Kristina Henriksson and Oscar Tenfält and Environmental Engineering Department of Chemical...

Water and Environmental Engineering Department of Chemical Engineering

Measurements of Hydrolysis in Moving Bed Biofilm Reactor Carriers

- Evaluation by means of Oxygen Uptake Rate Measurements

Master’s Thesis by

Kristina Henriksson and Oscar Tenfält

February 2011

Vattenförsörjnings- och Avloppsteknik Institutionen för Kemiteknik Lunds Universitet

Water and Environmental Engineering Department of Chemical Engineering Lund University, Sweden

Measurements of Hydrolysis in Moving Bed Biofilm

Reactor Carriers - Evaluation by means of Oxygen Uptake Rate Measurements

Master Thesis number: 2011-01 by

Kristina Henriksson and Oscar Tenfält Water and Environmental Engineering Department of Chemical Engineering

February 2011

Supervisors: Professor Jes la Cour Jansen

Ph.D. Eva Tykesson

Examiner: Associate professor Karin Jönsson

Postal address: Visiting address: Telephone:

P.O Box 124 Getingevägen 60 +46 46-222 82 85

SE-221 00 Lund. +46 46-222 00 00

Sweden, Telefax:

+46 46-222 45 26

Web address:

www.vateknik.lth.se

i

Summary Today the need for clean water is rapidly increasing as the world’s population grows

by each year. The new awareness about the decreased rainfall that is expected in large

parts of the world due to climate change and urbanization, which accumulate large

amounts of people in cities, calls for efficient wastewater treatment facilities. To remove

organic matter in wastewater activated sludge is the dominating biological treatment

method, in which the microorganisms consume organic substances suspended in the

solution. After the water is treated the microorganisms are settled and recycled in the

process. Another method that is becoming more common is the Moving Bed Biofilm

Reactor, in which the biofilm grows inside carriers of polyethylene, which in addition to

protecting the biofilm from friction forces, allow for a larger biomass in the treatment

process.

In this master thesis the aim was to determine the extent to which particulate organic

matter in wastewater is hydrolyzed in an MBBR carrier process by means of bacterial

respiration rates. A new method based on Oxygen Uptake Rate (OUR) measurements

was developed and important aspects concerning the method were identified. The

method was then used for measurements of hydrolysis during different operating

conditions. By comparing the bacterial respiration rates in wastewater containing

particulate organic matter and filtrated wastewater, the hydrolysis was qualitatively

determined. An important parameter is the oxygen level, which must be as high as

possible and similar in all reactors that are to be compared. Also, the biofilm used must

be equal and preserved throughout the experiment. The method was found to measure

hydrolysis accurately.

The method was finally tested in two experiments, which investigated the influence of

added particles and dissolved carbon on hydrolysis. The results suggest that the

hydrolysis is independent both of organic loading and the availability of easily accessible

carbon.

iii

Preface This report is the result of a master thesis in Environmental Engineering performed

at Water and Environmental Engineering, Department of Chemical Engineering, Lund

University. Hydrolysis of particulate organic matter in Moving Bed Biofilm Reactor

carriers was determined by Oxygen Uptake Rate measurements. The master thesis was

based on a literature review and laboratory experiments performed at AnoxKaldnes AB

in Lund. The work was supervised by Ph. D. Eva Tykesson at AnoxKaldnes AB and by

Professor Jes la Cour Jansen at Water and Environmental Engineering.

Several people have contributed to this master thesis and we would like to thank you

all for your support. We want to give special thanks to Eva Tykesson for initiating the

project, for introducing us to the pilot plant in Kävlinge, from which wastewater samples

and biofilm carriers were collected, and for her valuable comments and thoughts on our

work. Without the initiative of Eva Tykesson, this project would not have been realized.

Further, we want to thank Jes la Cour Jansen for guiding us through challenging

discussions, for supporting us whenever needed with comments and thoughts on our

work. Jes’ knowledge and dedication has helped us on numerous occasions.

Thanks to the staff at AnoxKaldnes AB for being so cheerful and helpful, and for

showing so much interest in our work. It has been a pleasure getting to know you all

while we have been working with our master thesis. We would like to thank Jenny for

guiding us through the COD as well as the TSS and VSS measurement procedures and for

supplying us with several wastewater samples and carriers from the pilot plant in

Kävlinge. We would also like to thank Carina and Maj-Elén for the introduction to the

laboratory at AnoxKaldnes and for the information on current safety regulations. Thanks

also to Magnus for interesting comments on our work and to Stig for supplying us with

the timer we used to start and stop the aeration in our experiments. A big thank you to

Petter, the photographer who took the picture on the front page.

Finally, a huge thank you to our families and friends who have supported us through

this challenging task.

Lund 2011-02-16

Kristina Henriksson and Oscar Tenfält

v

Table of contents

1 INTRODUCTION ................................................................................................................ 1

1.1 Aim ................................................................................................................................................ 1

1.2 Limitations ..................................................................................................................................... 2

2 BACKGROUND ................................................................................................................... 3

2.1 Particles in wastewater .................................................................................................................. 3

2.2 Moving Bed Biofilm Reactor ........................................................................................................... 3

2.2.1 Biofilm kinetics ................................................................................................................................ 4

2.3 The fate of particles ....................................................................................................................... 5

2.4 Hydrolysis ...................................................................................................................................... 5

2.5 Enzymatic hydrolysis ...................................................................................................................... 5

2.5.1 Location of hydrolysis ...................................................................................................................... 6

2.5.2 Rate of hydrolysis ............................................................................................................................ 7

2.6 Oxygen Uptake Rate ...................................................................................................................... 7

3 MATERIALS AND METHODS ......................................................................................... 9

3.1 Sampling ........................................................................................................................................ 9

3.1.1 Storage of samples .......................................................................................................................... 9

3.2 Experimental procedure .............................................................................................................. 10

3.2.1 Experimental setup ....................................................................................................................... 10

3.2.2 Execution of the experiments ....................................................................................................... 11

3.2.3 Processing of data ......................................................................................................................... 11

3.2.4 Measurements of COD .................................................................................................................. 12

3.2.5 Measurements of total suspended solids and volatile suspended solid ....................................... 12

3.2.6 Measurements of volatile fatty acids (VFA) .................................................................................. 12

3.3 Calculations ................................................................................................................................. 12

3.3.1 Oxygen consumption .................................................................................................................... 12

3.3.2 Oxygen dependence ...................................................................................................................... 13

4 METHOD DEVELOPMENT .......................................................................................... 15

4.1 Experiment 1 - Introduction to the Oxygen Uptake Rate method ................................................ 15

4.1.1 Purpose ......................................................................................................................................... 15

4.1.2 Expected results ............................................................................................................................ 15

4.1.3 Changes to setup ........................................................................................................................... 15

4.1.4 Procedure ...................................................................................................................................... 15

vi

4.1.5 Results from the introductory experiment ................................................................................... 16

4.1.6 Summary of experiment 1 ............................................................................................................. 17

4.2 Experiment 2 - Importance of the oxygen level ............................................................................ 17

4.2.1 Purpose ......................................................................................................................................... 17


4.2.3 Changes to setup ........................................................................................................................... 18

4.2.4 Procedure ...................................................................................................................................... 18

4.2.5 Results and from the second experiment ..................................................................................... 19


4.3 Experiment 3 - Oxygen dependence ............................................................................................. 20

4.3.1 Purpose ......................................................................................................................................... 20


4.3.3 Changes to setup ........................................................................................................................... 20

4.3.4 Procedure ...................................................................................................................................... 21

4.3.5 Results from oxygen dependence experiment ............................................................................. 21


4.4 Experiment 4 - Repeatability test ................................................................................................. 25

4.4.1 Purpose ......................................................................................................................................... 25


4.4.3 Changes to setup ........................................................................................................................... 25

4.4.4 Procedure ...................................................................................................................................... 25

4.4.5 Results from the repeatability experiment ................................................................................... 26


4.5 Experiment 5 - Hydrolysis experiment ......................................................................................... 31

4.5.1 Purpose ......................................................................................................................................... 31


4.5.3 Changes to setup ........................................................................................................................... 32

4.5.4 Procedure ...................................................................................................................................... 32

4.5.5 Results from the repeatability experiment ................................................................................... 32


4.6 Evaluation of the method ............................................................................................................ 37

5 HYDROLYSIS EXPERIMENTS ..................................................................................... 41

5.1 Experiment 6 - Significance of particles ........................................................................................ 41

5.1.1 Purpose ......................................................................................................................................... 41


5.1.3 Changes to setup ........................................................................................................................... 41

5.1.4 Procedure ...................................................................................................................................... 41

5.1.5 Results from the investigation of particles significance ................................................................ 42


5.2 Experiment 7 - Influence of dissolved carbon ............................................................................... 44

5.2.1 Purpose ......................................................................................................................................... 44

5.2.2 Changes to setup ........................................................................................................................... 44

vii

5.2.3 Procedure ...................................................................................................................................... 44

5.2.4 Results from the investigation of dissolved carbons influence ..................................................... 45


6 DISCUSSION .................................................................................................................... 51

7 CONCLUSIONS ................................................................................................................ 53

8 SUGGESTIONS FOR FURTHER RESEARCH ............................................................ 55

9 REFERENCES ................................................................................................................... 57

APPENDIX A - REACTOR CONTENTS .......................................................................... I

APPENDIX B - COD MEASUREMENTS ...................................................................... III

APPENDIX C - OXYGEN CONSUMPTION .................................................................... V

APPENDIX D - TEST OF OXYGEN METERS ............................................................ VII

APPENDIX E - TSS/VSS AND VFA MEASUREMENTS ............................................ IX

APPENDIX F - HYDROLYSIS OF PARTICLES .......................................................... XI

APPENDIX G - ARTICLE ............................................................................................. XIII

1

1 Introduction Clean water, which is essential for both human and animal life, is today a limited

resource in large areas of the world. By 2008 the world population reached 6.7 billion

people according to WHO´s World Health Statistics (2010), and by 2011 we are, by UN’s

medium estimate, expected to become 7 billion people on earth (United Nations, Dep. of

Economic and Social Affairs, 2010). The world’s growing population places new

demands on the supply of clean water as well as on treatment measures.

Today most people reside in urban areas and the requirements for wastewater

treatment are rapidly increasing. In wastewater treatment plants, combinations of

physical, biological and chemical methods are used to purify the water. The biological

treatment step reduces nitrogen, phosphorous, bioavailable carbon and other organic

contaminants with the aid of microorganisms (Gillberg et al., 2003). The most common

and well-known biological wastewater treatment process is the activated sludge process

(Jonstrup et al., 2010). Basically, the activated sludge process is based on an aerated

tank, in which organic matter is degraded by suspended microorganisms (Jonstrup et al.,

2010). The sludge is then separated in a clarifier and excessive sludge is removed from

the process (Jonstrup et al., 2010). In order to retain the bacterial fauna in the process,

some of the separated sludge is recycled to the aerated tank (Jonstrup et al., 2010).

Although still less common than activated sludge processes, the Moving Bed Biofilm

Reactor (MBBR) is becoming increasingly popular. By 2000, some 100 wastewater

treatment processes around the world utilized the MBBR concept (Ødegaard et al.,

2000). To date, more than 500 MBBR processes based on the use of carrier elements are

found in more than 50 countries (AnoxKaldnes, 2009). In difference from in activated

sludge systems, the biomass in MBBR processes is retained.

In order to determine the extent to which particulate organic matter in wastewater is

hydrolyzed in an MBBR carrier process, a new method based on Oxygen Uptake Rate

(OUR) measurements is developed. There are four experimental approaches in research

concerning the quantification of hydrolysis, look at enzymes, hydrolytic fragments, bulk

parameters or bacterial respiration rates (Morgenroth et al., 2002). Using one of the first

two approaches, the mechanisms of the process can be determined often using artificial

substrates that facilitate to isolate a specific mechanism (Morgenroth et al., 2002). The

result is therefore often not usable in real conditions where the substrate has a mixed

composition. The second two can give information on the whole process but not give

information on specific parts of the process (Morgenroth et al., 2002). In this thesis a

method to measure hydrolysis using the bacterial respiration rates will be developed.

According to Morgenroth et al. (2002) it is useful to do the research using a mixed

substrate, a mixed bacterial culture and to compare biofilm based processes with the

traditional activated sludge.

1.1 Aim

The main focus of this master thesis was the development of a new method for

determination of the extent to which particulate organic matter in wastewater is

2

hydrolyzed in an MBBR carrier process. The applicability of the method to measure

hydrolysis in the MBBR process was then to be evaluated. After testing the applicability

of the method, it was to be applied to different operating conditions. The importance of

three different concentrations of particles with regard to hydrolysis as well as whether

hydrolysis is inhibited at elevated levels of dissolved organic matter was then to be

investigated.

1.2 Limitations

Hydrolysis occurs under versatile circumstances but this master thesis focuses on

enzymatic hydrolysis in aerobic wastewater treatment processes based on an MBBR

process using carriers.

3

2 Background

2.1 Particles in wastewater

Wastewater is typically discharged from a number of diverse sources and as a result

it contains a variety of pollutants, including organic matter. Generally, the organic

material constitutes about 40-60% proteins, 25-50% carbohydrates and 10% lipids

(Haldane & Logan, 1994). Depending on their size, these compounds can be divided into

four fractions - soluble (<0.08 µm), colloidal particles (0.8-1.0 µm), supercolloidal

particles (1-100 µm) and sedimenting particles (>100 µm) (Gillberg et al., 2003). In the

following sections, organic matter will simply be regarded as the sum of two fractions -

particulate organic matter (particles) and dissolved organic matter. Any organic

material with a size greater than 0.8 µm is referred to as particulate. Particles larger

than 100 µm are to a large extent removed in the primary sedimentation, which often is

placed before the biological treatment in wastewater treatment plants (Morgenroth et

al., 2002). Therefore particles mainly in the size range 0.8 to 100 µm are discussed in

this chapter.

