cultures receiving 100 r n g . ~ - ' linoleic (Cig:t) acid. Only saturated LCFA

byproducts were obsen-ed during P-osidation of oleic (Ci8:i) acid and none were

observed for stearic (Cis:o) acid.

3 . I)I cornparison to oleic (CIPiS attd stearic (Cl& acids, lirloleic (CIP:J acid had rhe

gi-entesr irdtibirory efecr oti g/mose ferrnentatior~. Under the conditions examined,

inhibition of glucose fermenters caused by 100 mg^' linoleic ( C 1 4 acid was

approximately the same as cultures receiving a total LCFA concentration of 300

mg^" The inhibition was found to increase with increasing concentration of LCFA

mixtures. Also, inhibition of glucose degradation was similar for oleic (Cig:l) and

stearic ( C l g : ~ ) acids. LCFA mixtures containing 300 r n g ~ ~ ' total LCFAs were more

inhibitory to glucose fermenters than 100 mg^“ total LCFAs.

4. It~di\lidztal LCFAs and mixtures of LCFAs inhibired brrg~rate fermerlrariort. Inhibition

of butyrate fermentation increased in the following order: stearic (C18:0) acid < oleic

(Cl*.*) acid < linoleic ( C l g : ~ ) acid < mixtures of linoleic (C1g:*) , oleic (Ci8:i) and stearic

(C 18.0) acids.

5 . Itzhibiriot~ calrsed by itidividuai LCFAs and mixed LCFA on ace~iclmtic methatzogetls

rïas sigqficatlt itr comparison to corrtroi citiitrres. Acetate (C2) consumpt ion was

affected by individual LCFAs and mixed LCFAs. The inhibitory effect is exerted

abo\,e a threshold concentration and anaerobic systems will be affected by this

inhibition.

6 . Il ~hihitio,~ catrsed by individllal atld mixed LCFAs oi I hdvdrogetlorrophic tnethmogrt IS

\im sfaristica/& sÏg?lrficuttt ïtl cornparison to controi ctr fiures b tri fikeb has ktlle

practical itnplicatiotr. Hydrogen consumption was affected by individual and mixed

LCF.As however; the inhibition is not believed to be severe enough to affect the

overall operation of an anaerobic system receiving LCFAs.

12.0 ENGiNEERING SIGNIFICANCE AND SUGGESTIONS FOR FUTURE RESEARCH

Based on data fiom the degradation studies, Iinoleic (C1g12) and oleic acids were

degraded faster than stearic (Cl~o) acid. Hence, the slower degrading stearic (18:0) acid

will control the solids retention time (SRT) used to design an anaerobic treatment system.

If the system is designed based on degradation of linoleic (C18:z) or oleic (Cls:l) acids.

stearic ( C I ~ O ) acid will accumulate in the reactor and be washed out in the effluent.

The effect of temperature on the degradation and inhibition of oleic (C1gll) acid was

investigated by Hwu ( 1997). H w ( 1 997) reponed increasing LCFA degradation rates

two fold by a 10 O C temperature change from 40 to 50 OC however, increasing reaction

rates also increased inhibition. Although increasing degradation rates are observed at

elevated temperatures, understanding the reIationship between degradation and inhibition

is important for the operation of anaerobic systems treating effluents containing LCFAs.

Additional research is required to examine degradation and inhibitory effects of LCFA

mixtures.

Separation of aceticlastic methanogens to eliminate LCF.4s inhibition is a major

design issue to be resolved by engineering design of anaerobic systems. Since LCFAs

are adsorbed ont0 biomass (Hwu (1 997), it is conceivable a two-stage reactor could be

used to treat emuents containing LCF.4s. The two-stage reactor concept was onginally

proposed by Hanaki et al., ( 1 98 1) however, no one has investigated use of this

technology. In the first acetogenic @-oxidation) reactor, LCFAs would be adsorbed ont0

biomass and degaded to acetate and shorter chah LCFAs. Acetate fiom the first reactor

would then be transferred to the second methanogenic reactor where it is converted to

methane and carbon dioxide. Use of this approach removes the inhibitory effects of

LCFAs on aceticlastic methanogens by separating the hydrogen producing acetogens

from the aceticlastic methanogens. Further devefopment of the two-stage approach will

require determining design and operational parameters.

