Factors influencing the rate and stability of the anaerobic digestive process : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biotechnology at Massey University

Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author.

FECTORS I}IFLI'EDTCINC TBE RITE AND SEABII.TS OF TgE NNAEROBTC DTGESTIoIT PROCESS

A

thesi-s presented

in partial fulfilment of

the requirements

for the

^degree

of Dgctor of

philosophy

in

BiotechnoloEy

at

^Massey

University

AI{DREI| TOEN I'BITSoII 1986

11

ABSERJACI

Three factors affecting the rate and stability of the methane

fermentation of a readily-hydrorysable feedstock vrere

investigated. The aim of this work was to d.everop improved.

processes and control- strategies to facilitate economic treatment of ind.ustrial wastes by anaerobic d.igestion

A comparison was made between the performance of a continuousry- fed digester and semi-continuous digesters srug fed every second

day. A semi-synthetic medium eri.th grucose as the major carbon

and energy source was used and seed materiar was transferred.

between the digesters, which were operated under similar loading conditions. The continuous digester repeated.ly failed even ^when

nnaral-oA .+ /l l

v..elsLes su *ilution and loading rates much lower than the maxi-mum values commonly reported. rn contrast, the semi-

continuous units provided satisfact,ory perfonnance and could be

easily and rapidly recovered from retard.ed. operation. Faj-Iure of the contj-nuous digesters rdas characterised by a steady farl in volatile suspended solids concent,ration forlowed by a rapid.

accumulation of acetate, and was attributed to a deficiency in the medium of one or more essential nutrients. These erere

l:roughi to be provided in t.he semi-continuous diqester bv lvsis of acidogenic bacteria or luxury uptake from the ^medium.

Degradation of acetic and propionic acids vras investigated. in batch culture. rncreasing the concentration of either acid. from lol l-evefs decreased the rate of utilisation of the acid, but the proposed inhibitory role of un-ionised acids r.ras not conclusively supported. rncreasing: the initiar acetate concentration above 1000 to 1500 mg.r-1 significantly red.uced the rate of degradation

^€ ^-^^l ^Jl^l ^! c^n

v! lvlvt,rvlrdus added at 500 mg.l-r. -- when acetate was add.ed aE

2000 mg.r-' the rate of i propionate utirisation was approximately half of that when ace!.ac.e was present at 500 rng.l-1 or l-ower.

11t

In batch culture experiments, addition of ^up to 3.2 ^nM cysteine- hydrochloride or sodium sulphide, or ⁴ .4. ml'[ sodj.um

thioglycollate did not inhibit total gas production from samples drawn from the continuous dig'ester. ^However the rate of ^methane production in effluent samples from a semi-continuous dig:ester was inhibited by 25 t to 30 t by addition of 3.2 ^mM cysteine or sulphide. rnhibition ^was attributed to tbe sulphide ion. ^Sodium thioglycollate did not inhibit ^methane production from acetate but propionate degradation was markedly reduced, wit,h increasingt inhibition ^noted with increasing incubation tirne.

The work adds to a considerable body of investigation into ^the factors influencing: anaerobic digestion and. the unresolved problem of process stability in long-term operation of conventional stirred t.ank digesters has again been highlighted.

Indicators and possible causes of process failure have been

suggested and further development of these should assist in the continuing increase in the rate of treatment while ^ensuring acceptable working rnargins of safety for the Process.

I\/

ACKNOIVIADGEIIEIIIS

I wish to acknowledge the following:

* _Professor R.L. Earle, Dr V.F. Larsen and Dr G.J. Mandelsorr,

for their supervision and assist,ance.

* l'1r .Tohn Alger and Mr Derek Cou1ing, for their assistance in building and maintaining eguipment.

* Fellow staff members and post-graduate students of the Biotechnology Department,, for many useful discussions. I particularly wish t,o thank Dr Tony Paterson for his ^guidance on the use of various computer packages and his help with printing this thesis.

* The Department of Scientific and Industrial Research, for sponsoring this research.

* _{Nicky and} Sa1ly, for proof-reading at a critical tfune.

