changes to bacterial community make-up in a two-phase anaerobic digestion system

Journal of Chemical Technology and Biotechnology J Chem Technol Biotechnol 75:500±508 (2000)

Changes to bacterial community make-up in atwo-phase anaerobic digestion systemBahar Kasapgil Ince1* and Orhan Ince2

1Institute of Environmental Sciences, University of Bogazici, 80815, Bebek, Istanbul, Turkey2Department of Environmental Engineering, Istanbul Technical University, 80626, Maslak, Istanbul, Turkey

(Rec

* CoE-ma

# 2

Abstract: Changes to microbial populations in a two-phase anaerobic digestion system were studied

over 34 weeks. Numbers of auto¯uorescent methanogenic and non-methanogenic bacteria decreased

signi®cantly during start-up, but did not change markedly either in the acid reactor or the up¯ow

anaerobic ®lter for the remainder of the study. Although the proportion of auto¯uorescent

methanogens increased in the acid reactor, the numbers of viable methanogens decreased 590-fold.

The numbers of viable methanogens increased 10-fold in the port, decreased 10-fold in the ef¯uent and

there was almost no change in the drain of the up¯ow anaerobic ®lter. The data indicated that bacterial

attachment in the up¯ow anaerobic ®lter gave a 90% COD removal and a methane yield of 0.33m3

CH4kgÿ1 COD removed at an organic loading rate of 7kg COD mÿ3dayÿ1. Epi¯uorescence microscopy

of the seed sludge revealed a diverse methanogenic population of equally dominant groups of medium

rods and ®laments with Methanococcus, short rods, long rods and Methanosarcina also present. The

medium rod-shaped species remained the most dominant group in the acid reactor. As the volatile

fatty acid concentration increased in the acid reactor the number of Methanosarcina and ®lament

species decreased, becoming the least dominant groups. At the end of the operation, Methanococcus

species were the dominant group in the up¯ow anaerobic ®lter having been washed from the bio®lm.

# 2000 Society of Chemical Industry

Keywords: species composition; two-phase anaerobic digestion; completely mixed reactor; up¯ow anaerobic®lter; enumeration of methanogenic species

1 INTRODUCTIONTwo-phase anaerobic treatment systems are widely

used for treatment of many industrial wastewaters.

Two kinetically dissimilar groups of bacteria, acido-

gens and methanogens, are physically separated in two

reactors. This separation allows optimization of both

the hydrolysis±acidi®cation and acetogenesis±metha-

nogenesis phases, theoretically ensuring maximum

ef®ciency of the overall system.

Ef®cient full-scale application of these systems

requires effective control during start-up and steady-

state operations. This is because parameters in each

reactor such as pH, temperature, substrate concentra-

tion, hydraulic retention time, turbulence and shear

in¯uence the number and the composition of the

microbial populations. A well operated acid reactor

should ideally contain few methanogens. Optimum

conditions for acidi®cation severely retard methano-

genic activity but do not eliminate all methanogens,

which are sensitive to the operating conditions but may

persist in a dormant or semi-dormant state. Even if the

acid reactor is free from methanogens, populations of

H2-utilizing homoacetogens and/or sulfate reducers,

act as a partial H2 sink and reduce the pH.

Previous studies have shown the importance of

eived 27 April 1999; revised version received 2 February 2000; accep

rrespondence to: Bahar Kasapgil Ince, Institute of Environmental Scieil: [email protected]

000 Society of Chemical Industry. J Chem Technol Biotechnol 02

various bacterial species to the development of stable

bio®lms. Bacterial attachment or growth can signi®-

cantly affect the rate of acidi®cation and

methanogenesis.1±3 Since acetic acid is the precursor

of methanogens, acetate-utilizing methanogens such

as Methanothrix soenhngenii, Methanosarcina barkeri,and Methanosracina mazei may be important.1Metha-nothrix species have a high af®nity for acetate and

predominate under conditions of low acetate concen-

tration while Methanosarcina prevails at high acetate

concentrations.4Methanothrix is often the dominant

species in full scale reactors, possessing the ability to

attach to surfaces and other bacteria.5Methanothrixsoenhngenii grew both as rods and long ®laments,