Whether organic matter is available to microorganisms is dependent on the size and

composition of the material. It is common to distinguish between slowly biodegradable

organic matter and easily biodegradable organic matter, of which the latter often has a

mass less than 1000 amu1 (Morgenroth et al., 2002). The slowly biodegradable, on the

other hand, can be presumed to be in the range 1000 amu to 100 µm (Morgenroth et al.,

2002). This way of classifying organic matter indicates whether microorganisms in the

wastewater may utilize it directly by uptake through the cell membrane, as 1000 amu is

the upper limit for direct uptake (Confer & Logan, 1998; Morgenroth et al., 2002).

Another way of defining the upper limit for direct uptake is by particle size. According to

Janning et al. (1997), particles smaller than 0.0001 µm can cross the cell membrane of

microorganisms.

2.2 Moving Bed Biofilm Reactor

The AnoxKaldnes™ MBBR is used in wastewater treatment processes such as

nitrification, denitrification and BOD-removal (AnoxKaldnes, 2009). The technology is

based on the use of carrier elements made of a polyethylene that has a density slightly

lower than that of water (AnoxKaldnes, 2009). Thereby the carriers are easily kept in

suspension by aeration in aerobic processes or by mixing with stirrers in anoxic or

anaerobic processes (AnoxKaldnes, 2009).

Although most biological wastewater treatment processes are based on activated

sludge, the MBBR concept is becoming increasingly popular and to date it has been

applied to more than 500 wastewater treatment processes around the world

1 Atomic mass unit

4

(AnoxKaldnes, 2009). The biofilm is constituted by extracellular polymeric substances

(EPS) holding together a diverse culture of microorganisms, whose composition reflect

the chemical composition of the wastewater (Janning, 1998).

The application of the biofilm principle normally yields a higher biomass to volume

ratio than systems based on activated sludge (la Cour Jansen, 2011). Thus, in order to

achieve a certain treatment, an MBBR process requires less volume than corresponding

activated sludge process. Furthermore, the design of the carriers limits the impact from

physical stress on the biofilm which thereby allows for higher flows through the process.

There is always some loss of biomass with the effluent, in particular from the exposed

surface layer of the biofilm. Most of the biofilm is however retained in the MBBR as a

result of the carrier design (AnoxKaldnes, 2009). The main drawback with biofilm

processes in general is that only a small fraction of the biofilm is active. In MBBR,

however, the use of carriers compensate for part of this effect by allowing more biomass

and thereby more surface area per unit volume.

2.2.1 Biofilm kinetics

The substrate removal kinetics in biofilm applications is strongly dependent on the

concentration of substrate in the wastewater being treated. This is illustrated in Figure

2.1, which shows the development of the kinetic description from a 1’order expression

at low concentrations to a 0’order expression at very high concentrations. The transition

from low to very high substrate concentration is described with a ½’order expression.

Figure 2.1: The kinetic description with reaction rate as a function of the substrate concentration (Henze et al, 1997).

As seen in the figure, the substrate removal rate is limited by the substrate

concentration only at low concentrations where a small change in concentration gives a

proportional change in the degradation (Ødegaard et al., 2000). At high substrate

concentrations the rate is limited by the diffusion of substrate into the biofilm. Thus, as

the concentration increases the kinetics begins to shift from being concentration

dependent to being diffusion dependent and eventually the kinetics becomes in-

dependent of the substrate concentration, this is described by ½’order kinetics

5

(Ødegaard et al., 2000). At very high substrate concentrations the enzymatic efficiency

restrains the removal rate - 0’order dependence (Ødegaard et al., 2000).

Although the kinetics in biofilm processes is normally described as above, it should be

pointed out that the composition of the substrate in the wastewater will determine its

kinetic characteristics (Ødegaard et al., 2000). Diffusion is believed to be the most

important mass transfer phenomenon and is thus normally considered in kinetic

descriptions (Larsen, 1992). Other transport mechanisms such as advective transport,

on the other hand, are usually not regarded (Larsen, 1992).

2.3 The fate of particles

The majority of carbon input to wastewater treatment plants constitutes particulate

organic matter in the form of slowly biodegradable organic matter (Insel et al., 2003).

Particles entering a MBBR are either degraded by micro-organisms in the biofilm or pass

straight through the process. The particles may be completely degraded and taken up by

microorganisms but they could also be partially degraded and then released back into

the bulk liquid. The size of the organic fragments resulting from the degradation

determines whether the microorganisms in the process may utilize the degradation

products directly.

A fraction of the partially degraded particles will join the undegraded particles that

pass straight through the process, most of the partially degraded particles are however

likely to come in contact with the biofilm again for further degradation. Completely

degraded substrate is transported through the bacterial membrane, where it is used for

respiration and production of new biomass. Almost 50% of the energy in the substrate is

bound in new biomass (Jonstrup et al., 2010). Biomass eventually detaches from the

carrier surface mainly due to shear forces and degradation in the interior of the biofilm

(la Cour Jansen, 2011). Thus, to some extent, biodegradation transforms organic matter

in influent water to particles of biomass.

2.4 Hydrolysis

The process in which an organic molecule is split into smaller parts by addition of

water is called hydrolysis (Morgenroth et al., 2002). The water molecule is divided into a

hydrogen and hydroxyl ion and the hydroxyl ion performs a nucleophilic attack on a

bond, splitting the molecule in two parts (Ellervik & Sterner, 2007). The hydrogen and

hydroxyl ion then binds to each part of the divided molecule (Ellervik & Sterner, 2007).

In the context of wastewater treatment, hydrolysis includes all processes contributing to

degradation of organic matter (Morgenroth et al., 2002).

2.5 Enzymatic hydrolysis

In a MBBR process, degradation of particulate organic matter is aided by extracellular

enzymes produced by the microorganisms present in the process. Complete degradation

of individual particles as well as partial degradation of several particles is catalyzed by

extracellular enzymes. Further, out of 197 identified extracellular enzymes, 145 have

6

been found to be hydrolytic (Schomburg et al., 1997 cited in Morgenroth et al., 2002,

p.28). The hydrolytic enzymes are either attached to the bacterial membrane or

suspended in the bulk liquid (Confer & Logan, 1998). Complete degradation of more

complex particulate organic matter such as polysaccharides may require a mixture of

hydrolytic enzymes (Haldane & Logan, 1994). The large number of hydrolytic enzymes

is therefore essential for the microorganisms in wastewater to be able to utilize a wide

range of organic compounds.

Bacterial production of enzymes is normally stimulated by environmental factors

such as substrate availability, but in some cases the production of enzymes carry on

regardless of external effects (Larsen, 1992). Further, Goel et al. (1999) found that the

synthesis of enzymes is influenced by the oxygen level, while already synthesized

enzymes are unaffected by the availability of oxygen. The hydrolysis rate is however

independent of the oxygen level according to the results presented by Goel et al. (1999).

2.5.1 Location of hydrolysis

Organic particulate matter that is too large to diffuse into the biofilm is degraded

either at the biofilm surface by enzymes that are membrane attached or in the bulk

liquid by extracellular enzymes excreted by the bacteria.

Rohold & Harremoës (1993) investigated the importance of residence time by means

of OUR and found that a decrease in retention time leads to a decrease in OUR. The

interpretation was that there was a wash out of extracellular enzymes from the bulk

during the experiments, reducing the hydrolytic activity and thus the OUR (Rohold &

Harremoës, 1993). Based on this interpretation, Rohold & Harremoës (1993) concluded

that hydrolysis of particulate organic matter takes place in the bulk liquid. However,

since OUR only measures complete hydrolysis followed by microbial respiration, a wash

out of hydrolytic fragments would have the same effect as a wash out of enzymes. Thus,

a decrease in OUR is not proof of a lower hydrolysis rate, it could as well be explained as

loss of hydrolytic fragments from the system, leading to a lowered respiration.

Confer & Logan (1998) measured merely 3-7 % of the total hydrolytic activity in the

bulk, leading to the conclusion that hydrolysis takes place almost exclusively at the

biofilm surface. Further, Confer & Logan (1998) found the hydrolysis rate to be much

higher at the biofilm surface than at the surface of sloughed biofilm or in cell-free

environments. Several other studies performed in natural waters during 1983-1989

support these findings (Confer & Logan, 1998). The extracellular enzymes released to

the bulk liquid are always at risk of being prematurely washed out of the reactor due to

the hydraulic retention time, while those attached to the membrane are retained during

the entire lifetime of the cells. Thereby, from an energy efficiency point of view, it is

better for bacteria to hold on to enzymes than to release them into the bulk. Also,

enzymes released to the bulk liquid can be utilized as carbon source by bacteria (Confer

& Logan, 1998).

According to Boltz & La Motta (2007), organic particulates attach to the biofilm

surface due to bioflocculation. Further, Boltz & La Motta (2007) regard bioflocculation in

biofilm reactors and particle bioflocculation in activated sludge reactors as similar

7

processes and thereby validate the hypothesis that the EPS in the biofilm bind particles

with results from studies on activated sludge performed by La Motta et al. (2003, 2004).

Bioflocculation is a process in which chemical bonding arises between the EPS and the

organic particulates (Boltz & La Motta, 2007). The particles are then hydrolyzed by

extracellular enzymes and the hydrolytic fragments are, depending on their size, either

taken up by the microorganisms in the biofilm or released to the bulk solution (Confer &

Logan, 1998). This is consistent with the results from several other experiments, which

demonstrated that hydrolysis of proteins and polysaccharides occurs in contact with

biofilm or sludge flocs in activated sludge (Confer & Logan, 1998). However, with

increasing molecular weight the diffusion into the EPS becomes limiting, which results

in a relocation of the hydrolytic activity from the membrane surface to the EPS

constituting the biofilm surface (Dimock & Morgenroth, 2006). Dimock & Morgenroth

(2006) suggests that the microorganisms presumably release enzymes into the EPS. By

excreting extracellular enzymes into the EPS, the microorganisms would increase the

rate at which high molecular compounds are hydrolyzed and consumed. Also, the EPS is

flexible and can enfold larger particles, which are thereby accessible to hydrolysis by the

enzymes in the EPS (Dimock & Morgenroth, 2006).

2.5.2 Rate of hydrolysis

Hydrolysis is limiting for substrate degradation as the rate at which it proceeds is

lower than the rate of uptake of fully degraded substrate by microorganisms (Okutman

et al., 2001). Morgenroth, et al. (2002) highlights the importance of particle size for

bioavailability, mass transfer and attachment of particles. Further, Dimock &

Morgenroth (2006) performed experiments, which demonstrated that particle size plays

an important role in the rate of hydrolysis in activated sludge processes.

During degradation of particulate organic matter smaller particles are formed and

released back into solution, increasing the surface to volume ratio of the substrate

available for hydrolysis, making it more accessible to microorganisms (Dimock &

Morgenroth, 2006). Smaller particles are thereby hydrolyzed more rapidly than larger

particles. Size seems to have smaller influence on the rate of hydrolysis degrading

macromolecules, as the size primarily affects the diffusion to the biofilm surface. Larger

molecules have a small diffusion coefficient making the overall degradation rate slower

(Kommedal et al., 2006). The physical properties, such as diffusion, gain more

importance in the degradation rate as the hydrolytic fragments disperse into the bulk

liquid (Haldane & Logan, 1994). Dimock, & Morgenroth (2006) showed an increase of

hydrolysis rate with time, which could perhaps be explained by the increase in

hydrolytic fragments in the bulk liquid as the process proceeds. Priest, (1984) as cited

by Janning et al. (1997), found that the hydrolysis rate could be diminished due to

enzyme inhibition, as a response to addition of readily available carbon.

2.6 Oxygen Uptake Rate

Of the organic substances consumed in a wastewater treatment process one part is

used for biomass production while the other is used for respiration. The oxygen used for

8

respiration can be determined by measuring the decrease in oxygen level in the process

when no air is supplied. By calculating the slope of the oxygen concentration curve

during several unarerated intervals, the Oxygen Uptake Rate (OUR) and oxygen

consumption connected to respiration is found. OUR can be used to determine for

instance the performance of a treatment plant and wastewater characteristics and

combined with additional analytical methods more information can be retained about

the processes (Hagman & la Cour Jansen, 2007).

An important estimation made is that the substrate able to penetrate the bacterial

membrane is immediately utilized for respiration or biomass production and that no

accumulation or storage of PHA or glycogen occurs (Dimock & Morgenroth, 2006).

Dimock & Morgenroth (2006) performed experiments with acetate, BSA2 and egg whites

and only observed internal storage of PHA when using acetate. It is therefore unclear

whether the influence of storage is of significance in this master thesis since wastewater

is composed of diverse material. Whether a specific substance is stored and if it

constitutes a significant part of the organic material in the wastewater is hard to

determine.

2 Bovine serum albumin

9

3 Materials and methods This chapter includes the procedures that were undertaken in the experimental part

of the master thesis. However, since the main goal was to find a method that can be used

to measure hydrolysis of particles in wastewater, several different experiments were

performed. All basic information on the experimental work is covered by the following

sections while details specific on individual experiments are found in the next chapter, 4

Method development.

3.1 Sampling

Kaldnes carriers of model K3 and wastewater were sampled from AnoxKaldnes’ pilot

plant at the wastewater treatment plant in Kävlinge on the day of the experiment. The

carriers were collected in plastic containers and in order to preserve the carriers, the

containers were filled with effluent water. Plastic bottles with a volume of 1 l were used

for all wastewater samples. Further, the inflow of wastewater to the pilot tank was

measured manually with a plastic pitcher holding 3 l of water and a stop watch. All flow

measurements are found in Appendix E.

Figure 3.1: The hydrolysis tank at the wastewater treatment plant in Kävlinge is the sampling location.

3.1.1 Storage of samples

Particulate COD in wastewater samples is hydrolyzed and consumed by micro-

organisms in the wastewater as long as oxygen is available. Therefore, fresh wastewater

rather than wastewater that has been stored is preferred for experimental purposes.

Whenever samples had to be stored before use, they were put in a refrigerator keeping a

temperature of 4 °C. The microbial activity in the samples was thereby slowed down,

reducing the loss of substrate in the samples. Filtered samples have less biomass present

in the bulk water and were thereby not as affected by consumption during storage.