LCF-4 byprodua inhibition, another issue arising from this study, was observed when

cultures were fed with linoleic (Clg:~) acid. Accumulation of palmitic (C16:0) and myristic

(C 1 4 0) acids b y products may have influenced acetate degradation since large quantities of

both LCFA byproducts were observed in cultures receiving 50 and 100 r n g - ~ - ' linoleic

acid. Koster and Cramer (1 987) examined aceticlastic methanogenic inhibitory

effects due to rnyristic (C14.0), lauric ( C 1 4 , capric (Clo:~) and capryiic (Go) acids and

found iauric ( C i t : ~ ) acid to cause more severe inhibition. Further research to evaluate the

impact of C 16 to C8 LCFAs rnixqures on anaerobic organisms will assist in

understanding their synergistic effects on acetogenesis and methanogenesis. In addition,

understanding why LCFA byproducts accumulate and remain undegraded for long

periods \vil1 require further investigation.

Throughout this study the degradation and inhibitory effects of Iinoleic (Cig:~), oleic

(C I I I : l ) and stearic (C acids between 10 to 100 r n g ~ - ' were exarnined using

approsimately 1500 r n g - ~ - I VSS. In anaerobic systems these low biomass concentrations

may not represent typical operating conditions and additional work is required to examine

the effect of increasing the biomass to LCFA ratio. Additionally, in some effluents, the

influent oil concentration may be greater than 20,000 r n g - ~ - ' (Borja and Banks, 1994).

.L\ssuming a dissolved air flotation pretreatment facility is used with an 89 - 98 % oil

removal efficiency (Johns, 1995), the oil concentration remaining to be treated is between

400 to 2200 rng*~-' . This untreated oil fraction still exceeds the LCFA concentrations

used in these studies and additional research is required to investigate the impact of

LCFA concentrations greater than 100 rng-~" .

In effluents such as for example, fkied foods processing, LCFAs and carbohydrates are

present. Data fiom this study have shown LCFAs to inhibit acidogenesis, acetogenesis

and aceticlastic methanosenesis under the conditions examined. Therefore, during

treatment of these effluents, it is likely organisms mediating the degradation of glucose

and its byproducts will be inhibited at some threshold LCFA concentration.

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Appendia A

LCFA Free Energy Calculations

Table A. 1 : Constants A and B for AG; calculations C hemical

Group ,C H3

CH2 COOH

pans CH=CH "' ~ e a n ( i 999) AG; = A + (B/lOO)ST T (K) = 25 OC + 273 OC = 294 K

AG;, kcal- mole"

4 - 3 4 2.05

-89.87 22.89

A "' kcal-mole"

B "' kcal-mole" -K"

-10.94 1 2.22 -5.19 1 2.43 -98.39 1 2.86 17.19 1 1.92

Coinpoiind

"' Pree energy values are calculated bascd on the nuniher of functional groups iii each LCFA.

CHj(CH2)4CH=CHCH2Cii=CH(C142)7C00H CHi(CH2)sCH=CH(CH2)7COOH

r

AGl? (kcal-inole") CHI "' 1 CH^ "' 1 COOH "' 1 ( ' 1

AG/' gaseous 1 (2 ) coinpound (kcal-mole- )

" Free energy values reported in Table A. 1 .

-4.34 -4.34

24.58 24.58

-89.87 -89.87

45,78 22-89

-23.85 -46.74

conditions.

'l'atilc A.3: Estiniatioii o f coristants for Aiitoiric's cquat ion Vapor I'rcssure, nini Hg Compound CHj(CH2).lC001i Cl-13(CH2)r,COOH CIH3(CH2)nCOOH CHj(CH2)loCOOH CH1(CH2)l 2COOH CHJ(CH~),&OOH CCIx(CH2)16COOH CHj(CH2)~CH=CH(CH2)7COOH "' CH~(CH2)~CH=CHCH2CH=CH(CH2)1C001-I "' CH~(CH~)ICH=CH(CH~)~COOH ("

Antoine's equation for vapor pressure log 10 P = -0.05223(a/T) + b """~ssuine same constants a and b as for stcaric (Ciw 0) acid (CI-~,(CHI)I(,COOH) (no data available). '"~ssume same constants a and b as for palniitic (C16:iJ acid (CH3(CI-12)irCOOH) (no data available). '4"5' Values for a and b were estimated frorn solving Antoine's equation under the two given teinpcrature

I I I O

l'crtipcrat ure (K) a

874 10.39 8759 1.28 119120.74 73591 -59 755 16.00 749 17.70 83024.8 1 83024.8 1 83024.8 1 749 17.70

344.4 365.3 3 98. 0 394,O 4 15.0 426.6 446.7 446.7 446.7 426.6

b ( 5 )

13.26 12.52 15.63 9.76 9.50 9.17 9.7 1 9.7 1 9.71 9.17

372.5 397.0 425.2 439.0 463.8 478.8 498.0 498.0 498.0 478.8

Table A.4: Estiniation of Henrv's constant

"' Calculated from Antoiiie's equat ion for vapor pressure (sec Table A. 3) log 10 P = -0.OS223(a/T) + b (Dean, 1999)

'2' Ralston and Hoerr ( 1943) "' Henry's constant = vapor pressure (atm) 1 solubility (molesi.'). '""" Assume solubility the samc as stcariç (Cl n:o) acid (CH3(CH2),(,COOH). '" Assume solubility the sanie as palinitic (Clc:ii) acid (CI.li(CH2)l.lCOOH).