* Brenda and Isaac, and other friends and farnily, for their support and encouragement,. this work is dedicated to them.

TABLE OF coxEH|rs

ABSTRACT

ACKNOWLEDGEMENTS

TABLE OF CONTENTS

TABLE OF FIGURES TABLE OF TABLES ABBREVIATIONS CHAPTER ONE CHAPTER TWO

2.L Introduction 2.2 Microbiology fermentation

PREAMBLE

LITERATURE REVIEW

and biochenietry of the methane

2.2.1

Introduction

2.2.2

The fermentative bacteria

2.2.2.t

Hydrolysis

of

biopolyuers

2.2.2.2

Pnoduction and

utillsatton

of

f ermentati.on lntermediates

2.2.3

The hydrogen-producing acetogenic bacterla

2.2.4

The

role of

hydrogen

ln

regulating the fermentation

2.2.5

The methanogenic bacteria

2.3

Kinetic analysis

of

the nethane ferrnentatlon

2.3.t

Introduction

2.3.2

^COD

flux in

the ^methane fermentation

2.3.3

^Reaction rate analysis

of

the rate-1lnitlng

2.3.4

Modelling step

of

nicrobial ^growth

ln

anaerobic

digestion

2.4

Anaerobic reactor designs

2.4.L

fntroduction

2.4.2

The conventional single tank ^process

2.4.3

The anaerobic contact ^process

2.4.4 .

The _(UASBR}upflow anaerobic sludge blanket reactor

2.4.5

The anaerobic upflow

filter

2.4.5

The downflow stationary

fixed-fllm

reactor

( DSFFR )

2.t+.7 The attatched-fi1n fLuidised-bed reactor

(AFFBR) and the attatched-fllm expanded bed

reactor ^(AFEBR)

2.4.8

Novel anaerobic ^processes

Page

1i

1v v

ix

xii

xv

1 3

13 14 3 4 4 7 7 8

35 36 36 L7 22 22 23 25 27 31 31 33 34 35

35

v1

2.5 Envlronmental factors affecting the rate ^and stability of the methane fernentatlon

2.5.1 Introduction 2.5.2 Temperature

2.5.3 pH and alkalinity

2.5.4 Oxi-dation-reduction potential ^(ORP) and

anaerobiosis

2.5.5 Retention time and organic loadlng rate ^(OLR) 2.5.6 Nutritional requirenents and toxic compounds

2.5.6.7 fntroductlon

2 .5.6.2 ^Ammonia

2.5.6.3 Sulphur conpounds

2.5.6.\ A1ka1i and alkaLi-earth netal cations

2 .5.6.5 Heavy metals

2.5.6.6 Other growth-promotlng factors 2.5.6.7 Other toxic compounds

2.5.7 Fermentation intermediates and end-products

2 .5.7 .L Introduction 2.5.7 .2 ^Carbon dioxide

2.5.7 .3 Volatile f atty acids ^(vFA)

2.6

^Summary

CHAPTER THREE

Page 37 37 37 38 41 42 45 46 47 4B

5t

52 54 55 55 55 56 58

5t

63 63 63 63 63 64 56 65 66 67 67 nnII 72 73 75 77 79 79 79 B1 81 81 81 B3 B4 B4 B4 B4 B5 B5 86 B6 METHODS AND MATERIALS

3.1 ^Materlals

3. 1.1 Gener.al chenicals 3.1.2 ^Gases

3.1.3 Chromatography materlals 3.1.4 Microbial ^{growth ne<lium} 3.1.5 ^Glassware

3.2 Analytical ^proceduree

3.2 .1 lntroduction

3.2 .2 ^pH value

3.2.3 Volatile fatty aclds ^{(vFA) by} 3.2.\ ^Volatj-1e faEty acids by direct 3.2.5 ^{Chemical oxygen demand (COD)} 3.2.6 Biologlcal ^{oxygen demand (BOD)} 3.2.7 Solids compositlon

