Wat. Res. Vol. 35, No. 12, pp. 2833–2840, 2001# 2001 Elsevier Science Ltd. All rights reserved

Printed in Great Britain0043-1354/01/$ - see front matterPII: S0043-1354(00)00585-6

EVALUATION OF NEW METHODS FOR THE MONITORING

OF ALKALINITY, DISSOLVED HYDROGEN AND THE

MICROBIAL COMMUNITY IN ANAEROBIC DIGESTION

LOVISA BJORNSSON*, MARIKA MURTO, TOR GUNNAR JANTSCHand BO MATTIASSON

Department of Biotechnology, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden

(First received 30 June 2000; accepted in revised form 5 December 2000)

Abstract}New methods for spectrophotometric alkalinity measurement, dissolved hydrogen monitoringand for obtaining a fingerprint of the microbial community were evaluated as tools for process monitoringin anaerobic digestion. The anaerobic digestion process was operated at organic loading rates of 1.5, 3.0and 4.5 g volatile solids l�1 d�1 and subjected to pulse loads of carbohydrate, lipid, protein and a mixedsludge substrate. The spectrophotometric alkalinity monitoring method showed good agreement withtraditional titrimetric alkalinity monitoring and has the advantage of being easy to modify to on-linemonitoring applications. The on-line monitoring of dissolved hydrogen gave valuable information aboutapproaching process overload and can be a good complement to the conventional monitoring of volatilefatty acids. Changing process conditions were also reflected in the microbial fingerprint that could beachieved by partitioning in two-phase systems. The investigated methods showed potential for applicationin increasing our understanding of the anaerobic digestion process as well as for being applicable formonitoring in the complex environment of full-scale anaerobic digestion processes. # 2001 ElsevierScience Ltd. All rights reserved

Key words}anaerobic digestion, monitoring, dissolved hydrogen, Pd-MOS, alkalinity, CCD

INTRODUCTION

The organic waste produced by municipalities,industry and agriculture has a potential energy value,which may be exploited by anaerobic digestion and

methane production. However, instability duringboth the start-up and operation of the anaerobicdegradation process can be problematic. Anaerobic

digestion involves a complex series of microbiallycatalysed degradation steps. The instability is mainlydue to the interdependence of the different microbial

groups involved in the degradation (Gujer andZehnder, 1983). Process instability can be avoidedby operating the anaerobic digestion process farbelow maximum capacity. Another, more economic-

ally viable, way of circumventing this problem is toimprove the monitoring and control of the process inorder to allow waste treatment at a higher rate. Ideal

monitoring methods should be on-line, robust andgive early indications of imbalance in the microbialstatus of the system. The measured parameter could

either be an indirect status indicator, such as theconcentration of a metabolite in the anaerobic

degradation, or a direct indication of the microbial

status of the system, such as the number or themetabolic activity of the microorganisms involved.Some of the most commonly used indirect process

indicators include the gas production rate, the gas

composition, pH, the alkalinity and the concentra-tion of volatile fatty acids (VFAs) (Ahring et al.,1995; Moletta et al., 1994). The monitoring of gas-

phase parameters has, in several earlier investiga-tions, proved insufficient in indicating process statusand was not considered in this investigation (Frigon

and Guiot, 1995; Pauss et al., 1990a). This work wasfocused on liquid-phase parameters, since the condi-tions in the liquid reflect the microbial environment

where the important reactions in anaerobic digestionoccur. pH as a process indicator is stronglydependent on the buffering capacity, or alkalinity,of the system (Ahring et al., 1995). The main

buffering species in an anaerobic digester are theVFAs and the bicarbonate. Total alkalinity (TA)measured by titration to a pH endpoint of 4.3, as

suggested by APHA et al. (1985), includes both thesespecies. This has been considered as an insensitiveparameter for indicating process instability since an

increase in VFA concentration will cause a decreasein bicarbonate concentration, resulting in a fairlyconstant TA-value (Hill and Jenkins, 1989; Jenkins

*Author to whom all correspondence should be addressed.