10

3.2 Experimental procedure

3.2.1 Experimental setup

The experiments were based on an arrangement with two reactors run in parallel, see

Figure 3.2. Mantled glass beakers with an inner diameter of 9 cm functioned as the

reactors in which 1 l of wastewater was aerated by aquarium pumps and stirred by

magnetic mixers using fleas with a length of 7 cm. Diffuser stones were connected to the

tubings through which air was supplied in order to achieve gentle aeration with fine air

bubbles in the reactors.

Figure 3.2: Experimental setup: 1. magnetic stirrer, 2. 1 l glass beaker, 3. Probe, 4. HQ40d meter, 5. aquarium pump, 6. Flow meter.

Even though no diffusers are used in the pilot plant, sufficient amounts of air diffuse

into the water since the air bubbles have to travel several meters to reach the surface. In

the laboratory scale reactors, on the other hand, the water column was just 20 cm. Thus,

to achieve even distribution of oxygen in the reactors, the residence time as well as the

contact area to volume ratio was increased by using fine air bubbles. The airflow and

subsequently the aeration intensity in the reactors were controlled with rotameters

from Dwyer® and the airflow was measured with flow meters from TSI Instruments

Ltd. Further, the water and carrier elements were stirred by agitators during the

experiments, see Figure 3.2. When the aeration was turned on, it also contributed to the

agitation.

The consumption of oxygen during the experiments was studied using OUR

methodology, see 2.6 Oxygen Uptake Rate. HQ40d Digital Multi-Parameter Meters from

Hach® were used to measure the oxygen concentration in the reactors during the

experiments.

11

3.2.2 Execution of the experiments

50 carriers were added to each reactor and then, in order to determine the

endogenous respiration in the reactors, the first part of the experiments, the pre-period,

was run with water that had no external carbon source. External carbon was then added

and the consumption of oxygen resulting from increased microbial respiration was

studied. When the level of respiration returned to that of endogenous respiration, the

external carbon had been consumed and the experiments were terminated. The

consumption of particles takes several days, thus the particles were not fully degraded

during the experiments, but when the degradation is so slow that the oxygen

consumption is not detectably higher than the endogenous respiration, the experiment

is over. All experiments were carried out at room temperature.

Filtering

1.6 µm glass fiber filters were used to filter wastewater samples. Thus, according to

the definition of particulate and dissolved COD presented in 2.1 Particles in wastewater,

hydrolysis of colloidal particles and soluble organic matter too large for direct uptake by

microorganisms was excluded in the experimental part of the master thesis. However,

for practical reasons, COD with a size greater than 1.6 µm will be referred to as

particulate while COD with a size less than 1.6 µm will be referred to as dissolved in 4

Method development and 5 Hydrolysis experiments.

Oxygen concentration measurements

Oxygen concentration measurements were taken with ten second intervals in all

experiments except the first one, in which measurements were taken every thirty

seconds. Further, the HQ40d meters have a maximum storage capacity of 500 data

points, thus data had to be transferred to a USB memory during the experiments. By

timing the data transfers with aerated intervals, loss of important data was avoided. The

data transfers are seen as gaps in the graphs illustrating how the oxygen concentration

varies with time during the experiments presented in 4 Method development and 5

Hydrolysis experiments. A drawback with the oxygen meters is that bubbles might get

trapped in the probe measuring the oxygen level, influencing the measurements.

3.2.3 Processing of data

The data logs from the oxygen concentration measurements were inserted into Excel

and the maximum OUR was calculated for each unaerated interval in the experiments.

Due to differences between the experiments concerning the characteristics of the data, it

was difficult to apply a general rule concerning the choice of data for the calculations.

Five to ten data points were chosen from each unaerated interval and, to the greatest

extent possible, the OUR was calculated at high oxygen levels. The OUR was then plotted

against time and, based on the level of endogenous respiration, the amount of oxygen

consumed during respiration of the carbon source was determined. The oxygen

dependence was also calculated according to the method described in 3.3.2 Oxygen

Dependence. The addition of an external carbon source, and thus from which point and

12

on the oxygen consumption was calculation, is marked as a vertical black line in the OUR

graphs.

3.2.4 Measurements of COD

COD was measured in the inlet and outlet water from the pilot plant as well as in the

reactors during the experiments. Generally, both unfiltered and filtered water samples

were taken from the pilot plant, while filtered water samples exclusively were taken

from the reactors right after addition of external carbon and at termination of the

experiments. Dr. Lange cuvette test LCK 114 was then used for analysis of the COD

content in the water samples. The results from all COD measurements are found in

Appendix B.

3.2.5 Measurements of total suspended solids and volatile suspended solid

The TSS and VSS content of the biomass in wastewater samples were determined by

using a series of steps. Firstly, the wastewater was filtered through a 1.6 µm filter with

known weight. Then, in order to evaporate the water in the filter, the filter was dried in

an oven at 105 °C for 24 hours. The filter was weighted once again and the TSS content

was calculated as the difference in weight before usage and after drying divided by the

volume of filtered wastewater. Thereafter the filter was put in an incinerator at 550 °C

for 24 hours, only leaving ash and inorganic matter. The VSS content was then found as

the difference in weight between the dried and incinerated filter divided by the volume

of filtered wastewater. The procedure is described in the Swedish Standard SAP 9.

3.2.6 Measurements of volatile fatty acids (VFA)

In experiment 4 and 5, the VFA was measured once every half hour from the moment

carbon was added until the experiment finished. The results are found in Appendix E.

3.3 Calculations

3.3.1 Oxygen consumption

When determining the oxygen consumption, each calculated OUR was assumed to be

constant for a certain period of time. The first time interval ranged from the moment

external carbon was added in the experiments to the point in time located in between

the two following OUR. The remaining time intervals then ranged according to the same

pattern, which was from in between the first and second OUR after addition of carbon to

in between the third and fourth and so on. After the time intervals had been established,

the increase in OUR from the reference level (endogenous respiration) due to addition of

external carbon was calculated:

Whenever the OUR was equal to or lower than the reference level, OURref, ∆OUR was

set to zero. The oxygen consumption was then calculated as the sum of the first ∆OUR

13

multiplied with the first time interval, the second ∆OUR multiplied with the second time

interval and so on;

n is the number of OUR data points after the addition of external carbon in the

experiments.

3.3.2 Oxygen dependence

The oxygen dependence of OUR was qualitatively determined based on the results

from experiment 3, 4 and 5. OUR as well as average oxygen concentrations were

calculated based on sets of five oxygen concentration measurements. Linear regression

was then used to determine whether there is a significant relationship between OUR and

the oxygen concentration. Further, trends in the oxygen dependence as well as shifts

between oxygen limitation and carbon limitation were studied. All graphs and tables are

found in the next chapter, Method Development. Numbers with poor correlation

coefficients are regarded as irrelevant and subsequently they are in brackets.

15

4 Method development The purpose of the method development was to find a way to measure hydrolysis in

wastewater from AnoxKaldnes’ pilot plant. The main idea is that the Oxygen Uptake

Rate, OUR, is lower in a filtrated wastewater sample than in an unfiltered sample of the

same volume, due to the absence of particles in the filtrated sample. Thereby it should

be possible to measure the contribution to the OUR by the particles by comparing the

filtrated and unfiltrated wastewater. This chapter includes five experiments designed to

measure the hydrolysis as closely as possible. At the end of the chapter, parameters that

were identified as especially important are discussed.

4.1 Experiment 1 - Introduction to the Oxygen Uptake Rate method

4.1.1 Purpose

The initial experiment was a trial and error experiment aiming at introducing the

OUR method, but more importantly it was performed to identify relevant factors

influencing the results.

4.1.2 Expected results

Prior to the experiment, the importance of factors such as filtration, aeration

intensity, stirring intensity and storage time in the cool room was unknown. Therefore,

the results from this experiment were expected to only give an indication of the

possibility to measure hydrolysis of particulate organic matter in an MBBR process.

4.1.3 Changes to setup

The setup was as described in 3 Materials and methods, with the exception that no

flow meters were used to measure the air flow from the pumps. Further, the two

reactors were run with the following carbon sources:

Reactor 1A - 0.25 l of filtered outlet water and 0.25 l of filtered inlet wastewater

Reactor 1B - 0.25 l of filtered outlet water and 0.25 l of unfiltered inlet wastewater

4.1.4 Procedure

1 l of inlet wastewater, 1 l of outlet wastewater and 100 carriers was collected at

Kävlinge. The container with influent wastewater was placed in the cool room in order

to slow down the microbial activity in the water. When the carriers were added to the

reactors, no effort was put into adding an even mix of carriers from the top, middle and

bottom layer in the plastic container, in which they were brought. The first 50 were put

in one reactor and then the other 50 were put in the other reactor. 0.5 l of the effluent

wastewater was then filtered and split between two reactors - 0.25 l in each. Finally, to

each of the reactors, 0.5 l of tap water was added and the aeration and stirring started.

The aeration was kept on for 14 minutes and then it was turned off for 6 minutes

manually throughout the entire experiment.

After the pre-period started, 0.25 l of the inlet water was filtered for use in reactor 1A.

The inlet water was then placed in the cool room again for the remaining time of the one

16

hour long pre-period. Once the pre-period ended, the filtered water was added to

reactor 1A while 0.25 l of unfiltered water was added to reactor 1B.

4.1.5 Results from the introductory experiment

Figure 4.1 shows how the oxygen concentration in the reactors changed throughout

the experiment. The oxygen concentration reached a higher level in 1A than in 1B

already during the pre-period, which was probably the result of higher aeration

intensity in 1A than in 1B during the experiment. What caused the difference is unclear,

but one possible explanation is that the pump used to aerate 1A had a higher capacity

than the one used in 1B. Another one is that the diffuser stone used in 1B had less

porosity than the one used in 1A. After addition of carbon, the oxygen concentration

decreased rapidly in both reactors due to increased microbial activity.

Figure 4.1: Oxygen concentration as a function of time.

OUR

The oxygen was consumed at a higher rate in 1B than in 1A during the first unaerated

interval after the carbon was added. This observation is confirmed by Figure 4.2, in

which the OUR in the two reactors during the experiment is presented. At addition of

carbon, the OUR in 1B reached a significantly higher level than in 1A. There was only a

clear difference in OUR between the two reactors during a short period of time after the

carbon was added though.

17

Figure 4.2: OUR as a function of time.

Calculations of the oxygen consumption in the two reactors do, however, suggest that

twice as much oxygen was consumed in 1B than in 1A during the experiment, see

Appendix C - Oxygen consumption. This could indicate the microbial activity in 1B

increased due to respiration of hydrolyzed particles when unfiltered wastewater was

added to the reactor.

4.1.6 Summary of experiment 1

This experiment was successful in terms of acquiring knowledge how to utilize the

OUR method when studying hydrolysis of particles in an MBBR process. The relevance

of the results however, is arguable due to a number of uncertainties. These uncertainties

were identified as important factors that need to be taken into consideration in order to

attain results that can be compared. It was found important to have the same oxygen

level in both reactors, but another experiment is needed to determine an appropriate

level.

4.2 Experiment 2 - Importance of the oxygen level

4.2.1 Purpose

The aim with this experiment was to determine whether the OUR is dependent on the

oxygen level in wastewater. Two different oxygen levels - 6 mg O2/l and 3 mg O2/l - were

chosen for the study. Furthermore, the possibility of having shorter aerated and

unaerated intervals than in the first experiment was evaluated by measuring the oxygen

concentration every ten seconds instead of every thirty seconds that was the case in last

experiment.


The oxygen concentration graphs based on the results from the initial experiment

revealed a nonlinear decrease in oxygen concentration, see Figure 4.1. The slope of the

curve decreases gradually during the descents representing the unaerated intervals of

18

the experiment, indicating oxygen limitation in the reactors at lower oxygen levels.

Based on these observations, the OUR was expected to be lower in the reactor kept at 3

mg O2/l than in the reactor kept at 6 mg O2/l.


No changes were made to the basic setup of the experiment and the two reactors

were run with the same carbon source, which was 0.5 l of unfiltered inlet water from the

pilot plant. The reactor run at the higher oxygen level is referred to as 2A, while the one

run at the lower oxygen level is referred to as 2B.

Reactor 2A - 0.5 l unfiltered inlet wastewater 5< O2<6 mg /l.

Reactor 2B - 0.5 l unfiltered inlet wastewater 2< O2<3 mg/l.

4.2.4 Procedure

1 l of influent wastewater and 100 carriers were sampled at Kävlinge and brought

back to the laboratory. Before the experiment started, the carriers were aerated in glass

bottles filled with tap water for ten minutes to achieve saturation of the biofilm. This

was necessary since no pre-period was run prior to the addition of carbon source to the

reactors in this experiment. Otherwise, changes in the oxygen level in the reactors would

be the result of a combination of saturation of the biofilm and microbial activity, of

which only the latter was of interest in this experiment. The wastewater as well as the

saturated carriers was equally split between two reactors and like in last experiment no

effort was put into achieving an even distribution of carriers from the top, middle and

bottom layer in the plastic container, in which they were brought. 0.5 l of tap water was

then added to each of the reactors and the aeration was turned on. Outlet water was

assumed to be unnecessary in this experiment since the biofilm got access to the

external carbon source already at the very beginning of the experiment.

When the experiment started, the oxygen level was close to 5 mg O2/l in both reactors

so it had to be adjusted to the desired levels of 6 and 3 mg O2/l. The intention was to

reach the higher level in reactor 2A with aeration and to reach the lower level in reactor

2B with a mixture of air and nitrogen gas. According to the flow meters used in the

experiment, the flow of air to 2A and 2B was 1.18 l/min and 0.95 l/min, respectively.

Lowering the oxygen level in reactor 2B with nitrogen gas was however unsuccessful

because the valve to the tube containing nitrogen gas that was connected to reactor 2B

was not properly opened during the whole experiment, which prevented the nitrogen

gas from reaching the reactor. Thus, the oxygen level was lowered to 3 mg O2/l by

microbial consumption instead of by nitrogen gas. The oxygen concentration was then

manually controlled during the one and a half hour long experiment so that the intervals

5 to 6 mg O2/l and 2 to 3 mg O2/l could be studied. Measurements of the oxygen

concentration were taken every ten seconds.

19

4.2.5 Results and from the second experiment

The oxygen concentration in the reactors as a function of time is found in Figure 4.3.