3.2.8 ^cas composition 3.2.9 Alkalinity

3.2.1O ^{Ammonia nitrogen} 3.2 ^{11 Dissolved} sulphide 3.2.L2 ^Glucose

3.2.L3 Oxidation-reduction potential 3.3 Continuous digestion experiments

3.3.1 ^Equipment and i.nstrumentation 3.3.2 Operating conditlons

3.4 Semi-continuous digestJ-on experiments

3.4.1 Equipnent and instrumentatlon ^'

"lJ2 i q Rafnh

3.4.1.1 ^{Microferm dlgester} 3.4.1,.2 ^{Biogen digester} Operating conditions digestion experiments

Equipment and instrumentation 3.5.1.1 ^Serum bottle cultures

chromatography

tltration

(ORP)

vil

3.5.1.2

^{Flask digesters}

3.5.2

^{Operating conditlons}

3.6 Statistical

analysis

of

data

CHAPTER FOUR CONTINUOUS AND SEMI-CONTINUOUS DIGESTION EXPERIMENTS

4.1

Introduction

4.2

Experi.mental method 4.2

.t

^Equipment

4.2

.2

Experimental ^progra.nme

4.2.3

Growth mediun

4.2.4

Inoculun sources and preparation

4.2.5

Start-up ^procedure

4.2.6

Sanpling

4.2.6.I

Continuous digestion experinents

4.2.6.2

Serni-continuous digestlon experinents

4.2.7

Analytical procedures

4.3

Results and discuseion

4.3.1

Continuous digestion

4.3.1.1

^Run 1 ^(Conr)

4 .3.t

.z

^Run 2 ⁽ cDR2 )

4.3.1.3

^Run 3 ^(con3)

4.3.2

Semi-continuous dlgestion

4 .3 .2

.L

Run 1 ⁽ SCDR1 )

4.3.2.2

^Run 2 ^(SCDR2)

4.3.3

Evaluati-on

of

the

nutritional

^gtatus

of

the standard growth medium

4.3.4 Effect of

^nedlun addltlons on batch

digestion of

contlnuoue dlgester liquor

4.4

Overall discussion

4.5

Conclusions

CHAPTER FIVE DEGRADATION OF VOLATILE FATTY ACIDS THE METHANE FERMENTATION

5.7 Introductlon

5.2 Experimental method

5.2.7 ^Equipment

5.2.2 Inoculum sources and preparation 5.2.3 Culture conditions and preparation 5.2.4 Saropling and analytical ^procedures 5.2.5 Analysis of results

5.3 ^Results

5.3.1 Experiment ¹

5.3.1.1 Effect of ^{acetate on} utllisation of propionlc acid

5.3.1.2 Effect of pnopionate on utllisation of acetic acid

5.3.2 Experlment ²

5.3.2.7 Effect of ^{acetate on} utilisation of propionic acid

5.3.2.2 Effect of propionate on utilisation of acetic acid

rN

Page 87 B7 87 88

B8 88 88 88 90 90 90

9t

9L 91 92 92 92 92 94 105

t07 r07

TL2 118 124 L26 135

t37

137

t37

L37 138 138 139 L39 140 140 141 r47 150 150 755

5.3.3

^Estimation

5.4

Discussion

5.5

Conclusions

CHAPTER SIX

APPENDIX THREE

APPENDTX FOUR

APPENDTX FIVE

vili

page

of

the ^ps;fprrm specific ^growth

rate

L6t

151 158 EFFECT OF SULPHUR.CONTAINTNG _REDUCING

AGENTS ON THE METHANE FERI{ENTATION

170

t70t7t

77L L73 174 L74 190 194 796 204 224

6.7

Introduction

6.2

Experinental method

6.2.!