Tel.: +46-46-222-8324; fax: +46-46-222-4713; e-mail:

lovisa.bjornsson@biotek.lu.se

2833

et al., 1983; Quimby and Whittle, 1992). Instead, a

number of methods based on titration have beendeveloped in order to discriminate between bicarbo-nate alkalinity and VFA alkalinity (de Haas andAdam, 1995; Moosbrugger et al., 1993). The partial

alkalinity (PA) as measured by titration to anendpoint pH of 5.75 has been suggested, whichindicates changes in the bicarbonate concentration

(Jenkins et al., 1991; Ripley et al., 1986). Measuringthe PA can be one way of indirectly measuring theVFA accumulation. On-line methods have been

developed for the monitoring of bicarbonate con-centrations, based on acidification of the sample withsubsequent determination of the carbon dioxide flow

or pressure (Hawkes et al., 1993; Rozzi and DiPinto,1994). In the present study, a new approach to PAand TA monitoring was investigated, employing thespectral properties of pH indicators (Jantsch and

Mattiasson, submitted).On-line methods for measuring the concentration

of total VFAs have been suggested by Powell and

Archer (1989), using a titration technique, and byRozzi et al. (1997) employing a biosensor withdenitrifying bacteria. On-line monitoring of the

individual VFAs has been performed by vonZumbusch et al. (1994), who measured acetic andpropionic acids. The method suffered, however, from

problems associated with membrane fouling. Aninteresting alternative is to monitor the dissolvedhydrogen concentration, since this is known to beclosely related to the accumulation of VFAs (Cord-

Ruwisch et al., 1997; Schink, 1997). Several methodshave been developed for on-line monitoring ofdissolved hydrogen in anaerobic digesters, including

the hydrogen/air fuel cell detector (Pauss et al.,1990b), membrane-covered electrodes for directliquid-phase hydrogen measurements (Kuroda

et al., 1991; Strong and Cord-Ruwisch, 1995) and atrace reduction gas analyser (Cord-Ruwisch et al.,1997). In the present study, a new approach todissolved hydrogen monitoring was investigated

including hydrogen transfer from the liquid througha Teflon membrane, and gas-phase detection with aPd metal oxide semiconductor (Pd-MOS) sensor

(Bjornsson et al., in press).Direct process indicators are concerned with the

number or status of different microbial groups in

anaerobic digestion. As reviewed by Oude Elferinket al. (1998), these methods can include directenumeration or microscopic studies of the micro-

organisms. The molecular-based methods include theuse of membrane lipids (Hedrick et al., 1991), geneticprobes (Raskin et al., 1994) or immuno-techniques(S�rensen and Ahring, 1997) for characterisation ofmicrobial communities. In the present study, amethod aimed at obtaining a ‘‘fingerprint’’ of themicrobial community in the process was investigated.

By performing multi-step partitioning of themicroorganisms in an aqueous polymer–polymertwo-phase system, the microorganisms will be frac-

tionated according to size and surface properties and

the obtained distribution pattern will give a finger-print of the culture (Murto et al., submitted).The aim of this study was to evaluate the potential

of the new methods as indicators of process changes

in anaerobic digestion. It was considered importantto evaluate the methods upon process changes in acomplex environment since many full-scale applica-

tions today include the co-digestion of different kindsof waste. The strategy was to use a laboratory-scalemixed culture fed with a base load of a mixed sludge

substrate from a large-scale digester. Pulse loads ofdifferent organic composition were then added tosimulate the variations in load and composition that

might occur in large-scale co-digestion processes.

MATERIALS AND METHODS

Laboratory-scale digester

The laboratory-scale reactor had a working volume of 3 land was maintained under mesophilic conditions (378C). Amixed culture from a full-scale biogas plant was used asinoculum. The contents of the reactor were continuouslymixed with a mechanical stirrer (KeboLab, Spanga,Sweden) operating at 180 rpm. The substrate was kept in astirred vessel at 48C. For feeding of the substrate, whichcontained particulate organic matter, a semi-continousfeeding mode was used. A piston pump (Watson-MarlowAlitea AB, Stockholm, Sweden) fed 5ml of the substrate tothe reactor each time, and the loading rate was adjusted bychanging the feeding interval. The effluent was collected in abottle, and gas from the reactor and the effluent bottle wascollected in gas-tight bags.