On average close to equally long aerated and unaerated intervals were required to keep

the oxygen concentration within the desired range in both reactors.

Figure 4.3: Oxygen concentration as a function of time in 1A-B.

OUR

Figure 4.4 indicates the microbial activity in 2B was suppressed due to the lower

oxygen concentration leading to a lower oxygen consumption in 2B, than in 2A, during

the experiment. However, a small part of the difference in OUR between the reactors

could be explained by uneven distribution of the inlet water and thus the COD used in

the experiment.

Figure 4.4: OUR as a function of time in 1A-B.

From the results presented in Figure 4.4, the oxygen consumption in 2A was found to

be about 4.5 mg O2 higher than that in 2B (Appendix C).

20


The hypothesis that OUR is dependent on the oxygen concentration in the wastewater

was verified by this experiment. Most oxygen was consumed in reactor 2A, in which the

oxygen concentration was kept within the range 5 to 6 mg O2/l. It is best to keep the

oxygen level in the reactor as high as possible to avoid oxygen limitation in the

experiments.

4.3 Experiment 3 - Oxygen dependence

4.3.1 Purpose

To try to find a way to interpret the experimental data from the experiments acetate

was added as a carbon source to the experimental set up of the next experiment. Acetate

is a carbon source the microorganisms can utilize directly, and it was added in a high

concentration creating a surplus of carbon. Both acetate and oxygen is transported to

the microorganisms by diffusing into the biofilm and when the acetate is in surplus the

access oxygen becomes limiting.

A parallel reactor containing unfiltered inlet water was also included in order to put

the OUR from the acetate reactor into perspective with something that is similar to the

previous experiments. The goal of the experiment was to find a relationship that can be

used to describe the oxygen limitation.

A reflection from experiment 2 was that the aeration intensity is important for the

comparability of OUR between two reactors. To achieve the same level of oxygen in both

reactors a flow meter was to be used to adjust the flow of air to the same intensity.

Another reflection was that the aerated time interval was too short during

experiment 2, thus needed to be prolonged to give the oxygen level in the reactor

enough time to reach its maximum again after the unaerated periods.


The OUR graph of acetate was expected to be constant, where the oxygen

consumption rate is constant until the acetate is totally consumed. An estimation of how

large the active biomass is could be made from the results of this experiment.

A problem could arise if the acetate concentration chosen increases the microbial

consumption of oxygen to such an extent that the oxygen concentration quickly reaches

zero during the unaerated intervals. To prevent this from occurring either the

concentration of acetate can be lowered or the biomass can be reduced, lowering the

activity in the reactor.


The reactors in this experiment contained the following carbon sources:

Reactor 3A - 85 mg acetate

Reactor 3B - 0.5 l unfiltered inlet water

21

The aeration in the two reactors was measured with a flow meter and the flows were

adjusted to the same intensity before the experiment started. The aeration was

calibrated using flow meters and rotameters to set the initial air flow to 2.5 l/min in

each reactor. The time intervals of aeration were prolonged from the ones used in

experiment 2. A timer was used to start and stop the aeration.

4.3.4 Procedure

1 l outlet water was used as bulk liquid during the pre-period in the two reactors. A

highly concentrated acetate solution had been prepared in advance. The strong

concentration prevents degradation by microorganisms as well as reduces the volume

that has to be added to the reactors. On the day of the experiment the acetate solution

was diluted with water before it was added to the reactor. The reactor contained 85

mg/l acetate after the addition.

The timer was set to intervals of 13 minutes and 15 seconds aeration, followed by a 5

minutes and 50 seconds long unaerated period.

The experiment was run for a total of 4 hours. 50 K3 carriers were placed in each

reactor and then the outlet water was added. The first hour was a pre-period, where 1

liter of outlet water was used. After the pre-period, 500 ml was removed from each

reactor and 500 ml of the solution containing the carbon source was added. Samples of

the reactor bulk were taken every half hour for measurements of VFA, starting after the

addition of the carbon source. COD samples were taken as described in 3 Materials and

methods.

4.3.5 Results from oxygen dependence experiment

In Table 4.1 the results from the COD samples from the experiment are shown.

Table 4.1: The COD measured on the raw water and before and after the experiment. Filtrated sample only contain dissolved particles smaller than 1.6 μm.

In Figure 4.5 the oxygen levels in the reactors are presented. The oxygen level in the

two reactors was not the same although the aeration was calibrated shortly before the

experiment started, see Figure 4.5. The pre-period had a higher level than the post-

period, where the carbon source had been consumed, indicating that there was carbon

that the microorganisms could utilize in the outlet water used as medium in the pre-

period. The lengths of the descents of the oxygen concentration in Figure 4.5 were

22

longer after the addition of unfiltered inlet water. This could be explained by the variety

of carbon sources that was included in raw wastewater which provides for a larger

range of microorganisms. A bigger part of the biomass is active when for instance

ethanol is present, because some bacteria that are unable to degrade acetate may utilize

ethanol. The oxygen level in the acetate reactor didn’t decrease as much as was

presumed when planning the experiment. The narrow increase shortly before the fourth

decent was due to the decreased volume when 500 ml of the outlet water in the pre-

period was pumped out and the probe of the oxygen meter was out of the water for a

short time period until the carbon source was added to the reactors.

Figure 4.5: The oxygen concentration in the two reactors plotted against the time.

The maximum oxygen uptake rate is presented in Figure 4.6. The fourth point in 3A is

in the level of the OUR in the pre-period although the carbon source already was added.

Perhaps it indicates that the addition was too close to the unaerated period. From the

fifth point and on in Figure 4.6, the OUR in the acetate reactor was constant at an

elevated level until the acetate had been consumed. This result is comparable to similar

experiments conducted with acetate in activated sludge (Hagman & la Cour Jansen,

2007).

The fourth point of the unfiltered inlet water series was very high compared to the

other points, probably due to the fact that there were numerous easily degradable

organic compounds in the raw wastewater. Ethanol can for instance be degraded by

microorganisms that don´t use acetate as a substrate and therefore a larger fraction of

the biomass present in the reactor was active and the OUR became very high during the

first minutes. After that the OUR in 3B quickly decreased to close to pre-period levels,

however since they were elevated they give a false impression of the amount of time it

took to consume the inlet water. The use of outlet water in the pre-period seems to have

cause problems in the method by adding a carbon source to the microorganisms in the

pre-period.

23

Figure 4.6: The maximum OUR in the two reactors plotted against time.

The pre-periods in Figure 4.6 were not on the same level despite the fact that the

oxygen level, bulk liquid and biomass should be identical. One or several of these factors

must have differed between the reactors. The oxygen level was clearly not equal in the

two reactors but that does not exclude that other errors occurred as well.

Oxygen dependence

In Figure 4.7 and Figure 4.8 the OUR is plotted against the oxygen concentration in

each decent to see if a relationship between the parameters could be found. The

descents are numbered from 1 to 13 in both reactors. The carbon sources were added

before the fourth unaerated interval. In the acetate reactor, Figure 4.7, the 5th – 7th

unaerated periods tend to have larger OUR than the other intervals. However, according

to Table 4.2, which shows the slopes of the linear descents in Figure 4.7, there was not a

clear oxygen dependence during the degradation of the acetate in this experiment.

Figure 4.7: The OUR in the acetate reactor is plotted against the oxygen concentration. The legend shows which of the unaerated periods that is plotted.

Table 4.2: Slope of the trends in OUR during consumption of external carbon in reactor 3A.

24

In Figure 4.8 the unaerated periods from the unfiltered reactor is plotted against the

oxygen concentration and there is a linear relationship between the OUR and the oxygen

concentration during descent number 4 and 5, see Table 4.3. After that the OUR was

very similar to the pre- and post-periods where there was no COD in the reactor. The

COD added to the reactor seems to have been small because it was quickly consumed,

only two descents, 4 and 5, have an elevated OUR value. There seems to have been a shift

from an initially high oxygen dependence in descent number 4 to a significantly lower

dependence in number 5. It would indicate stronger oxygen dependence while the

surplus of acetate was large in the beginning of the experiment, which is consistent with

the theory of carbon and oxygen dependence.

Figure 4.8: The OUR in the unfiltered reactor is plotted against the oxygen concentration. The numbers define which of the unaerated periods that is plotted.


The OUR in the acetate reactor showed similarities to comparable experiments using

activated sludge. The oxygen dependence was clearer in the unfiltered reactor than in

the acetate reactor. In the unfiltered reactor the oxygen dependence was larger when a

carbon source was present, indicating an oxygen limited process, whereas the other

intervals was limited by carbon supply.

Improved aeration is essential to the method development, to make it possible to

compare reactors to each other, and must be further developed.

To do this the calibration of the aeration must be further developed. The diffuser

stones must distribute equal air bubbles. The outlet water most definitely contained

carbon in this experiment because the pre-period had a higher oxygen level than the

post-period. Water would be a more appropriate pre-period medium, where only the

carbon in the biofilm would affect the oxygen level before the carbon source is added.

The biomass should be evenly distributed between the two reactors and statistically the

probability of identical biomass in both reactors increase if the carriers are collected

from the pilot by the same person and directly placed into a plastic box intended for

each reactor.


25

The time used for the aerated intervals was sufficient for the reactor using unfiltered

wastewater, but too long for the acetate reactor which was aerated faster.

4.4 Experiment 4 - Repeatability test

4.4.1 Purpose

The purpose of this experiment was to test the repeatability of the experimental

work. By adding equal amounts of the same substrate to two reactors run in parallel, the

possibility to achieve the same OUR in the reactors was investigated. Further, changes in

the performance of the biomass on the carriers could be studied by running the

experiment twice. In order to limit the influence of the oxygen concentration, the same

oxygen level was to be kept in the reactors. Also, the reactors had to be set up the same

way and in order to ensure the reliability of the data, close to equally calibrated oxygen

meters were to be used.


The OUR was expected to be the same in the two reactors during both the first and

second run. However, the performance of the biomass and subsequently the OUR was

expected to be higher in the first run than in the second one. In terms of oxygen

concentration, the same level was expected in the reactors during both runs.


Based on the results from experiment 3 shown in Figure 4.5, it was clear that shorter

aerated intervals could be used. 9 minutes of aeration was found to be sufficient and

subsequently the aerated intervals were reduced from 14 to 9 minutes. Two pairs of

reactors were run and they all utilized 85 mg of acetate as carbon source. The ones in

the first run are denominated 4A and 4B, while those in the second run are denominated

4C and 4D. Only one flow meter was available, thus it was swapped between the reactors

when adjusting the aeration.

4.4.4 Procedure

200 carriers were collected at Kävlinge and brought back to the laboratory. Unlike in

experiment 1 and 2, the carriers were collected in separate containers - 50 in each.

Thereby, four batches with a similar composition of biomass were achieved. However,

there was a leakage of water from one of the boxes holding carriers during the trip from

the pilot plant. Together with carriers from one of the other boxes, the ones in the box

with the leakage were used for adjustment of the aeration equipment - pumps, diffusers

and rotameters. Half of the carriers in each box were used in one reactor, while the other

half was used in the other. Further, different Oxygen meters were tested until two close

to equally calibrated ones were found, see Appendix D. When the aeration equipment

had been adjusted to a satisfactory level, the flow of air to both reactors was about 2

l/min. This flow was maintained throughout the entire experiment.

Subsequent to the adjustment of the aeration equipment, the carriers in the reactors

were replaced with new ones. The carriers in the first of the two remaining boxes were

26

added to one reactor and the ones in the second box were added to the other reactor. 1 l

of tap water was then added to each reactor and a pre-period of about 40 minutes was

run. 5 ml from a 17 g/l acetate stock solution was then added to the reactors from a bulk

solution that had been prepared prior to the experiment and the experiment continued

for another hour.

After the first run, the reactors were emptied of water and then swapped places with

each other. The aeration equipment and oxygen meter used in 4A during the first run

was thereby used in 4D during the second run and, conversely, the same aeration

equipment and Oxygen meter was used in 4B and 4C. 1 l of tap water was then added to

each reactor and a second pre-period of 45 minutes was run. After addition of 5 ml from

the 17 g/l acetate stock solution to the reactors, the experiment was run for another 1.5

hours.

4.4.5 Results from the repeatability experiment

Variations in the oxygen concentration in reactor 4A and 4B during the experiment

are presented in Figure 4.9. There was a clear difference in oxygen concentration

between the reactors, which is explained partly by differences in the calibration of the

meters, see Appendix D. The differences in oxygen concentration were however too great

to be explained solely by the calibration. It seems like the oxygenation of reactor 4A was

better than that of 4B throughout the entire experiment.

Figure 4.9: The oxygen concentration as a function of time in 4A and 4B using 85 of mg acetate.

Figure 4.10, in which the oxygen concentration in 4C and 4D is shown, indicates a

smaller difference between the reactors in the second run than in the first. This is

explained partly by the swap of the reactors in between the first and second run. Due to

unequal calibration of the oxygen meters, the difference in oxygen level between the

reactors was amplified during the first run, see Figure 4.9. During the second run, on the

other hand, the difference was impaired as the better aeration equipment was used in

4C. The actual difference in oxygen level between 4C and 4D is thereby not obvious in

Figure 4.10.

27

Figure 4.10: The oxygen concentration as a function of time in 4C-D.

Interestingly, the flow meters showed no significant changes in the aeration of the

reactors during the experiment. This suggests the biomass in 4A and later 4D was more

active than that in the other reactors.

OUR

Figure 4.11 presents the maximum OUR in 4A and 4B throughout the experiment and

as seen the trends in the two reactors are similar. The seventh data point is however

higher in 4A than in 4B. As seen in Figure 4.9, more oxygen was consumed in 4A during

the seventh descent even though equal amounts of COD were added to 4A and 4B, see

Appendix B. This could be due to that the microbial activity in 4A and 4D was higher than

that in the other reactors.

Figure 4.11: The maximum OUR as a function of time in 4A-B.

The maximum OUR in reactor 4C and 4D is shown in Figure 4.12 and, as seen in the

figure, oxygen was consumed to the greatest extent in 4D.

28

Figure 4.12: The maximum OUR as a function of time in 4C-D.