Equiprnent and general procedure

6.2.2

fnoculum source and media preparation

6.2.3

Sanpllng and analysis 6.3 Results

6.4 Discussion 6.5 Conclusions

CHAPTER SEVEN REFERENCES

APPENDIX ONE

FINAL DISCUSSTON AND CONCLUSIONS

PROCEDURE USED TO CALCULATE THE

TOTAL VOLATTLE FATTY ACID ^(TVFA}

CONCENTMTTON

APPENDTX TWO ADDITIONAL EXPERTUENTAL DATA PERTAINING 226 TO THE CONTINUOUS AND SEMI-CONTTNUOUS

DIGESTION EXPERTMENTS

EXPERIMENTAL DATA FOR BATCH DIGESTTON EXPERIMENTS INVESTTGATTNG DEGNADATION

OF VOLATTLE FATTY ACIDS

229

SAMPLE GENSTAT _PROGRAI'! USED TO FIT THE 234 LOGTSTTC EQUATION TO EXPERTI{ENTAL DATA

FROM STUDTES ON DEGRADATION OF VOLI\TILE FATTY ACIDS

EXPERTMENTAL DATA FOR BATCH DIGESTTON EXPERIMENTS INVESTIGATING THE EFFECT OF SULPHUR-CONTAINING REDUCTNG AGENTS ON

THE METHANE FERI'IENTATTON

239

1x

TABLE OF FIGUNES;

page

2.I Outline of the majon netabolic ^ptocesses involved in 5

the nethane fermentation

2.2 Major pathways fon anaerobic uti.llsation of pynuvate ¹⁰ 2.3 ^COD flux in the nethane fernentation of complex waste ²⁴ 2.4 Proposed COD flux in the methane fernentation of ²⁶

carbohydrate waste

2.5

Configurations

for

the major anaerobic reactor designs

3.1

Experimental set-up

for

the contlnuoua

digestlon

⁸²

experiments

3.2

Experimental set-up

for

the senl-continuous

digestion

⁸⁵

experiments

4.1

Performance

of

the continuous dlgegter, run

1

errcl.*

(CDR1), from inoculatlon

to

day 103

4.2

Perfomance

of

the continuous digester, run

2

^(CDR2) encl.

4.3 Specific r

"ate ^data for ^{CDR2 encl.}

4.4

Effect

of

^additi-on

of

sulphur-containing

reducing

¹⁰⁰

agents

to

^CDR2; five-day avet:age data

4.5

ORP and TVFA data

for

^CDR2 durlng the onset

of

^LOz retarded digestion

\.6

Perforrnance

of

the continuous dlgester, run 3 ^(cDR3) encl.*

4.7 Specific rate daEa for ^CDR3 encl.*

4.8

Avenage tirne course

of total

^gas production

fron

¹¹¹

SCDRl

4.9

Performance

of

the seml-continuous Blogen

digester,

^enc1. *

( SCDR2 )

4.10 Average

tine

^course

of total

gas pnoduction

frorn

^I75

SCDR2

4.11 Acetate degradation

in

batch dlgesters

supplenented

^t25 with trace metals and 1ysed. ce11s

. :jj

* Iarge tl iagram enclosed inside back ^coven . _,--,'. i

. ':l

.i;

32

page

4.I2 Data from ^Hansson and Molin ^(1981a) showing methane ¹³³ producti.on from acetate in continuous-culture under

a N, atmosphere

5.1 Propionate utilisation in selected runs with ¹⁴² propionate added at 500 mg/I, experinent ¹

5.2 Proplonate uti.llsation ln duplicate runs with ¹⁴³ propionate added at 500 mg/I, experinent ¹

5.3 ^{Observed and} predicted propi-onate concentfatlons 145

for ^gelected runs, experiment ¹

5.4 Volumetric rate of proplonate utilisatlon in selected t46 runs wi-th proplonate added at 500 _{mg/\ ,} experiment ^1-

5.5 ^Acetate utilisation in selected runs with ¹⁴⁸ propionate added at 500 mg/I, experiment ¹

5.6 ^{Observed and} predicted potential acetate pool I49 concentrations for selected runs, expe::iment ¹

5.7 Propionate utllisati-on in selected runs with ⁷⁵⁷ acetate added at 500 mg/\, experiment ²

5.8 Propionate utilisatj.on 1n duplicate runs with 153

propionate added at 500 mg/I, experirnent ²

5.9 ^Observed and predicted propionate concentrati-ons ^L54 for selected runs, expefiment 2

!.10 ^Acetate utilisation in seLected runs with ^tS6 propionate added at 500 mg/I, experinent ²

!.11 ^Acetate utilisation in dupJ-icate runs with I57 propionate added at 500 mg/l , experi-ment 2