Methanosarcina species readily form agglomerates that

produce methane over the pH range of 5±8.6 In loosely

aggregated sludge, a thin ®lamentous methanogen

identi®ed as either Methanobacterium bryantii or

Methanospirillum hungatei was found to be dominant.7

Overall performance of anaerobic treatment systems

is totally dependent on the composition of microbial

populations in the anaerobic reactors. Determination

of changes in microbial populations and its effect on

performance at various operating conditions of a two-

stage anaerobic treatment system would be of con-

ted 8 February 2000)

nces, University of Bogazici, 80815, Bebek, Istanbul, Turkey

68±2575/2000/$17.50 500

Figure 1. Schematic configuration of two-stage anaerobic digestion system.

Changes to bacterial community in anaerobic digestion

siderable interest. This study, therefore, examined

microbiological aspects, including changes in the

number and composition of the methanogenic bacter-

ia, in both acid and methane reactors using epi¯uor-

escence microscopy and microbiological enumeration

techniques.

2 MATERIALS AND METHODS2.1 Description of two-phase anaerobic digestionsystemA schematic diagram of the laboratory-scale, two-

phase anaerobic digestion (TSAD) system used is

given in Fig 1. The system consisted of a completely

mixed anaerobic digester as the acid reactor (10dm3)

and an up¯ow anaerobic ®lter (31dm3) as the

methanogenic reactor. Temperatures in both the acid

and methane reactors were maintained at 33±36°Cthroughout the study. The pH in the acid reactor was

adjusted by automatic addition of HCl and NaOH and

maintained in the range of 5.5±6.0. The acid reactor

led to a 5dm3 volume pH neutralizing vessel where the

pH of the acidi®ed dairy wastewater was adjusted to

7.0±7.5 prior to the up¯ow anaerobic ®lter by

automatic addition of NaOH. The in¯uent and

ef¯uent ports of the up¯ow anaerobic ®lter were

positioned at 8cm from the base and 10cm from the

top respectively. Two further sampling ports were

placed at 2cm and 40cm from the base. Sampling

locations of the TSAD system are also shown in Fig 1.

The drain, situated 2cm from the base of the up¯ow

anaerobic ®lter, was connected to the recirculation line

to increase the methanogenic activity by recirculating

the biomass back to below the in¯uent port. The

up¯ow velocity (UV) was initially maintained at 5m

dayÿ1 in order to allow biomass attachment to the

J Chem Technol Biotechnol 75:500±508 (2000)

media. After start-up had been completed the UV was

increased to 15m dayÿ1 and maintained at the same

level for the remainder of the study. The medium used

was modi®ed unplasticized non-porous PVC pall rings

and was loosely packed into the ®lter column with a

porosity of 0.92.

2.2 Analytical methodsChemical oxygen demand (COD) analyses were

carried out using a Hach COD Reactor Model.

Volatile fatty acids (VFA) were determined using a

gas liquid chromatograph (GLC). Gas production was

measured using a wet gas meter while gas composition

was determined using gas chromatography (GC). A

Becker Model 403 GC with a thermal conductivity

detector having a Poropak Q stainless steel column

(1.5�4mm) operating at 55°C using helium as the

carrier gas (¯ow rate of 50cm3minÿ1) was used to

detect the methane in a 1cm3 sample of gas.

Throughout the study, pH, temperature, gas composi-

tion, gas production rate and VFA were monitored

daily and suspended solids (SS) and volatile sus-

pended solids (VSS) were measured two times a week.

All analyses were carried out according to StandardMethods.8

2.3 Feed and seedWastewater from a milk and cream bottling plant was

used as feed (Table 1). To establish an active

anaerobic bacterial population, acid and methane

reactors were seeded with 3dm3 and 15dm3 digesting

sludge (14700mg VSSdmÿ3) respectively from a

primary sludge digester of a local domestic wastewater

treatment plant.