Based on the results shown in Figure 4.12, it is apparent there were differences either

in the oxygenation or in the microbial activity, most likely in both, between 4C and 4D.

Furthermore, the OUR in the oxygen span 6-7 mg O2/l was determined and the results

are presented in Figure 4.13 and Figure 4.14.

Figure 4.13: The OUR as a function of time in 4A-B in the span 6-7 mg O2/l.

Except for the fifth data point, the OUR in 4A and 4B follows the same pattern in the

interval 6 -7 mg O2/l as when the maximum OUR was found. The same accounts for 4C

and 4D, in which the trends in OUR were unchanged when the oxygen span 6-7 mg O2/l

was studied.

29

Figure 4.14: The OUR as a function of time in 4C-D in the span 6-7 mg O2/l.

Based on the maximum OUR, the oxygen consumption was found to be higher in 4A

than in 4B. When the calculations were based on the OUR calculated in the interval 6 - 7

mg O2/l, on the other hand, the oxygen consumption was close to the same in both

reactors. Further, the oxygen consumption in 4C was also almost equal to that in 4A and

4B. This clearly demonstrates the importance of achieving equal aeration in two reactors

run in parallel. All numbers on the oxygen consumption are found in Appendix C.

Oxygen dependence

Figure 4.15 shows the oxygen dependence in 4A and Table 4.4 shows the slope of the

trends in OUR during consumption of external carbon in the reactor. According to the

table, there was no clear trend in the oxygen dependence during the consumption of

external carbon.

Figure 4.15: OUR as a function of the oxygen concentration during all descents in reactor 4A.


30

Figure 4.16 shows the oxygen dependence in 4B and Table 4.5 shows the slope of the

trends in OUR during consumption of external carbon in the reactor. Based on the

calculated slopes, there were no clear trends in the oxygen dependence in reactor 4B.

The oxygen dependence was however stronger in 4B than in 4A, perhaps as a result of

unequal aeration of the reactors.

Figure 4.16: OUR as a function of the oxygen concentration during all descents in reactor 4B.

Figure 4.17 and Figure 4.18 show the OUR as a function of the oxygen concentration

in reactor 4C and 4D, respectively. Table 4.6 and Table 4.7 show the oxygen dependence

during the consumption of external carbon in the reactors.

Figure 4.17: OUR as a function of the oxygen concentration during all descents in reactor 4C.

There was no clear difference between 4C and 4D regarding the oxygen dependence

during the consumption. In comparison with reactor 4A and 4B, however, there was

generally a lower oxygen dependence in 4D and possibly also in 4C.

Table 4.5: Slope of the trends in OUR during consumption of external carbon in reactor 4B.

Table 4.6: Slope of the trends in OUR during consumption of external carbon in reactor 4C.

31

Figure 4.18: OUR as a function of the oxygen concentration during all descents in reactor 4D.


It can be concluded that the reactors in pair is similar but that further development is

needed to make the result fully comparable. Even more equal oxygen meters must be

found and tested and the aeration must be equal in both reactors before the experiment

starts.

4.5 Experiment 5 - Hydrolysis experiment

4.5.1 Purpose

This experiment aimed at testing the applicability of the methodology that was

developed by experiment 1 - 4. Thus, results mainly from this experiment but partly also

from experiment 3 and 4 will be used for evaluation of the methodology. Four waters

with COD from different size fractions were to be characterized by the extent to which

hydrolysis and subsequent microbial respiration of particles took place. In addition to

testing the method, the experiment would thereby also increase the knowledge on the

impact of COD from different size fractions on the hydrolysis taking place in an MBBR

process.


The oxygen consumption was expected to be the highest in the unfiltered reactor. The

sedimented reactor was expected be the second highest and the filtrated to have the

lowest oxygen consumption of the reactors using wastewater. Hydrolysis is expected to

be measurable in the unfiltered reactor by comparison to the filtered.

Table 4.7: Slope of the trends in OUR during consumption of external carbon in reactor 4D.

32


No changes were made to the setup used in experiment 4 and the following carbon

sources were utilized:

Reactor 5A - 85 mg of acetate

Reactor 5B - 0.25 l of filtered inlet water from the pilot plant

Reactor 5C - 0.25 l of unfiltered inlet water from the pilot plant

Reactor 5D - 0.25 l of sedimented inlet water from the pilot plant

5A and 5B contained the least COD and were therefore run in parallel. 5C and 5D,

which contained more COD, were subsequently run in parallel.

4.5.4 Procedure

200 carriers and 4 l of inlet water were collected at Kävlinge by and brought back to

the laboratory. Like in experiment 4, the carriers were collected in separate containers -

50 in each. Before commencing the experiment, two equally calibrated oxygen meters

were found, see Appendix D, and the aeration equipment was adjusted the same way as

in experiment 4 so that an air flow of 2 l/min was achieved. The reactors where then

filled with 1 l of tap water and one box of carriers was added to each reactor. By adding

the carriers after the water, unnecessary physical stress to the carriers prior to starting

the experiment was avoided. The agitation was then started carefully and a pre-period

of almost one hour was run. When the pre-period ended, 250 ml of water was removed

from each reactor and 5 ml from a 17 g/l acetate stock solution was added to reactor 5A

together with 250 ml of tap water, while 250 ml of filtered inlet water was added to

reactor 5B. The experiment then continued for about 1.5 hours. At the end of the run, the

flow of air from the pump used to aerate reactor 5B had decreased to 1.4 l/min. Nothing

was done about this.

After the first run, the reactors were emptied and once again 1 l of tap water and one

box of carriers was added to each reactor. A one hour long pre-period was then run,

followed by removal of 250 ml of water from each reactor. Subsequent to the pre-period,

250 ml of unfiltered inlet water was added to reactor 5C and 250 ml of water from a 1 l

bottle of inlet water, in which the particles had sedimented for 4 hours, was added to

reactor 5D. After addition of external carbon, the experiment was run for 1 hour and 45

minutes longer.

4.5.5 Results from the repeatability experiment

Figure 4.19 shows how the oxygen concentration in reactor 5A and 5B changed

throughout the experiment. The oxygen concentration in both reactors varied within the

same interval during the entire pre-period, thus equal amounts of air was supplied

initially. After addition of carbon, the oxygen concentration dropped in both reactors as

a result of the excess of easily accessible carbon. Surprisingly, the oxygen concentration

reached a lower level in 5B than in 5A during the first descent seen in Figure 4.19.

Possibly, the biofilm in 5A was not accustomed to acetate before the addition.

33

Figure 4.19: The oxygen concentration in reactor 5A and 5B.

Already during the second descent after addition of carbon, it is evident 5B contained

the least fraction of easily accessible carbon. According to Figure 4.19, the oxygen

consumption in the reactor decreased gradually and after about 45 minutes from the

moment carbon was added to the reactors, the oxygen concentration reached the same

level as during the pre-period. It is uncertain, however, to which level the oxygen

concentration dropped during the third descent. The oxygen meter used to measure the

oxygen concentration in 5B was subject to a battery malfunction during the unaerated

interval, thus data from part of the third descent is missing. Possibly, most of the easily

accessible carbon in 5B was consumed already within 30 minutes. In contrast to 5B, 5A

experienced relatively constant oxygen consumption for 45 minutes and then suddenly

the oxygen concentration reached the same level as prior to the addition of carbon.

Figure 4.20: The oxygen concentration as a function of time in reactor 5C and 5D.

Figure 4.20 shows the trends in oxygen concentration in 5C and 5D and the results

indicate that less air was supplied to 5C than to 5D. Subsequently, the oxygen

34

concentration increased at a slower rate in 5C. Possibly a change in the settings of the

rotameter used to regulate the flow of air to 5C during the experiment explains this.

Also, the difference in oxygen level between 5C and 5D was greater than between 5A and

5B. The reason for this is unknown.

The respiration in 5C and 5D returned to the level of endogenous respiration after

about one hour from the moment carbon was added. Further, the trends in oxygen

concentration as well as the length of the descents were quite the same in both reactors

during the run. This implies that the extent, to which oxygen was consumed in 5C and

5D, was also quite the same.

OUR

As seen in Figure 4.21, after addition of external carbon the OUR in 5A was constant

at an elevated level for 45 minutes and then decreased instantaneously while it

decreased gradually in 5B.

Figure 4.21: The OUR as a function of time in reactor 5A and 5B.

Figure 4.22 shows how the OUR changes throughout the second run, in which

unfiltered and sedimented inlet water from the pilot plant were used as carbon sources.

Like the differences in oxygen concentration between 5C and 5D, the differences in OUR

between the reactors are probably due to a combination of uneven aeration of the

reactors and differences in the composition of the COD. However, during the first 30

minutes after addition of carbon to the reactors, the OUR was clearly higher in 5C than in

5D. This could be explained by hydrolysis, but also by differences between the reactors

regarding the amount of dissolved COD, see Appendix B.

35

Figure 4.22: The OUR as a function of time in reactor 5C and 5D.

According to calculations of the oxygen consumption, close to twice the amount of

oxygen consumed in 5B was consumed in 5A. About 30 % more oxygen was consumed

in 5C than in 5D according to calculations. All numbers on the oxygen consumption are

found in Appendix C.

Oxygen dependence

Figure 4.23 shows how the OUR in 5A varied with the oxygen concentration in the

reactor during the experiment and Table 4.8 shows the slopes of the trends. During

consumption of the easily accessible acetate, the OUR was clearly dependent on the

oxygen concentration in the reactor. Furthermore, the oxygen dependence increased

gradually during the fourth, fifth and seventh descent. According to Table 4.8, the

oxygen dependence during consumption of acetate peaked during the seventh descent.

Figure 4.23: OUR as a function of the oxygen concentration during all descents in reactor 5A.


36

According to Figure 4.24 and Table 4.9, the OUR in 5B was most dependent on the

oxygen level during the fourth and fifth descent. Like in 5A, the oxygen dependence in

5B increased gradually after addition of external carbon and it peaked during the fifth

descent. Further, comparison of the results in Table 4.8 with those in Table 4.9 reveals a

significantly stronger oxygen dependency during consumption of acetate than during

consumption of filtered inlet water from the pilot plant.

Figure 4.24: OUR as a function of the oxygen concentration during all descents in reactor 5B.

Figure 4.25 shows the OUR in 5C as a function of the oxygen level in the reactor

during the experiment and Table 4.10 shows the slopes of the trends. The microbial

activity in 5C was most limited by the availability of oxygen during the fifth and sixth

descent. Also, the oxygen dependency in 5C is more apparent than that in both 5A and

5B, as seen in Table 4.10.

Figure 4.25: OUR as a function of the oxygen concentration during all descents in reactor 5C.

Figure 4.26 shows how the OUR in 5D varied with the oxygen level in the reactor

during the experiment. The biomass was clearly limited by the amount of oxygen in the

Table 4.10: Slope of the trends in OUR during consumption of external carbon in reactor 5C.

Table 4.9: Slope of the trends in OUR during consumption of external carbon in reactor 5B.

37

reactor during the fifth and sixth descent, as seen in Table 4.10. It seems like the

respiration in 5D was less dependent on the oxygen level than that in 5C. This suggests

the unfiltered inlet water perhaps contained more readily available carbon than the

sedimented water.

Figure 4.26: OUR as a function of the oxygen concentration during all descents in reactor 5D.


Even though the COD content in reactor 5B was higher than that in 5C, the oxygen

consumption was the highest in 5C, see Appendix B and Appendix C. This supports that

hydrolysis of particulate COD occurred in 5C. The baselines representing the

endogenous respiration are not the same in all reactors, despite the efforts in handling

the biofilm in all reactors identically. This suggests that the conditions for the biofilm in

the different reactors were not identical, as desired. Too rough aeration causes

sloughing of biomass from the carriers and thereby affects the performance of the

biofilm. The oxygen dependence increased with increasing amount of readily accessible

carbon in the reactors. As seen in Appendix C, different oxygen consumption was the

result of addition of COD from different size fractions.

4.6 Evaluation of the method

In experiment 5 the oxygen consumption is larger in the unfiltered sample compared

to the filtrated sample despite the fact that the dissolved COD is higher in the filtrated

sample. The most probable explanation for this is that the particles in the unfiltered

wastewater have been hydrolyzed to small enough fragments to be utilized by the

microorganisms. This result show that it is possible to measure hydrolysis using the

OUR method. However, hydrolysis of particles that results in larger fragments than the

microorganisms can use will not be measured by this method. To utilize the full

potential of the methodology developed, it is important to consider several aspects. This

following discussion covers the ones identified as most important in experiment 1 to 5.

Table 4.11: Slope of the trends in OUR during consumption of external carbon in reactor 5D.

38

Oxygenation is the single most important factor. It contributes to the turbulence

mixing the carriers vertically in the reactor. In terms of comparability, equal aeration of

reactors run in parallel is desired since the methodology is based on the study of

differences in OUR and oxygen consumption, which both are strongly correlated to the

oxygen level in the reactor.

Further, convection increases the extent to which organic particles are hydrolyzed by

transporting particles from the bulk liquid to the biofilm surface and by thinning out the

stagnant layer just outside the biofilm surface. Too rough agitation in the laboratory

scale reactors causes sloughing of biomass from the carriers, thus the agitation intensity

must be kept at a moderate level. It is also important to achieve a high oxygen level in

the reactors during the experiments due to the oxygen limitation in the biofilm. In

experiment 4 it is shown that the oxygen dependence is lower when the oxygen level is

high in the reactor.

The OUR should to the greatest extent possible be calculated in the same oxygen

interval when comparing results between reactors due to the clear oxygen dependence

in the MBBR carrier process. In order to reach the microorganisms in the biofilm, the

oxygen has to diffuse into the biofilm. Otherwise it is hard to isolate the dependence of

carbon in the biofilm and accurately determine the hydrolysis. In experiment 4 it is

shown that the oxygen consumption from reactors with the same content are more

similar when the values are calculated in the same oxygen interval, see Appendix C.

Further, the calculations of the oxygen consumption during the experiments are based

on a quite uncertain assumption, which is that each calculated OUR in the experiments is

constant for a certain period of time. In reality, it is unknown how the OUR varies

between the data points in the OUR graphs presented in this report.