5.12 Observed and predicted potential acetate pool 158

concentrations for selected runs, experinent ²

5.13 Acetate utilisation in selected runs with acetate 59

added at ²⁰⁰⁰ mg/\, experlment ²

5.14 Acetate utilisation ln dupllcate runs with acetate ¹⁵⁰

added at ²⁰⁰⁰ mg/a, experiment ²

6.7 Total gas production in batch digesters, experiment 1 775

6.2 Total ^gas production in batch digesters, experiment 2 L76

6.3 Methane productlon for batch digesters in experinent 3 ^t78 6.4 ^Methane production for batch digesters in experiment 3 ^L79 6.5 Methane productlon in batch digesters, experiment 4 183

r1

6.6 Methane

6.7 Methane

6.8 Methane

6,9 Methane

production production production production

dlgesters, dlgesters, digesters, dlgesters,

experinent experiment experlnent experinent in batch

in batch in batch in batch

5 6

7 8

Page 184 185 185 188

xil

TABLB OF TABI,E'S

page

2.7

Stoi-chiometny and change

in

free energy values

for

¹⁵

propionate _and butyrate degradatlon reactione

2.2

Stoichiometry _{and change}

in

free enetgy values

for

^LT

pyruvate degradation reactlons

2.3

Stoichionetry and change

in

free energy values

for

^Lg the major nethane fornation reactions

2.4

Kinetic parameters describing the methane

fernentatl-on

30

2.5

Conparison

of typical

organlc loadLng rates

and

⁴⁵

treatment efflciencies

for

various anaerobic reactor designs

3.1

Conposition

of

the growth nedium used,

in contlnuous

^d5

and seml-continuous dlgestlon experlments

3.2

Composition

of

the vitamin solution used

to

66

supplenent the standard growth rnedium

3.3

Conposition

of

the

volatile fatty

acld

standard

69

solutions

3.4

Estination

of

the precislon

of volatile fatty acid

TO

concentration neasurement

3.5

Eetimation

of

the accuracy

of

the volatiLe

fatty

_7!

acid rneasurement by gas chronatography

3.6

Performance characterlstics

for

chemlcal

oxygen

,U demand measurement

3.7

Estimatj-on

of

the precision

of

biological

oxygen

7j

demand measurement

3.8

Estination

of

the pnecision

of

solids

conpositlon

TT

!oeasurement

3.9

Performance characteristics

of

^gas

compostion

80 measurement

4.1

Overview

of

experimental programme

for continuous

89

and semj--continuous digestion experinentg

4.2

Comparison

of

speciflc rate data

for continuous

104

digestion, run

2

^(CDR2) ana other studies using glucose-based media

x1L1

page

4.3 Summary of steady-state data for the senl-continuous ¹⁰⁹ Microferm d.j.gester ^(SCDR1)

4.4 Summary of steady-state data for the semj.-continuous ¹¹³ Biogen digester ⁽ SCDR2 )

4.5 Major performance characteristics for SCDR2 at Lt6 steady state and for ^CDR3 from day 7O to day 96

4.6 Conparison of the composition of ^yeast extracts from ^LzO various manufacturers

4.7 Comparlson of the standard growth mediun ^L22

supplemented with cysteine-HC1 and other nedia used

in studies of the methane fermentation

5.1 Experinental conditions for batch dlgestion ^138a experlnents

5.2 Coefflclents of the logistic equation for acid ¹⁴⁴ utilisation in exoerinent ¹

5.3 Coefficients of the Loglstlc equation for acid I52 utilisation in exoeriment ²

6.I Batch digester protocol showing component volumes t|t 6.2 Experimental conditions fon the batch digestion I72