Compound Vapor pressure, atm " )

Solubility (water) L - 1 (2 )

Solubil it y, niolesl"

Henry's Constant a t iw~mole~ ' (')

Tiiblc A.5: Estiiiiiiiioii of ioiiizcd LCFA frcc ciicrgv of foriiiiiiioii

H = Henry's constani, valiics iakcn froiii Tiiblc A.4. ('' D G ~ ( 1099) "'K. for CC, Io Clfi wcrc csiiiintcd froni il linciir ~Wi ipo l i i t i ~ i i of K. niliics for C: Io C,, iicids. K ii VII~ICS for Cl(, iiiid Cl ,' iirc ~ISSIIIIIC~ IO bc flic siiiiic.

"' Ka valiics for CI* LCFAs iirc iissiiiiicd io bc ilic sariic. '" CCrclciila~cd viiliics iirc bciwccii 7 io IO %i grciiîcr t l i i i i i viiliics rcpricd by (Thiiiicr O( trl. , 1077).

R l i i ( l / l i ) kciil -itiolc"

-8.70 - 10.99 -7.44 -7.57 -7.2 1 -7.81 -7.82 -7.82 -7.2 1

Ili( 1/11)

-14.00 - 18.57 -12.58 -12.79 -12.17 -13.20 -13.21 -13.22 -12.17

Coiiipoiiiid

CH~CH~)I,COOH CHdCH2)&OOH CH3(CH2)&OOH CH3(CH2)I ,COOH CHj(CH2)I.&30H CHdCH2hnCC)OH CH3(CH2)iCH=CH(CH2)7COOH CH3(CH2)4CH=CHCH2CH=CH(CHZ)7C00H CH3(CH2)sCH=CH(CH2)7COOH

IW '" (atrn~~mole.'). '

4.151E-o7 8.59 1 E-0') 3.452E-O0 2.789E-O0 5.159E-06 1. 11411~-00 1.835E-00 I ,822E-06 5.159E-O6

AG;' ;\q. iiriioiiizcd kciil~iiiolc~'

-VO.OZ -88.82 -111.17 -77.20 -72.74 -09.25 -50.40 -3 1.67 -53.94

K,, """

I.35E-O5 I .3OE-O5 1.2%-OS 1.2OE-OS

I E - O ' I .OOE-05 '" 1.00E-OS 'v 1 ,(HIE-OS (" 1 , l 5 - 0 5 '

RTlrr (K,J k ~ i i l . i i i ~ l ~ . '

4 -64 -6.66 -6.fi11 -0.7 1 4.73 - 6 . 1 -0. 11 l -0.8 1 4 . 8 i

AG," i q ionizcd kciil~iiiolc"

-97.25 -95.47 -87.85 -83.9 1

-79.47 " -76.06 (" -57.27 -38.49 -60.76

AG;' ;iq, ioiiixcd k.f.iiiolc~'

-4OO,9 -399.4 -367.5 -35 1 ,O

-332.5 -830,8 -239.6 -161,O -254.2

Appendix B

Example LCFA Mass Balance Calculations

Table B. 1 : Mass balance calculations for cultures receiving 100 rng~-' linoleic (Cisz) acid at time 5 1 davs

1 Parameter 1 Measured concentration 1 Linoleic acid units 1 Carbon units 1

[ Total 1 - 104.1 6.77 1 Based on total stoichiornetry shown in Table 2.17.

1

Linoleic acid Palmitoleic acid Palmitic acid Mvristic acid

For example.

LA conc. = PA conc. x ( i mole/MW PA) s ( 1 mole LN1 mole PA) x (MW LNmole LA)

r n g ~ - ' 68.8 5.97 22.8 2.88

Acetate was not included because of variable stoichiometery depending on the extent of the reaction.

r n g - ~ - ' 68.8

rnrnole C-L" 4.47

6.69 25.3 3.61

0.43 1 -64 0.23