501

Table 1. Chemical characteristics of wastewater used

Parameter Concentration (mg cmÿ3)

COD 2000±6000

BOD5 1200±4000

SS 350±1000

VSS 330±940

PO4-P 20±50

TKN 50±60

Alkalinity (as CaCO3) 150±300

Total fatty matter 300±500

Sodium 170±200

Potassium 35±40

Calcium 35±40

Magnesium 5±8

Ferrous 2±5

Cobalt 0.05±0.15

Nickel 0.50±1.00

Manganese 0.02±0.10

pH (units) 8±11

BK Ince et al

2.4 Microbiological studies2.4.1 Sample preparationAll enumeration studies were completed immediately

after sampling.9 Strict anaerobic techniques were

observed throughout all medium preparations and

sample handling.10,11

2.4.2 Direct microscopic countEnumeration of the total bacteria and total auto¯uor-

escent methanogenic bacterial populations in the

samples was made using a Zeiss D-7082 Epi¯uores-

cence microscope ®tted with a 50W high pressure

mercury lamp.1,12,13 The samples were diluted and

homogenized to give counts of between 100 and 400

for each ®eld of view and counted using an Improved

Neubauer Chamber. This had a depth of 0.1mm and

an area of 1mm2. Zeiss �63 water immersion lenses

Figure 2. Changes in number ofmethanogens (microscopic count) andnon-methanogens in two-stageanaerobic digestion system.

502

were used with a �10 eyepiece. Detailed counting

procedures have been described previously.14

2.4.3 Counts of viable methanogensThe Most Probable Number (MPN) technique was

used to count viable methanogenic bacteria in the

samples.6,15 The sample preparation took place inside

an anaerobic cabinet. The inoculated media were

statically incubated at 35°C for 4±6 weeks. The

growth was recorded as the number of positive tubes

by detection of methane in the head space using GC.

All positive tubes were also examined by epi¯uores-

cence microscopy to con®rm the MPN results. The

numbers of positive tubes at different dilutions were

used to obtain the most probable numbers of

methanogenic bacteria in the samples from the

Probability Tables.8

3 RESULTS3.1 Performance of two-phase anaerobic digestionsystemOptimum operating conditions such as pH, tempera-

ture, hydraulic retention time (HRT) and organic

loading rate (OLR) in acid reactor were predetermined

in a subsequent study.16 The TSAD system was then

operated using the results of that study. Variations of

�5% in the ef¯uent COD concentration of the up¯ow

anaerobic ®lter were taken as an indication that a state-

state was obtained. Overall TSAD system gave almost

90% COD and 95% BOD removal ef®ciencies at an

overall OLR of 5kg COD mÿ3dayÿ1 with an HRT of 2

days. The acid reactor was operated up to an OLR of

23kg COD mÿ3 dayÿ1 with an HRT of 0.5 days

whereas the up¯ow anaerobic ®lter performed well up

to OLR of 7kg COD mÿ3 dayÿ1 with an HRT of 1.5

days without having any sign of instability throughout

40 weeks of operation. Good separation of the acid


Figure 3. Changes in morphology ofmethanogens and volatile fatty acid(VFA) concentration in acid reactor.


and methane phases was achieved, as indicated by

methane yields of 0±0.15m3 CH4kgÿ1 COD removed

and 0.30±0.34m3 CH4kgÿ1 COD removed respec-

tively. Although high concentrations of n-butyric and

n-valeric were produced in the acid reactor only acetic

and propionic acids were detected in signi®cant

concentrations in the ef¯uent from the up¯ow

anaerobic ®lter. The VFA produced in the acid reactor

were effectively consumed by the methanogens in the

up¯ow anaerobic ®lter. The operation and perfor-

mance of the TSAD system has been discussed

elsewhere.16,17

3.2 Changes in species compositionFigure 2 shows the changes in the number of

auto¯uorescent methanogens and non-methanogens

in the acid reactor, drain, port and ef¯uent from the

up¯ow anaerobic ®lter. Figures 3 and 4 show the

variations in the morphology of auto¯uorescent

methanogens with the VFA concentrations in the acid

reactor and in the ef¯uent from the up¯ow anaerobic

®lter. The numbers of Methanosarcina and ®lamentous

Figure 4. Changes in morphology ofmethanogens and volatile fatty acid(VFA) concentration in effluent fromupflow anaerobic filter.