experiment s

6.3 Actual and theoretical yields of nethane fron batch ¹⁸⁰ digesters in experiments 3 to ^B

6.4 Residual volatile fatty acids ln batch digester with ¹⁸¹

added thioglycollate; experiment 3, ^tun5

6.5 Residual volatile fatty acj-ds in batch dlgester with ^L87

added thioglycollate; expeniment 8, run3

6.6 Residual dlssolved sulphide concentrations in ¹⁸⁹ selected batch digesters

A2.1 Gas production from SCDR1 at varying tines fron feed ²²⁶ addition

A2.2 Gas production from SCDR2 at varying tines fron feed ²²⁷ addition

A2.3 Acid concentratlons in batch digesters supplemented 228

with trace metals or lysed cell-s

Al.1 Data for experiment ¹ 229

A3.2 Predicted volumetric rates of proplonate degradation ^23O for batch digester, experiment ¹

xtv

AJ. J ^Data for

45. 1 Data for

AJ.2 Data for 45.3 Data for 45.4 Data for 45.5 Data for 45.6 Data for 45.7 Data

for

A5.8 Data for

experiment experinent experiment experiment experinent experinent expeninent expeniment experi_rnent

2 1

2 3

4

5 5 I

8

Page

23t

239 239 240 240 24L 24L 242 242

EBEREMTATTONS

Abbreviations of volatile fatty acid names:

Ac acetic aci-d

Pr propionic acid iBu iso-butyric acid Bu butyric acid iVa iso-valeri-c acid Va valeric acid

Acids will be reffered t,o by the suffLxes r-ate' and '-ic ^acidtr interchangeably.

A subscript "i' ^denotes the intlal acid concentration.

Abbreviation of units:

atm

atmosphere

s'

^gramne

hr

hour

kcal

kilocalorie

I litre

mg:

milligrarune

min

minute

mI nilli1itre nm

millimetre

mM

milU:noles per

litre

mo1

mole

mv ni-llivolt

r.p.m.

revolutions per minute

pf

microlitre

xvf-

other abbreviations:

a coefficient, of the logistic equation (dirnensionless) ATP adenosine triphosphate

b coefficient of the logistic eguation ^(day-l) BOD' five-d.ay biological oxygen demand (*g.f-l) CDR1 continuous digestion, run L

CDR2 cont.inuous digestion, run ² CDR3 continuous digestion, run 3

COD chemical oxygen denand (*S.f-1)

CODr chemical oxygen dernand removed ^(mg.1-1)

CODR' specific rate of chenical oxygen demand removal (9 coDr.g vss-1.d"y-l)

cyst cysteine-hydrochloride

E" electrode potential relative to the saturated calomel

electrode ^(mV)

Eh electrode potential relative to the standard hydrogen

electrode ^(mv)

HRT hydraulic retention time ^(days) i.d. internal diameter

K coefficient of the logistic equation ^(g.1-1) K" half-saturation constant for sustrate utilisation

(n. ,_r )

NAD nicotinamide adenine dinucleotide

NADH reduced nicotinamide adenine dinucleotide

oLRs specific organic loading' rate ⁽⁹ COD.g VSS-1.d.y-l) OLR' volumetric organic loading rate ^(g COD.t-1.aay-1)

ORP oxidation-reduction potential

pCOZ carbon dioxide partial ^{pressure (bar)}

rs rate of substrate utilisation (g substrate.l-1.hr-1) rsrmax maximum rate of subst,Eate utilisation

f,x race of biomass growth (g biomass.l-1.d"y-1) s substrate concentration (n.t-t)

S total sulphur concentration ^(mM or ng.t-l) SCDR1 semi-continuous digestion, runl

SCDR2 semi-continuous digestion, run 2

Irvi1

s.d.

standard deviation

sRT

solids retention

tir@

^(days)

suJ.p

sodium sulphide

t

^t,l-me

thio

sodium thioglycoJ.J.ate

TSS

tot.al suspend.ed soLLds tg.f-1)

Tvl'A total volatire fatty

acid, concentration as acetate

(rng. r-1y

uwA

un-ionised

volatite fatty

acid concentration as acetate

(mg. t-1)

\rFA volatile fatty acid

(concentratlon)

(rg.f-l) vSS votatite

suspended soHds

(S.f-l)

Y*s

bLornass

yietd coefficlent

(g VSS.S COD}

p

specLfj.c gro$rth

rate

(aa"-f,

Fma*

^maximurn opecific _{Atrowth rate}