Operating conditions and substrate

The organic material was quantified as volatile solids(VS), and the organic loading rate (OLR) was calculated asgram VS per litre reactor per day (gVS l�1 d�1). Thelaboratory-scale process was run consecutively at threeOLRs, 1.5, 3.0 and 4.5 g VS l�1 d�1. As substrate at thesebase loads, a mixed sludge from a full-scale anaerobicdigester was used. This complex substrate was a mixture ofsludge from a municipal wastewater treatment plant and acarbohydrate-rich, food-processing waste. The substratehad a VS concentration of 3%, of which on average 50%was starch and 20% fatty acids (4.2 g l�1 lactic acid, 1.9 g l�1

acetic acid and 0.3–0.4 g l�1 of propionic-, butyric-, andvaleric acid).At each base load level, the process was subjected to pulse

loads of 3 g VS l�1 using organic material of differentchemical composition (Fig. 1). The sludge substrate wasused also in pulse loads. d(+)glucose (KeboLab, Spanga,Sweden) was used for the carbohydrate pulses. Olive oil(LabKemi, Stockholm, Sweden) was used in the lipid pulses,and whey powder (90% proteins, Twin Laboratories Inc.,New York, USA) was used for the protein pulses. Towardsthe end of the experimental period, the process wasoverloaded with a carbohydrate pulse of 9 g VS l�1.

Sampling

Alkalinity, VFAs and pH were measured off-line.Samples were taken daily at the base load levels and every10–30min during 24 h after pulse loads. Ammonia concen-trations were analysed off-line in connection with theprotein pulses. Hydrogen was continuously sampled on-linewith readings at 7.5min intervals. The samples for microbialfingerprinting were taken several times at base load levels to

Lovisa Bjornsson et al.2834

evaluate the reproducibility of the method, and 6–10h afterevery pulse load.

Analytical methods

Total solids (TS) and volatile solids (VS) were determinedaccording to APHA Standard Methods (1985). Theammonia concentrations were measured with the Langemethod LCK 303 (Dr. Bruno Lange GmbH, Dusseldorf,Germany).Samples for alkalinity measurements were centrifuged at

6000 rpm for 3min and the supernatant was used for furtheranalysis. The titrimetric alkalinity was evaluated as PA bytitration to pH 5.75, and the TA by titration to pH 4.3 withstandardised 0.1M HCl using a TitraLabTM 80 titrator(Radiometer, Copenhagen, Denmark) and expressed as mgCaCO3 l

�1. For the measurements of PA and TA byphotometry, the sample was added to a mixture of diluteHCl and a pH indicator; the absorbency was then measured(Jantsch and Mattiasson, submitted). The indicator wasmethyl red for PA measurements and bromphenol blue forTA measurements with colour change in the pH region 4.2–6.2 and 3.0–4.6, respectively. The initial sample to acid ratioor the acid concentration were adjusted to obtain a final pHof 4.3 for the TA and 5.75 for the PA sample. Changes inthe sample alkalinity will then influence the final pH of themixture, which will be reflected in the measured absorbance.PA and TA values expressed as mg CaCO3 l

�1 werecalculated from calibration curves obtained with standardsolutions containing known concentrations of NaHCO3 andNaCH3COO. Absorbance measurements were performed in1-ml plastic cuvettes with a Pharmacia Biotech Ultrospec1000 spectrophotometer.VFAs were analysed with HPLC, Varian Star 9000

(Varian, Walnut Creek, USA), with a Biorad column, 125–0115 (Hercules, USA). The column temperature was 658C,sulphuric acid (1mM) was used as mobile phase and theliquid flow was 0.8mlmin�1. Peak detection was achievedwith UV absorption at 208 nm. The samples were acidified,centrifuged, stored at �208C and filtered before analysis.The dissolved hydrogen was sampled on-line by transfer

from the liquid through a Teflon membrane. The samplewas injected to a semiconductor sensor, and the responsegiven as a voltage output. No direct quantification of thedissolved hydrogen in the digesters was performed, butcalibration of the method with dissolved hydrogen in water(Bjornsson et al., in press) gave detection limits for themethod at 580 and 160nM, before and after the sensitivitychange on day 110.