species remained low compared with other species as

the VFA concentration increased in the acid reactor

(Fig 3). However, after week 10, the number of

Methanococcus species increased from about

1.6�105mgÿ1 VSS to 6.8�106mgÿ1 VSS while the

number of Methanosarcina species reduced to

3.5�103mgÿ1 VSS from about 6.0�106mgÿ1 VSS

(week 5) in the ef¯uent from the up¯ow anaerobic

®lter (Fig 4). Morphological variations of the auto-

¯uorescent methanogens in the drain and the port of

the up¯ow anaerobic ®lter are also presented in Figs 5

and 6. All bacterial counts were expressed per mg VSS

instead of countscmÿ3 in order to avoid the effect of

changes in the concentration of biomass in the reactor.

The changes in the SS and VSS concentrations in the

TSAD system can be seen in Fig 7. During the

operation, the total number of auto¯uorescent metha-

nogens remained at between 0.01±3% and 2±13% of

the total population in the acid reactor and the up¯ow

anaerobic ®lter respectively, as seen in Fig 8.

As seen from Fig 9(a) the wash out did not adversely

affect the COD removal ef®ciency of the up¯ow

503

Figure 5. Changes in morphology ofmethanogens in drain of upflowanaerobic filter.

BK Ince et al

anaerobic ®lter. Figure 9(b) shows that as the OLR

increased, the number of auto¯uorescent methano-

gens washed out from the ef¯uent did not increase. In

contrast, the number of auto¯uorescent methanogens

in the ef¯uent remained below 1.0�107mgÿ1 VSS. As

seen from Tables 2±5, for most of the operating

period, the numbers of viable methanogens were in a

range of 105±107mgÿ1 VSS in the samples taken from

the drain and the port of the up¯ow anaerobic ®lter,

whereas they were found to vary between 104±

105mgÿ1 VSS and 104±106mgÿ1 VSS in the ef¯uent

from the anaerobic ®lter and the acid reactor

respectively.

Figure 10 shows the variations in the ratio of the

number of total auto¯uorescent methanogens (micro-

scopic count) to the number of viable methanogens

(MPN) during the operation. Although Fig 8 shows a

sharp increase in the ratio of auto¯uorescent metha-

Figure 6. Changes in morphology ofmethanogens in port of upflowanaerobic filter.

504

nogens to total bacteria in the acid reactor, as seen

from Table 2 and Fig 10, the number of viable

methanogens decreased and remained almost un-

changed after week 30 and the ratio of the total

auto¯uorescent methanogens to the viable methano-

gens, therefore, increased dramatically.

The reason for an increase in the ratio of auto¯uor-

escent methanogens to total bacteria in the acid

reactor after week 9 (Fig 8) might be due to some

time needed by the predominant methanogens in the

inoculum sludge in order to acclimatize themselves to

the operating conditions such as low pH and short

HRT. The same explanation can be applied to the

decrease in the numbers of viable methanogens in the

acid reactor during the ®rst 9 weeks of operation after

which the numbers of viable methanogens increased

and remained unchanged during the last 10 weeks.

Table 3 shows an increase in the number of viable


Figure 7. Changes in suspended solids (SS) (— —) and volatilesuspended solids (VSS) (--*--) concentrations in (a) acid reactor (b) drain(c) port and (d) effluent of upflow anaerobic filter.

Figure 9. (a) Changes in total number of methanogens in effluent and CODremoval efficiency (b) OLR of upflow anaerobic filter against operating time.


methanogens in the drain of the up¯ow anaerobic ®lter

which was supported by a continuous decrease in the

ratio of total auto¯uorescent methanogens to viable

methanogens, as shown in Fig 10.

In order to regain the settled biomass at the bottom

of the up¯ow anaerobic ®lter, which contained high

numbers of viable methanogens, recirculation from

the drain line back to the ®lter was carried out more

frequently. Although an increase in the number of

Figure 8. Changes in ratio ofmethanogens to total bacteria in two-stage anaerobic digestion system.


viable methanogens in the port of the up¯ow anaerobic

®lter was observed from week 10, the number of viable

methanogens in the ef¯uent decreased approximately

10-fold remaining low and only slightly changed

during the remainder of the study (Tables 4 and 5).