To ensure that the quality of the biofilm as well as that an equal amount of biofilm,

and thereby the same microbial activity, is achieved in reactors run in parallel, it is

important to collect the carriers carefully and to treat them all in the same way

throughout the entire handling. The carriers are sampled from the pilot tank by the

same person and put into separate containers, collecting the same number as the

number of reactors used in the experiment. Thereby, as all batches of carriers are

handled the same way, differences between biomass in the reactors due to the human

equation are minimized. Also, the statistical probability of equal biomass increases when

the carriers are sampled from a large container and put directly into the reactor.

Furthermore, the agitation of the reactors is started gently in order avoid initial

sloughing of biomass from the carrier surface. The biofilm is always subject to friction

due to the carriers collide with each other, the wall of the reactors, the aeration

equipment and with the probe of the oxygen meter.

During storage of wastewater samples, some COD is always consumed by suspended

bacteria in the water. All reactors in the experiment should therefore preferably be run

simultaneously. By filtrating the wastewater samples used in the experiment shortly

39

after sampling, suspended bacteria are removed from the water and the consumption of

COD prior to use in the experiment is reduced. However, unless all wastewater samples

used in the experiment is filtrated there will be unequal conditions in the samples. This

is due to the degradation of COD in unfiltered wastewater that also leads to a lowered

oxygen level whereas filtration in addition to lowering the microbial activity in the

wastewater samples, also increases the oxygen level in the sample by allowing oxygen to

diffuse into the water.

The level of endogenous respiration is of great importance in the calculations of the

oxygen consumption in the experiments. Accurate determination of the level of

endogenous respiration increases the reliability of the results. This could be achieved by

increasing the length of the pre-period, thus allowing the biofilm to consume more of the

readily degradable carbon that comes with the carriers when they are collected. Another

approach is to put the carriers in oxygenated tap water for a few hours before and then

when the experiment starts the carriers are transferred to the reactors. Procedures to

lower the carbon content in the biofilm prior to starting the experiment are relevant

particularly when the wastewater has a high carbon content. The level of endogenous

respiration could possibly also be determined at the end of the experiment by extending

the time the experiment is continued after external carbon has been consumed.

41

5 Hydrolysis experiments

5.1 Experiment 6 - Significance of particles

5.1.1 Purpose

If the extent to which particles in wastewater are hydrolyzed is dependent on the

concentration of particles, measurable differences in OUR between wastewaters with

different particle content will possibly be observed. In this experiment, three

wastewaters with particles from 2, 1 and 0.5, l of wastewater were produced and

investigated. The aim was to determine whether the OUR increases with particle

content.


A higher particle concentration is believed to lead to an increased hydrolysis of

particles in wastewater. If that is wrong, there will be no distinction between the three

reactors that can be explained as an increased hydrolysis.


Three reactors were run in parallel instead of two, as described in Materials and

methods, and they contained the following carbon sources:

Reactor 6A - 300 ml of tap water and particles from 2 l inlet water

Reactor 6B - 300 ml of tap water and particles from 1 l inlet water

Reactor 6C - 300 ml of tap water and particles from 0.5 l inlet water

5.1.4 Procedure

5 l of influent wastewater and 200 carriers was collected at Kävlinge and brought

back to the laboratory. The carriers were kept in four plastic containers with effluent

wastewater - 50 in each container.

Back at the laboratory, the particles in a total of 3.5 l of inlet water were separated

from the water through filtering. About 125 ml of inlet water was filtered through each

filter. The particles were then gently washed off the filters with tap water into glass

beakers. Three beakers were used since three separate bulk solutions with particle

contents corresponding to 0.5, 1 and 2 l inlet water were desired. About 300 ml of tap

water was needed to wash off all the filters that the filtering of 2 l of inlet water

generated. The bulk solutions based on 0.5 and 1 l of inlet water were therefore each

diluted to a volume of 300 ml.

While filtering the inlet water, one single reactor holding 1 l of tap water and 50

carriers was used to adjust the air flow from the three pumps that were to be used in the

experiment. When the air flow had reached a satisfactory level, a pre-period with the

three parallel reactors that were to be used in the experiment was run. 1 l of tap water

and 50 carriers were used in each reactor during the pre-period as well. Since the

filtering required an additional 1.5 hour after the air flow had been adjusted, a 1.5 hour

long pre-period was run.

42

Once the filtering was finished, 300 ml of water was removed from each of the

reactors and the carbon sources were added. The experiment was then run for an

additional 4 hours.

5.1.5 Results from the investigation of particles significance

Figure 5.1 shows how the oxygen concentration in the three reactors varies during

the 6 hours long experiment. The low oxygen levels in reactor 6C are due to low capacity

of the pump used to aerate the reactor. In order to increase the oxygen level in the

reactor, the pump was replaced with a better one. This was done during the second

aerated interval of the pre-period, thus about half an hour after the initiation of the pre-

period. It was however not possible to reach the same air flow to 6C as to the other

reactors, which is clearly seen in the figure.

Figure 5.1: Oxygen concentration in the three reactors as a function of time.

Figure 5.2 shows the OUR in the reactors that was calculated from the descents in

Figure 5.1. The OUR was very high in the beginning of the pre-period, only to decrease to

the base level of endogenous respiration after about 30 minutes. This is explained by the

high content of COD in the outlet water in the pilot tank due to the high retention time

during the sampling, see Appendix B. Although the carriers are placed in tap water there

is carbon in the biofilm that are consumed during the first minutes of the pre-period.

However after the carbon is consumed the OUR decreases to pre-period levels.

43

Figure 5.2: OUR at the three different particle concentrations as a function of time.

Reactor 6C shows a significantly higher OUR at the third data point in Figure 5.2 than

the other two reactors. This is probably due to the oxygen deficiency and subsequent

constrained microbial activity in 6C during the first 30 minutes of the experiment,

caused by the insufficient aeration of the reactor. With equal aeration in all three

reactors, the trends in OUR during the pre-period would probably have been more the

same.

After the addition of carbon source to the reactors, the OUR stays higher in 6A than in

6B and 6C until almost 3.5 hours of the experiment has passed. When the filters were

washed, some dissolved COD came with the particles, thus dissolved COD was added to

all three reactors together with particles. The bulk solution containing particles from 2 l

of inlet water contained most dissolved COD, most likely since it required most filters for

preparation, while the bulk solution with particles from 0.5 l of water contained the

least, see Appendix B. Thus, more dissolved COD was probably added to reactor 6A than

to the two other reactors.


The OUR was clearly higher in 6A and this could be explained either by an increased

level of soluble COD or by the fact that the addition of more particles contributed with a

larger amount of easily degradable substrates. There was no apparent difference

between 6B and 6C. These observations supports that the hydrolysis of particles in the

wastewater is unaffected by the amount of particles present.

44

5.2 Experiment 7 - Influence of dissolved carbon

5.2.1 Purpose

The importance of dissolved organic matter for the hydrolysis of particles is to be

examined in this experiment. The hypothesis is that dissolved COD will prevent

hydrolysis from happening, and that less hydrolysis will be observed when more

dissolved COD is added.


The same setup as in experiment 5 was used and the reactors were run with the

following carbon sources:

Reactor 7A - 250 ml of filtered inlet water

Reactor 7B - 250 ml of filtered inlet water and particles from 1 l inlet water

Reactor 7C - 250 ml filtered inlet water and 60 mg of acetate

Reactor 7D - 250 ml filtered inlet water, particles from 1 l of inlet water and 60 mg of

acetate

Furthermore the reactors were run in pairs, first 7A-7B and then 7C-7D.

5.2.3 Procedure

Theory

Amount of particles

Experiment 6 was used as an estimation of how much particles should be added to

the reactors. The minimum amount that needs to be added is particles from 0.5 l of inlet

water according to calculations performed. Because 2 l would take too long time to

filtrate, we chose to add particles from 1 l filtrated inlet water.

Amount of dissolved COD

To estimate the amount of dissolved COD to add to the reactors, experiment 5 was

used as template. In experiment 5 the following times can be deducted from the input of

carbon source to the point in time where the activity is no longer detectable with the

OUR method:

250 ml filtered inlet water 45 minutes

250 ml unfiltered inlet water 45 minutes

85 mg acetate 1 hour

The experiment must keep to a total length of three to four hours including the pre-

period. The combination of carbon sources that is most time consuming is reactor 7D, in

which particles, filtered inlet water and acetate are mixed. If 250 ml filtrated inlet water,

60 mg/l acetate and particles from 1 l filtrated inlet water is added, the experiment must

run for two hours after the addition of carbon source.

45

Practical

6 l of influent wastewater and 200 carriers was collected at Kävlinge and brought

back to the laboratory. The carriers were kept in four plastic containers with effluent

wastewater - 50 in each container. According to measurements, the inflow to the pilot

plant was the same as in the previous experiment (0.3 l/s).

Back at the laboratory, the particles in a total of 2 l of inlet water were separated from

the water. The same filtering and washing procedures as described in Experiment 6 were

applied and two bulk solutions, each with a particle content corresponding to 1 l of inlet

water, were derived. About 250 ml of tap water was used to wash off all the filters that

the filtering of 1 l of inlet water generated. 1 of the 2 l of filtered inlet water was kept for

the experiment.

While filtering the inlet water, the first pre-period was run and as soon as the filtering

of inlet water was completed (after about one hour), 500 ml of water was removed from

reactor 7A and 7B and the carbon sources were added. The reactors were then run for

almost two more hours. After the first pair of reactors finished, a new pre-period with

the second pair of reactors was started and, like the first pre-period, it was run for about

an hour. Once again, 500 ml of water was removed from each of the reactors and the

carbon sources were added. The acetate was added as 5 ml of a 12 g/l acetate stock

solution. The experiment (including pre-period) finished after 3.5 hours. The COD values

are presented in Appendix B.

5.2.4 Results from the investigation of dissolved carbons influence

The oxygen concentration during the experiment is presented in Figure 5.3 and

Figure 5.4. The carbon sources were added after 0.8 hours in Figure 5.5 and Figure 5.6,

which is marked in the graphs as a black vertical line. The increase in oxygen

concentration shortly before the fourth interval was due to the extraction of water to

make room for the carbon source. The procedure left the oxygen meter in the air for a

few minutes, which is clearly seen in the graph. The aeration in the reactors was

struggling to increase the oxygen concentration which can be explained by the elevated

COD content in the outgoing water from the pilot tank that the biofilm was taken from,

see the measurements of COD on the outlet water that were taken in connection with

experiment 6 (Appendix B). The lengths of the descents in the pre-period in 7A and 7B,

Figure 5.3, were almost as long as the descents which also show that there was an

ongoing consumption although no carbon source was added. It could have been avoided

by storing the biofilm carriers in tap water for an hour before the experiment, extending

the pre-period or by increasing the residence time in the pilot tank. However it was not

possible to use any of these methods due to the tight schedule. The total time for the

experiment would have increased too much, and the pilot plant was adjusted for another

experiment at the time of sampling.

After the carbon source was consumed, the OUR returned to the level of the

endogenous respiration that was used as the base line in calculations of the hydrolysis.

The aeration was equal in the reactors which can be seen in the pre- and post-period,

where the oxygen concentration was very similar.

46

Figure 5.3: The oxygen concentration in reactor 7A and 7B as a function of time.

In Figure 5.4 the oxygen level is higher than in Figure 5.3 which is explained by the

fact that the carriers for this experiment were stored during the time it took for reactor

7A and 7B to run, because the same equipment was used. This gave the biofilm more

time to consume the carbon in the outlet water they were stored in and entered the pre-

period with less carbon in the biofilm. The pre-period medium was tap water which was

not contributing with any carbon, so the only carbon found in the pre-period was the

one found in the biofilm itself.

Figure 5.4: The oxygen concentration in reactor 7C and 7D as a function of time.

In Figure 5.5 and Figure 5.6 the OUR in reactor 7A-B and 7C-D respectively is

presented. The carbon in the outlet water can be seen in these graphs as well as that the

OUR was initially higher during the pre-periods in both graphs. The OUR during the pre-

period in 7C-D was lower than in 7A-B which supports the theory that some carbon in

the biofilm was consumed before the pre-period was started due to the consumption

during the wait.

47

Figure 5.5: The OUR-graph for reactor 7A and 7B.

In Figure 5.5 the OUR exhibits a pattern well known from the previous experiment

with a peak after the addition of carbon source and a diminishing OUR up to the base

line was reached. It is interesting to see the difference between the OUR in 7A compared

to 7B when all that differed was the added particle solution. It could be explained by the

hydrolysis of the particles, which lead to a larger OUR in 7B.

In Figure 5.6 the reactor containing particulates 7D shows a higher OUR than 7C

indicating that the hydrolysis was contributing with an extra oxygen consumption in

that reactor. It is not clear whether the soluble carbon added, acetate, had an impact on

the hydrolysis, because the method needs further repetitive studies to ensure that

quantitative analysis can be made using this method. However by the increasing OUR

after the addition of carbon source it is a good assumption that the acetate was

consumed first before the rest of the carbon sources was consumed.

Figure 5.6: The OUR-graph for reactor 7C and 7D.

48

Hydrolysis

There was a clear visible difference in OUR between the filtered reactor and the

reactor with the particles in both cases, see Figure 5.5 and Figure 5.6. This could

possibly be explained by a combination of two things, hydrolysis and a larger amount of

dissolved COD in the reactors with the particles. The dissolved COD in the reactors was

composed of an equal amount of filtrated inlet water in all reactors making up 106 mg/l

in 7A, whereas in 7B the particles contributd with an additional 12 mg dissolved COD/l

besides the substantial contribution in particulate COD, see Table 5.1 and Table 5.2. The

acetate contributed with 60 mg dissolved COD/l in 7C and 7D and the particles

contributed with 15 mg dissolved COD in 7D. To estimate the significance of the

difference in dissolved COD of the hydrolysis seen in Figure 5.5 and Figure 5.6, a few

assumptions were made. However, the assumptions must be used with caution in order

to allow as accurate conclusions as possible to be drawn.

Table 5.1: COD values of the inlet and outlet wastewater and of the particle solutions that was added to 7B and 7D Table 5.2: Dissolved COD in all reactors in mg/l. in mg/l.