The sample for microbial fingerprinting was pretreatedwith sonication to disintegrate flocs. The two-phase systemused for separation contained 6% Dx T500 (Pharmacia AB,Uppsala, Sweden), 6% PEG 4000 (Merck-Schuchardt,Hohenbrunn, Germany), 5mM Li2SO4 solution and 5mM phosphate solution at pH 7.0, and was prepared asdescribed by Murto et al. (submitted). The counter currentdistribution (CCD) apparatus consisted of a rotor with 60cavities. The cell suspension was mixed with the bottomphase and added to the first cavity while two-phase systemwas added in the other cavities. The bottom phase volumewas 0.6ml and the top phase volume was 0.8ml. Thevolume of the cavity in the lower part of the rotor was0.8ml. The bottom phases and interfaces were thus keptstationary in the lower half of the rotor while the top phaseswere moved in the upper half at each transfer. A cycleinvolved shaking (30 s), settlement (10min) and transfer.Fifty-eight transfers were carried out in a CCD-run. After acomplete CCD-run, the separate CCD-fractions werediluted with 0.1M glycine–sodium hydroxide buffer, pH9.0, in order to dissolve the two-phase system. Thedistribution of cells in the different fractions was analysedby measuring the cell density at an absorbance of 620 nm.By plotting the absorbance as a function of the fractionnumber, a cell distribution diagram was obtained.

RESULTS

The values of the monitored parameters at the

three levels of OLR prior to the pulse load sequencesare shown in Table 1.The response in VFA concentration during the

experimental period is shown in Fig. 1. At overloadthe VFA concentration increased to 3500mg l�1 (notshown). The response to the pulse loads at an OLR

of 1.5 g VS l�1 d�1 is shown in Fig. 2. The addition ofpulse loads as outlined in Fig. 1 is indicated by thevertical lines, and there is a 4-day interval betweeneach pulse load. The spectrophotometric TA mea-

surement initially deviated from the titration valuesdue to an erroneous callibration curve. This wascorrected at day 108. After this modification the

titrimetric and spectrophotometric alkalinity mea-surements showed good agreement. The sensitivity ofthe dissolved hydrogen method was too low at the

Fig. 1. The organic loading rate (OLR), the pulse loads and the response in volatile fatty acid (VFA)concentration during the experimental period. The mixed sludge substrate was used as the base load. Theorganic material in the pulse loads is indicated as follows; S=sludge substrate, C=carbohydrate, L=lipid,

P=protein.

Evaluation of new methods 2835

beginning of the experimental period, and at day 110the sensitivity was increased by allowing longer timefor the equilibration in the liquid-to-gas hydrogen

transfer.The response to the pulse loads at the base load of

3.0 g VS l�1 d�1 is shown in Fig. 3. The vertical linesindicate the addition of pulse loads, and there is a 4-

day interval between each pulse load. Change in pHand alkalinity was similar to that at the lower OLRlevel, but the VFA accumulation was significantly

higher as shown in Fig. 1. The spectrophotometricmethod did not respond to alkalinity below 1500mgCaCO3 l

�1 and at day 111, the acid concentration

was lowered to accommodate PA measurements in alower range. After modification it showed goodagreement with the titrimetric method. Comparing

VFA and dissolved hydrogen data showed that VFAaccumulation was similar both for the substrate andcarbohydrate pulses whereas the dissolved hydrogenresponse differed with a factor of 4.

On neither of the two first baseline levels did anyof the methods respond to the lipid pulses. Theammonia concentration in the reactor increased by

305 and 311mg l�1 (results not shown), respectively,after the two protein pulses. The increase in ammoniaconcentration was accompanied by increases in pHand alkalinity. The time required to reach the

maximum ammonia concentration was 22 h at theOLR of 1.5 g VS l�1 d�1 and 15.5 h at the OLR of3.0 g VS l�1 d�1. A comparison of VFA and hydrogen

data for the protein pulses shows that the VFAresponse is significantly higher at the higher OLR,whereas the hydrogen response is in the same range

at both levels.At the OLR level of 4.5 g VS l�1 d�1 the process

was subjected to a carbohydrate pulse of 3 g VS l�1

followed, after 11 h, by another carbohydrate pulseof 9 g VS l�1, to study the effects of process overload.The response of pH, alkalinity and dissolved hydro-gen is shown in Fig. 4. The spectrophotometric

Table 1. Mean values and standard deviations for the monitored parameters at different organic loading rate before each pulse loadsequence. The only volatile fatty acid (VFA) detected during steady-state operation was acetic acid (b.d.=below detection limit)

Organic loading rate pH Partial alkalinity Total alkalinity VFA Conc. Dissolved hydrogen(g VS l�1 d�1) (mg CaCO3/l

�1) (mg CaCO3/l�1) (mg l�1) (mV)

1.5 7.2� 0.1 2050� 40 2670� 40 27� 2 b.d.3.0 7.0� 0.1 1900� 40 2410� 40 34� 4 b.d.4.5 7.0� 0.1 1910� 20 2390� 30 36� 8 b.d.

Fig. 2. The response in dissolved hydrogen, alkalinity and pH to the pulse loads at a base load of 1.5 gVS l�1 d�1. TA=total alkalinity, PA=partial alkalinity, spectr=spectrophotometric method, titr=titri-

metric method.

Lovisa Bjornsson et al.2836

method was not in operation during this last part of

the experiment.The change in the microbial fingerprint at different

base loads, after the pulse loads at a base load of 3.0 gVS l�1 d�1, and at overload is shown in Fig. 5.

DISCUSSION

The reason for using the sludge substrate for pulseloads was to study the effects of a mixed substrate to

which the process was adapted. Carbohydrate wasused to give pulses with an easily degradablesubstrate, where the last steps in the degradation,acetogenesis and methanogenesis, would be expected

to be limiting. Lipid was used to study the effects of amaterial where the initial hydrolysis was the limitingstep in the degradation, and proteins were used to

study the effects of the degradation product ammo-nia. As expected, all pulse loads except the lipidpulses gave corresponding increases in the concen-

trations of VFA. At a higher OLR, the VFAaccumulation was higher and faster due to highermetabolic activity of some of the microbial groupsinvolved in the degradation. This illustrates the often

observed limitations in the last steps of the anaerobicdegradation process, the acetogenic degradation ofpropionate and butyrate and the methanogenesis

from hydrogen and acetate. The ammonia producedby protein degradation increased the bufferingcapacity in the reactor. The potential of the

investigated methods to monitor the effects of the

pulse loads was thus dependent both on the totalOLR and the substrate used. There is still a lack ofsensors for monitoring the degradation of com-pounds where the initial hydrolysis is rate limiting.

Undegraded lipids can give clogging problems in thedigesters, and have been shown to have a bactericidaleffect on the anaerobic digestion (Rinzema et al.,

1994).

Alkalinity measurements

TA measurements have earlier proved unreliablefor process monitoring (Hill and Jenkins, 1989;Jenkins et al., 1983), which was confirmed in this

study. It was also shown that the correlation betweenPA and the accumulation of VFAs is stronglydependent on the organic composition of the

substrate. For sludge substrate and carbohydratepulses the correlation between VFAs and PA wasgood. Following protein pulses, the VFA accumula-

tion was in the same range, but the alkalinity and pHboth increased due to the formation of ammoniaduring anaerobic protein degradation. The methodof process monitoring by PA measurement is highly

empirical and is only useful if the history of thereactor is known and the influent is of a relativelystable composition. Within these limitations the

spectrophotometric alkalinity measurement methodwas as good as the conventional titrationmethod at detecting process disturbances. The new

Fig. 3. The response in dissolved hydrogen, alkalinity and pH to the pulse loads at a base load of 3.0 gVS l�1 d�1. TA=total alkalinity, PA=partial alkalinity, spectr=spectrophotometric method, titr=titri-

metric method.

Evaluation of new methods 2837

spectrophotometric method described in this paperavoids any titration procedure and the potentialerrors that are introduced by the use of a pHelectrode. The method could be automated if a

suitable sample pre-treatment method could befound. A continuous flow injection analysis (FIA)system that can minimize analysis errors by produ-

cing many measurements within a short period oftime could be used.

Determination of dissolved hydrogen

The pulse of sludge substrate at the OLR of3.0 gVS l�1 d�1 gave a fast accumulation of hydrogenwhereas the accumulation of VFAs reached its

maximum after 7 h. The metabolisation of pureglucose caused a successive build-up of hydrogenand VFAs, both reaching the maximum concentra-tions after 4 h. The glucose pulse at an OLR of 4.5 g

VS l�1 d�1 gave a 4 times higher hydrogen accumula-tion, but a VFA accumulation in the same range,both reaching the maximum after 2.5 h. For the

protein pulses the hydrogen accumulation was inthe same range irrespective of the OLR level, but theconversion rate of the protein was higher at the

higher OLR. This pattern of hydrogen accumulation

gives additional information about the microbiallimitations and degradation pattern that cannot beconcluded from the VFA accumulation alone. Adissolved hydrogen concentration of more than