It was assumed that the 12 and 15 mg COD was made up of molecules that are easily

accessible to the microorganisms and if they were consumed fully the oxygen

consumption would be described by the equation below, where S is the overall

consumption in the reactor giving the amount of oxygen consumed per added mg COD in

the filtrated reactors during consumption of wastewater.

The results are shown in Table 5.3. The difference in COD could explain at maximum

4 mg of the oxygen consumed in the 7B and 5 mg in 7D. This is to be compared to the

difference in consumed oxygen the two reactor pairs of 6 mg O2 in 7A-B and 12 mg O2 in

7C-D, see Table 5.4. The COD difference could represent 65 % of the hydrolysis seen in

Figure 5.5 and 45 % in Figure 5.6. That means that 35 % and 55 % respectively is

demonstrated was due to hydrolysis. However, a repetitive experiment is needed to

verify the finding and to quantify the numbers and the statistical uncertainty.

Table 5.3: The difference in added carbon source between the filtered and reactor containing particulates is shown as COD and O2 in (mg/l).

49

In Table 5.4 the oxygen consumed in all reactors are also presented. The oxygen

consumption was almost twice as large in 7C-D than in 7A-B and this is consistent with

the fact that twice as much COD was added to 7C-D. This leads to the conclusion that the

hydrolysis was not inhibited by the acetate.

Table 5.4: The consumed oxygen in each reactor is presented in the middle column and the difference between the reactor pairs is shown in the last column.


Hydrolysis was detected both in 7B and 7D using the OUR-method. It can be

concluded that the hydrolysis is not inhibited by the addition of an easily accessible

carbon source because the difference in the oxygen consumed is larger rather than

smaller as was expected should the hydrolysis be inhibited.

51

6 Discussion The findings in experiment 5 support the belief that hydrolysis may be detected with

the methodology developed. The oxygen consumption in the unfiltered wastewater, 5C,

was found to be higher than that in the filtrated, 5B, even though COD measurements

demonstrated more dissolved COD was added to 5B than to 5C (145 mg COD/l versus

128 mg COD/l). It is therefore possible the difference in oxygen consumption between

the reactors was the result of hydrolysis and consequently respiration of particulate

COD. Possibly the explanation for the lower COD content in 5C is that the easily

accessible COD in the reactor was subject to more extensive degradation by suspended

biomass during the three hours storage than that in 5B, which was filtered and thereby

free of microorganisms.

To be able to compare the oxygen consumption between two reactors, it was found

important to use OUR values based on the same oxygen concentration intervals in both

reactors. Experiment 4, using the same acetate mixture in both reactors, the maximum

OUR as well as the OUR in the interval 6 to 7 mg O2/l was determined, clearly

demonstrated this. More air was supplied to 4A than to 4B, which resulted in a higher

maximum OUR, calculated at the highest oxygen level, in 4A than 4B and consequently

the oxygen consumption was higher in 4A than in 4B. On the other hand, when the OUR

was calculated in the interval 6 to 7 mg O2/l in both reactors, there was no significant

difference in the oxygen consumption between the reactors. The oxygen consumption in

4D was also close to equal to that in 4A and 4B. These findings proved that the single

most important factor influencing the comparability of oxygen consumption and OUR

values between reactors is the oxygen level. Thus, since the OUR values was calculated

in the same oxygen interval in 5B and 5C, the observed differences in oxygen

consumption were probably due to hydrolysis and not to differences in the oxygen

concentration between the reactors. This further support the suggestion that hydrolysis

was detected in experiment 5.

The main argument against the conclusion that hydrolysis can be seen using the

model described in this thesis is that the finding is not statistically verified by

performing repetitive experiments proving that the result is analogous in more than one

experiment. This is something that clearly needs to be further investigated to verify the

results in this thesis.

However, in experiment 7 it is demonstrated that in spite of a small increase in COD

the reactor containing particulates compared to the filtrated reactor the hydrolysis is

detectable both with and without acetate. The added acetate could not be proven to

inhibit the hydrolysis, but further comparison of the results must await repetitive

studies.

Also, by taking further efforts to ensure important parameters, such as an equally

high oxygen concentration in both reactors during the experiment and preventing

sloughing of biomass out into the bulk liquid, the accuracy of the measurement can be

http://tyda.se/search/consequently

http://tyda.se/search/consequently

52

improved further. The oxygen level during the experiments is found to be the most

important factor to be able to compare reactors against each other. Also the amount of

suspended biomass in the bulk during the experiment should be minimized to isolate

the hydrolysis performed by the biofilm, but since the biomass in the wastewater is

removed by filtering the suspended biomass originates only from the biofilm sloughing

that occurs during the experiment. It looks like the biofilm is exposed to rougher

conditions in batch experiments than in the pilot. It is shown by the biomass that is

sloughed from the carriers during the experiments. They are clearly covered in less

biomass after four hours in the lab than after weeks in the pilot tank although the

aeration is rougher there.

53

7 Conclusions The method developed was to be used to measure hydrolysis in an MBBR process

using carriers by comparing the bacterial respiration rates in wastewater containing

particulate organic matter and filtrated wastewater. The process is subject to oxygen

limitation because the oxygen has to diffuse into the biofilm to reach the

microorganisms and the oxygen level was identified as an important aspect when

comparing reactors. Also the biofilm used in reactors run in parallel must be equal and

to the greatest extent possible be preserved throughout the experiment. Taking these

aspects into consideration, it is possible to detect hydrolysis in an MBBR carrier process

using OUR methodology.

After developing the method it was tested in two experiments investigating the short-

term influence of added particles on hydrolysis and whether hydrolysis is inhibited at

shorter periods of time with elevated levels of dissolved carbon. The hydrolysis was

found to be unaffected by the amount of particles in the wastewater and not to be

inhibited at elevated concentrations of a readily available dissolved substrate.

55

8 Suggestions for further research Before using the method developed to quantify hydrolysis of particles in an MBBR

process, the reliability of the method should be statistically verified by repetitive

experiments. A repetitive study using experiment 7 as a template is recommendable.

The results can be used to statistically verify that hydrolysis can be detected by OUR

methodology, but also to investigate whether the method can be used to quantify the

hydrolysis it detects.

If it is possible to quantify the hydrolysis, further studies of different operating

conditions can be conducted. An interesting approach would be to find the amount of

hydrolysis performed in an existing wastewater treatment process. It is possible to

investigate the importance of substrate loading, temperature and residence time for any

given wastewater.

Further research could aim at mapping the relationship between hydrolysis in batch

experiments and continuous systems. It would help applying the findings from studies

on batch experiments to full scale continuous processes.

An experiment similar to experiment 7, but using filtrated inlet wastewater instead of

acetate would be interesting. It would help to further determine the relationship

between the availability of easily accessible carbon and hydrolysis using a naturally

occurring carbon source in wastewater treatment processes.

57

9 References

AnoxKaldnes. (2009, April). AnoxKaldnes™ MBBR - An overview of the technology. Lund.

Boltz, J. P., & La Motta, E. J. (2007). Kinetics of Particulate Organic Matter Removal as a

Response to Bioflocculation in Aerobic Biofilm Reactors. Water Environment

Research: A Research Publication of the Water Environment Federation , 79 (7), pp.

725-736.

Confer, D. R., & Logan, B. E. (1998). Location of Protein and Polysaccharide Hydrolytic

Activity in Suspended and Biofilm Wastewater Cultures. Water Research , 32 (1), pp.

31-38.

Dimock, R., & Morgenroth, E. (2006). The influence of particle size on microbial

hydrolysis of protein particles in activated sludge. Water Research , 40 (10), pp. 2064-

2074.

Ellervik, U., & Sterner, O. (2007). Organisk Kemi (2nd edition). Lund: Studentlitteratur.

Gillberg, L., Hansen, B., Karlsson, I., Nordström Enkel, A., & Pålsson, A. (2003). About

Water Treatment. Helsingborg: Kemira Kemwater.

Goel, R., Mino, T., Satoh, H., & Matsuo, T. (1999). Modeling Hydrolysis Processes

Considering Intracellular Storage. Water Science and Technology , 39 (1), pp. 97-105.

Hagman, M., & la Cour Jansen, J. (2007). Oxygen Uptake Rate Measurements for

Application at Wastewater Treatment Plants. Vatten , 63, pp. 131-138.

Haldane, G. M., & Logan, B. E. (1994). Molecular Size Distributions of a Macromolecular

Polysaccharide (Dextran) During Its Biodegradation in Batch and Continuous

Cultures. Water Research , 28 (9), pp. 1873-1878.

Henze, M., Harremoës, P., la Cour Jansen, J., & Arvin, E. (1997). Wastewater Treatment

(2nd ed.). Lyngby: Springer Verlag.

Insel, G., Orhon, D., & Vanrolleghem, P. A. (2003). Identification and Modeling of Aerobic

Hydrolysis - Application of Optimal Experimental Design. Journal of Chemical

Technology and Biotechnology , 78 (4), pp. 437-445.

Janning, K. F. (1998). Hydrolysis and oxidation of particulate organic matter in biofilters.

Lyngby: Technical University of Denmark, Department of Environmental Science and

Engineering.

Janning, K., Mesterton, K., & Harremoës, P. (1997). Hydrolysis and Degradation of

Filtrated Organic Particulates in a Biofilm Reactor under Anoxic and Aerobic

Conditions. Water Science and Technology , 36 (1), pp. 279-286.

Jonstrup, M., Murto, M., & Björnsson, L. (2010). Compendium in Environmental

Biotechnology. Lund: Lunds Tekniska Högskola, Department of Biotechnology.

Kommedal, R., Milferstedt, K., Bakke, R., & Morgenroth, E. (2006). Effects of initial

molecular weight on removal rate of dextran in biofilms. Water Research , 40 (9), pp.

1795-1804.

la Cour Jansen, J. (2011, February). Professor. Personal contact. Department of Chemical

Engineering. Lund University, Lund.

58

La Motta, E. J., Jiménez, J. A., Josse, J. C., & Manrique, A. (2004). The Role of

Bioflocculation on COD Removal in the Solids Contact Chamber of the TF/SC Process.

Journal of Environmental Engineering , 130, pp. 726-735.

La Motta, E. J., Jiménez, J. A., Parker, D., & McManis, K. (2003). Removal of Particulate

COD by Bioflocculation in the Activated Sludge Process. Water Pollution VII; WIT Press

, pp. 349-358.

Larsen, T. A. (1992). Degradation of colloidal organic matter in biofilm reactors. Lyngby:

Technical University of Denmark, Department of Environmental Engineering.

Morgenroth, E., Kommedal, R., & Harremoës, P. (2002). Processes and Modeling of

Hydrolysis of Particulate Organic Matter in Aerobic Wastewater Treatment - a review.

Water Science and Technology , 45 (6), pp. 25-41.

Okutman, D., Övez, S., & Orhon, D. (2001). Hydrolysis of settleable substrate in domestic

sewage. Biotechnology letters , 23 (23), pp. 1907-1914.

Priest, F. G. (1984). Extracellular enzymes. Van Nostrand Reinhold .

Rohold, L., & Harremoës, P. (1993). Degradation of Non-diffusible Organic Matter in

Biofilm Reactors. Water Research , 27 (9), pp. 1689-1691.

United Nations, Dep. of Economic and Social Affairs. (2010, 11 10). United Nations: World

Population Prospects, the 2008 Revision. Retrieved 12 31, 2010, from United Nations:

http://esa.un.org/unpd/wpp2008/frequently-asked-questions.htm

(2010). World Health Statistics. World Health Organization.

Ødegaard, H., Gisvold, B., & Strickland, J. (2000). The Influence of Carrier Size and Shape

in The Moving Bed Biofilm Process. Water Science and Technology , 41 (4-5), pp. 383-

391.

I

Appendix A - Reactor contents

Table A.1: Reactor content in all experiments.

III

Appendix B - COD measurements

Table B.1: A compilation of all COD measurements that were taken in connection with the experiments. * - filtered sample.

Table B.2: COD measurements on particle bulk solutions after filtration and before addition to the reactors. * - filtered sample.

V

Appendix C - Oxygen consumption

Table C.1: Oxygen consumption during all experiments.

VII

Appendix D - Test of oxygen meters In order to detect deviations resulting from the calibration of the oxygen meters used

in experiment 4-7, the oxygen concentration in a water-filled vessel was measured with

the oxygen meters used in parallel in each experiment. However, no test was carried out

in connection with experiment 7 since the oxygen meters used in this experiment were

tested already in connection with experiment 5.

Meter 2 and 6, which were used in experiment 4, were tested in a beaker filled with

tap water and the results from the measurements are found in Figure D.1.

Figure D.1: Differences between the oxygen meters used in experiment 4 regarding measured oxygen concentration.

On average, meter 2 measured a 0.26 mg O2/l higher oxygen concentration than

meter 6.

Two new oxygen meters were chosen for experiment 5 and they were tested in a

reactor containing 1 l of tap water and 50 carriers. The results from the 1.5 hours long

test are shown in Figure D.2.


VIII

On average, meter 7 measured a 0.11 mg O2/l higher oxygen concentration than

meter 8.

Meter 6, 7 and 8 were used in experiment 6 and they were tested in a reactor holding

1 l of tap water and 50 carriers. Figure D.3 shows the results from the test.


According to the measurements, the average difference between meter 7 and 8 was

0.03 mg O2/l. The average difference between meter 6 and 7 was 0.23, thus the

difference between meter 6 and 8 was 0.20.

The observations presented in this appendix clearly show the uncertainty associated

with oxygen measurements. However, all deviations presented here are within or very

close to the margin of error of the oxygen meters, which is 0.20 mg O2/l.

IX

Appendix E - TSS/VSS and VFA measurements

Table E.1: TSS, VSS and flow measurements.

Table E.2: VFA measurements, experiment 4. Table E.3: VFA measurements, experiment 5.

XI

Appendix F - Hydrolysis of particles The rate of hydrolysis was estimated from COD measurements (Appendix B) and the

oxygen consumption in the experiments performed with filtered and unfiltered

wastewater - experiment 1, 5 and 7.