40 nM has been said to be crucial in the regulationof the flow of carbon during mineralisation oforganic matter (Pauss et al., 1990b). Higher hydrogen

concentrations will direct the electron flow frommethane production to the production of electronsinks such as butyrate, propionate, lactate or ethanol

(Mosey, 1983; Schink, 1997). Furthermore, degrada-tion of propionate and butyrate by the obligatesyntrophic acetogenic bacteria is hydrogen dependent

and will be inhibited at elevated hydrogen concentra-tions (Harper and Pohland, 1986). Thus, hydrogenaccumulation is a good indicator of imbalancebetween some of the most sensitive microbial groups

in the anaerobic digestion process.One advantage of this method, compared to other

on-line methods for dissolved hydrogen monitoring is

that the semiconductor sensor responds specificallyto hydrogen. This means that after transfer fromthe liquid, no further treatment of the sample is

necessary. The Pd-MOS sensor used can be poisonedby hydrogen sulphide (Hornsten et al., 1991), whichis often present in anaerobic digesters. However, the

stability of the sensor during the 3 months of on-lineoperation during this study indicated that thehydrogen sulphide could not penetrate the Teflonmembrane. The main problem of using the on-line

method was the microbial growth on the liquid-to-gas hydrogen transfer membrane, which was circum-vented by changing the membrane every 48 h, as

described by Bjornsson et al. (2000).

The microbial fingerprints

The method of partitioning in aqueous two-phasesystem is highly sensitive for the separation and

fractionation of biological particulates (cells, orga-nelles) on the basis of surface properties (Walteret al., 1985). The method has successfully been used,e.g. in fractionation of rat bone marrow cells

(Garcia-Perez et al., 1990). Another advantage isthat the conditions in a two-phase system are mild andcells that has been subjected to partitioning in a CCD

are viable and can, for example, be cultured (Walterand Johansson, 1994). Repeated measurements at thedifferent OLR levels showed good reproducibility in

the distribution pattern, but the absorbance in thedifferent CCD-runs varied (data not shown). Underchanging culture conditions, the CCD profiles werefound to change accordingly. A broader profile was

observed for the overloaded culture (Fig. 5(c)) thanwhen the culture had been run at a constant OLRof 1.5 g VS l�1 d�1 (Fig. 5(a)) and 3.0 g VS l�1 d�1

(Fig. 5(b)). A broader cell distribution reflectsheterogeneity in cell surface properties. Furthermore,different CCD-profiles were observed when the

Fig. 4. The response in dissolved hydrogen, alkalinity andpH to the pulse loads at a base load of 4.5 g VS l�1 d�1.

TA=total alkalinity, PA=partial alkalinity.

Lovisa Bjornsson et al.2838

reactor had been subjected to different carbon

sources (Fig. 5(d)–(f )). When the reactor wassubjected to lipid and protein perturbations arougher profile was observed than when subjectedto sludge substrate and glucose perturbations. The

patterns observed are rather complicated since theyreflect both differences in surface structure and, as aconsequence of this, differences in aggregation

behaviour. During sample pretreatment, the com-plete disintegration of flocs could not be achievedand aggregates could be found in the first CCD

fractions giving high absorbance. Whether these flocsare the result of very strong binding, or efficientreassociation, remains to be elucidated.

CONCLUSIONS

The methods evaluated in this investigationshowed good potential for use as monitoring toolsfor anaerobic digestion processes.

The spectrophotometric alkalinity method provedto be a method as valuable as the traditional titrationmethod for process monitoring. The potential of thismethod lies in the ease with which it can be modified

to on-line sampling and monitoring.The hydrogen monitoring method was used as an

on-line measurement system giving valuable informa-

tion as dissolved hydrogen accumulated in theprocess. This method can be used as a processmonitoring tool as well as for increasing the basic

understanding of the metabolism in the anaerobic

digestion process.It was shown that CCD profiles could be used to

obtain a fingerprint of the microbial population inmixed cultures and that the partition patterns showed

an interesting versatility upon changes in the processcondition. This opens up a new field of applicationsfor the CCD method.

Acknowledgements}This work was supported by theSwedish National Energy Administration, the NordicEnergy Research Programme and Borregaard IndustriesLtd. We thank Gisela Wendt-Jonsson for skilful laboratoryassistance. The friendly assistance of the staff at EllingeWastewater Treatment Plant is gratefully acknowledged.

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