Particulate COD in the sampled wastewater:

Dissolved COD consumed in the reactor with filtered wastewater:

The fraction of substrate used for microbial respiration in the reactor with filtered

wastewater, S, was calculated from ∆CODf,diss and the oxygen consumed in the reactor,

∆(O2)f,diss.

S was assumed to be the same also when particles are hydrolyzed and contribute to

the respiration. The COD consumed in the reactor with unfiltered wastewater was then

calculated as:

∆(O2)u is the oxygen consumed in the reactor run with unfiltered wastewater. The

particulate COD consumed in the experiment could not be calculated directly from

measurements of COD on unfiltered samples from the reactor because there was

sloughed biomass in the reactor.

The particulate COD that was completely hydrolyzed and used for respiration,

, was estimated from the total amount of consumed carbon in the reactor

containing particulates, ∆CODu, minus the dissolved carbon consumed in the unfiltered

reactor:

1

Measurements of Hydrolysis in Moving Bed

Biofilm Reactor Carriers - Evaluation by means

of Oxygen Uptake Rate Measurements

K. HENRIKSSON*, O. TENFÄLT**

Water and Environmental Engineering, Dep. of Chemical Engineering, Lund University, P.O, Box

118, SE-221 00 Lund, Sweden (E-mail: [email protected], [email protected])

Abstract The extent to which particulate organic matter in municipal wastewater is

hydrolyzed in an MBBR carrier process was determined with a new method based on

Oxygen Uptake Rate measurements. Different operating conditions were studied and

qualitative estimates of hydrolysis were made. The oxygen level in the reactors that

were compared was found to be important due to oxygen limitation in the biofilm. A

high oxygen level is desired and whenever reactors are compared, similar oxygen

should be achieved in the reactors. The biofilm used in the reactors should to the

greatest extent possible be handled equally. The method can be used to detect

hydrolysis in an MBBR carrier process. In excess of easily degradable dissolved

substrate, the hydrolysis was not inhibited.

Keywords: Hydrolysis, Moving Bed Biofilm Reactor, MBBR, Oxygen Uptake Rate, OUR, wastewater,

biofilm, carriers

Introduction By 2011 we are, by UN’s medium estimate, expected to become 7 billion people on

earth (United Nations Dep. of Economic and Social Affairs, 2010). This places new

demands on the supply of clean water as well as on treatment measures. In order to

clean wastewater, treatment plants use combinations of physical, biological and

chemical methods. Nitrogen, phosphorous, bioavailable carbon and other organic

contaminants are reduced in the biological treatment step with the aid of

microorganisms (Gillberg et al., 2003).

The most common and well-known biological wastewater treatment process is the

activated sludge process (Jonstrup et al., 2010). Other treatment processes that are

based on the biofilm principle are however becoming increasingly popular. Such a

process is the Moving Bed Biofilm Reactor (MBBR), which was utilized in some 100

wastewater treatment processes around the world by 2000 (Ødegaard et al., 2000).

Today, over 500 MBBR carrier processes are found around the world (AnoxKaldnes,

2009).

In order to determine the extent to which particulate organic matter in wastewater is

hydrolyzed in an MBBR carrier process, a new method based on Oxygen Uptake Rate

mailto:[email protected]

mailto:[email protected]

2

(OUR) measurements, as described by Hagman & la Cour Jansen (2007), was developed.

Hydrolysis of particulate organic matter in municipal wastewater was then studied

during different operating conditions.

Materials and methods Kaldnes carriers of model K3 and wastewater were sampled from AnoxKaldnes’ pilot

plant at Kävlinge wastewater treatment plant. OUR experiments were then performed

with a setup of two aerated and agitated reactors run in parallel. The reactors had an

inner diameter of 9 cm and they were filled with 1 l of wastewater. Aquarium pumps

supplied the reactors with air and in order to achieve appropriate oxygenation of the

water, the air entered the reactors through diffuser stones. The air flow and

subsequently the aeration intensity in the reactors was controlled with rotameters from

Dwyer®. HQ40d Digital Multi-Parameter Meters from Hach® were used to measure the

oxygen concentration in the reactors.

Two experiments are covered by this paper and the following external carbon

sources were used:

Experiment 1

1A - 0.25 l of filtered inlet wastewater from the pilot plant

1B - 0.25 l of unfiltered inlet wastewater from the pilot plant

Experiment 2

2A - 0.25 l of filtered inlet wastewater from the pilot plant

2B - 0.25 l of filtered inlet wastewater and particles from 1 l of inlet wastewater

2C - 0.25 l of filtered inlet wastewater from the pilot plant and 60 mg of acetate

2D - 0.25 l of filtered inlet wastewater, particles from 1 l of inlet wastewater,

60 mg of acetate

Although run on the same day, the reactors in experiment 1 were not run

simultaneously. The OUR and oxygen consumption in 1A and 1B can however still be

compared. In experiment 2, two pairs of reactors were run. The first pair was 2A and

2B, and the second pair was 2C and 2D.

Prior to adding external carbon to the reactors, 50 carriers and 1 l of tap water were

added to each reactor and a pre-period was run - 1A: 55 min, 1B: 65 min, 2A-D: 50 min.

During the pre-period, the level of endogenous respiration was estimated from the OUR

in the reactors. Subsequent to the pre-period, 0.25 l of water was removed from the

reactors and the external carbon was added. The first experiment was run for an

additional 1 hour and 40 minutes after addition of carbon. In experiment 2, the first pair

of reactors was run for two more hours, while the second pair was run for 2 hours and

40 minutes after addition of carbon.

The MBBR process is subject to oxygen limitation because the oxygen has to diffuse

into the biofilm to reach the microorganisms and the oxygen level is an important

3

aspect when comparing reactors. The oxygen concentration was therefore kept as high

as possible and within the same span in all reactors that were compared.

The particles added to reactor 2B and 2A were separated from the inlet water with

1.6 µm glass fiber filters. 125 ml of inlet water was filtered through each filter. The

particles were then gently washed of the filters with tap water into glass beakers.

Dr. Lange cuvette test LCK 114 was used to measure the COD in the inlet and outlet

water from the pilot plant as well as in the reactors during the experiments. Both

unfiltered and filtered water samples were taken from the pilot plant, while filtered

water samples exclusively were taken from the reactors right after addition of external

carbon and at termination of the experiments.

Determination of hydrolysis

The method developed measures hydrolysis by comparing the bacterial respiration

rates in wastewater containing particulate organic matter with that in wastewater

without particles. Differences in OUR between reactors with unfiltered and filtered

wastewater, respectively, were thus used for estimation of the hydrolysis of particles.

Measurements of COD and calculations of the oxygen consumption were used to

calculate the extent to which particles were hydrolyzed. Firstly, the fraction of microbial

respiration, S, was assumed to be the same when particulate COD is hydrolyzed and

used for respiration as when only dissolved COD is consumed;

Δ(O2)filt. and ΔCODfilt. is the consumption of oxygen and dissolved COD, respectively,

in the reactors without particulate COD - 1A, 2A and 2C. The total consumption of COD

in the reactors with particles - 1B, 2B and 2D - was then determined as:

Δ(O2)unfilt. is the consumption of oxygen in the reactors with particles. The particulate

COD that was hydrolyzed and used for respiration could then be determined from

ΔCODunfilt. and measurements on the consumption of dissolved COD in the reactors with

particles.

Results and discussion

Oxygen Uptake Rate

The OUR in reactor 1A-B, starting at the last unaerated interval of the pre-period and

reaching to the end of the experiment, is presented in Figure 1. The first data point in

4

the graph is the last OUR value in the pre-period in both reactors in the first experiment.

It is only meant to demonstrate the level of endogenous respiration.

As seen in Figure 1, the OUR in 1B reached a higher level than that in 1A when carbon

was added to the reactors. The OUR in 1B then stayed higher than in 1A for about 30

minutes, probably due to hydrolysis and subsequent consumption of particles in the

wastewater that was added to the reactor.

Figure 1: OUR in reactor 1A-B throughout experiment 1. The vertical black line marks the addition of external carbon and subsequently from which point the oxygen consumption was studied.

The OUR in 2A-B during experiment 2 is found in Figure 2. The whole pre-period of

50 minutes is included in the graph. Apparently the OUR was initially higher in 2B than

in 2A during the pre-period. Also, the level of endogenous respiration was not reached

in the reactors until at the end of the run. Possibly more COD was brought with the

biofilm added to 2B than with the biofilm added to 2A, thus yielding a higher OUR in 2B.

Figure 2: OUR in reactor 2A-B throughout experiment 2. The vertical black line marks the addition of external carbon and subsequently from which point the oxygen consumption was studied.

5

After addition of the carbon sources, the OUR reached a higher level in 2B than in 2A.

Since the only difference between the carbon sources was particles corresponding to 1 l

of inlet water, the higher OUR in 2B can be explained by hydrolysis.

The OUR in 2C-D during experiment 2 is found in Figure 3 and as seen the first data

point of the pre-period was significantly higher than the two following. Also, the OUR

was lower in the second reactor pair than in the first during the pre-period. This was

probably due to that some carbon in the biofilm utilized in the second run was

consumed while the first pair of reactors was run.

Figure 3: OUR in reactor 2C-D throughout experiment 2. The vertical black line marks the addition of external carbon and subsequently from which point the oxygen consumption was studied.

The OUR was higher in 2D than in 2C after addition of the carbon sources, although

the same amount of acetate was added to both reactors. This supports hydrolysis and

subsequent respiration of particles in 2D. The addition of readily degradable acetate

had no obvious impact on the hydrolysis of particles. However, the peak in OUR shortly

after addition of the carbon sources suggests that the acetate was consumed first.

Oxygen consumption

Based on the assumption that each calculated OUR was constant for the same period

of time, which was 15 minutes, the oxygen consumption due to respiration of external

carbon in all reactors was estimated. However, the first OUR data point after addition of

carbon was assumed to be constant for half that time as it is located right after the

addition of external carbon in the experiments. The oxygen consumption in each reactor

in the experiments is found in Table 1.

6

Table 1: Oxygen consumption due to respiration of external carbon in the reactors.

Hydrolysis

The oxygen consumption was higher in 1B than in 1A (Table 1) even though COD

measurements suggest that more dissolved COD was added to 1A than to 1B, see Table

2. It is therefore possible that the difference in oxygen consumption between the

reactors was the result of hydrolysis and subsequent respiration of particulate COD. A

likely explanation for the lower COD content in 1B is that the easily accessible COD in

the reactor was subject to more extensive degradation by suspended biomass than that

in 1A, since the wastewater in 1B was filtered more than three hours prior to addition

to the reactor.

In experiment 2, There was clearly a difference in OUR between 2A and 2B, as well as

between 2C and 2D. This could possibly be explained by a combination of two things;

hydrolysis and a larger amount of dissolved COD in the reactors with the particles.

Equal amounts of filtrated inlet water corresponding to about 106 mg dissolved COD,

see reactor 2A in Table 2, was added to all reactors. The additional 12 mg of dissolved

COD in 2B are due to the addition of some dissolved COD and perhaps also the particles.

60 mg of acetate was added to 2C-D and subsequently the reactors contained 60 mg of

dissolved COD more than 2A-B, see Table 2. Also, 15 mg of dissolved COD more was

added to 2D with the particles.

Table 2: COD measurements on filtered wastewater samples from the reactors in experiment 1 and 2.

Apparently, twice as much dissolved COD was consumed in 2C and 2D than in 2A and

2B. This is consistent with the differences in oxygen consumption between the reactors

shown in Table 1.

S in the reactors may be calculated with Equation 1 and the results are found in Table

3. Then, if the 12 and 15 mg of additional dissolved COD in 2B and 2D are assumed to be

7

molecules easily accessible to the microorganisms, the same S may be used to

determine the corresponding oxygen consumption, see Table 3.

Table 3: Differences in oxygen consumption between the reactors due to the differences in dissolved COD added.

The difference in COD could explain at maximum 4 mg of the oxygen consumed in the

2B and 5 mg in 2D. This is to be compared to the difference in oxygen consumption in

the two reactor pairs of 6 mg O2 in 2A-B and 12 mg O2 in 2C-D, see Table 1. The COD

difference could represent 65 % of the hydrolysis seen in Figure 2 and 45 % in Figure 3.

That means that 35 % and 55 %, respectively, is demonstrated to be due to hydrolysis.

However, repetitive experiments are needed to verify the findings and to quantify the

numbers and the statistical uncertainty.

Conclusions In order to quantitatively determine hydrolysis, it is necessary to statistically verify

the results from the experiments by repetitive experiments. However, the following

conclusions are drawn:

It is possible to detect hydrolysis using OUR methodology.

Hydrolysis of particles in wastewater is not inhibited at a short retention time

with elevated concentrations of readily available dissolved substrate.

Acknowledgements This article was based on a master’s thesis performed at Water and Environmental

Engineering, Department of Chemical Engineering, Lund University, Faculty of

Engineering. Ph. D. Eva Tykesson at AnoxKaldnes AB and Professor Jes la Cour Jansen at

Water and Environmental Engineering supervised the work. The authors would like to

thank them both for their guidance and support.

8

References

AnoxKaldnes. (2009, April). AnoxKaldnes™ MBBR - An overview of the technology.

Lund.

Gillberg, L., Hansen, B., Karlsson, I., Nordström Enkel, A., & Pålsson, A. (2003). About

Water Treatment. Helsingborg: Kemira Kemwater.

Hagman, M., & la Cour Jansen, J. (2007). Oxygen Uptake Rate Measurements for

Application at Wastewater Treatment Plants. Vatten , 63, pp. 131-138.

Jonstrup, M., Murto, M., & Björnsson, L. (2010). Compendium in Environmental

Biotechnology. Lund: Lunds Tekniska Högskola, Department of Biotechnology.

United Nations, Dep. of Economic and Social Affairs. (2010, 11 10). United Nations:

World Population Prospects, the 2008 Revision. Retrieved 12 31, 2010, from United

Nations: http://esa.un.org/unpd/wpp2008/frequently-asked-questions.htm

Ødegaard, H., Gisvold, B., & Strickland, J. (2000). The Influence of Carrier Size and Shape

in The Moving Bed Biofilm Process. Water Science and Technology , 41 (4-5), pp. 383-

391.

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