This was supported by an increase in the ratio of total

auto¯uorescent methanogens to viable methanogens

(Fig 10) and also a considerable decrease in the VSS

concentration between the port and the ef¯uent. This

might imply that the viable methanogens remained in

between the port and the ef¯uent levels attaching to

the bio®lm.

4 DISCUSSIONChanges in the numbers of methanogenic species

occurred in the TSAD system throughout the operat-

505

Table 2. Most Probable Numbers of methanogens in acid reactor showing95% confidence limits

Time

(weeks)

Methanogens

(numbermgÿ1 VSS)

95% con®dence limit

Lower Higher

1 1.7�107 5.5�106 5.2�107

2 6.5�103 1.7�103 1.6�104

3 2.5�104 7.4�103 5.8�104

5 6.1�103 1.5�103 1.7�104

7 1.1�104 3.6�103 3.0�104

9 1.4�105 4.5�104 3.5�105

11 5.1�105 1.7�105 1.6�106

12 6.1�105 2.1�105 1.7�106

18 1.1�106 3.5�105 2.8�106

31 4.3�104 1.5�104 1.2�105

35 3.5�104 1.2�104 1.0�105

40 2.9�104 1.0�104 8.3�104

Table 4. Most Probable Numbers of methanogens in effluent of upflow filtershowing 95% confidence limits

Time

(weeks)

Methanogens

(numbermgÿ1 VSS)


Lower Higher

1 1.4�106 4.0�105 4.4�106

3 1.6�105 5.2�104 4.4�105

4 1.5�105 4.8�104 3.6�105

6 1.7�105 5.8�104 4.8�105

8 2.2�105 5.5�104 6.3�105

10 5.4�104 1.7�104 1.6�105

12 5.1�104 1.3�104 1.6�105

18 9.6�104 3.2�104 2.5�105

31 1.2�105 4.8�104 4.4�105

35 1.6�105 5.3�104 4.2�105

40 1.3�105 4.3�104 3.4�105

BK Ince et al

ing period. The medium rod species remained the

most dominant group in the acid reactor throughout

the study followed by short rods and Methanococcusspecies whereas a signi®cant decrease in the numbers

of ®lamentous and Methanosarcina species occurred,

becoming the least dominant groups towards the end

of the study. Noticeable decreases in the numbers of

Methanococcus species were observed in the acid

reactor during the ®rst 7 weeks of operation after

which their numbers increased, becoming the third

dominant group at the end of the study. Increases in

the concentration of VFA in the acid reactor through-

out the operation might have caused the changes in the

dominant methanogenic species and their numbers.

Although the numbers of methanogenic species

were high in the inoculation sludge their numbers,

except Methanococcus species, signi®cantly decreased

in the drain of the up¯ow anaerobic ®lter in the ®rst 3

weeks after which their numbers gradually increased

for the remainder of the study. The only exception

were the numbers of ®laments and Methanosarcinawhich decreased drastically in the acid reactor. These

signi®cant decreases, which ranged from 40 to 400-

able 3. Most Probable Numbers of methanogens in drain of upflow filterhowing 95% confidence limits

ime

eeks)

Methanogens

(numbermgÿ1 VSS)


Lower Higher

1 1.4�107 4.5�106 3.4�107

2 5.1�106 1.7�106 1.6�107

3 1.0�105 3.4�104 2.4�105

5 6.3�105 2.1�105 1.6�106

7 6.7�105 2.2�105 1.6�106

9 4.3�105 1.5�105 1.2�106

1 1.4�106 3.8�105 3.5�106

2 2.4�106 7.8�105 7.3�106

8 4.5�107 1.5�107 3.0�108

1 3.7�107 1.3�107 1.1�108

5 1.3�107 4.2�106 3.5�107

0 4.9�106 1.6�106 1.3�107

Table 5. Most Probable Numbers of methanogens in port of upflow filtershowing 95% confidence limits

Time

(weeks)

Methanogens

(numbermgÿ1 VSS)


Lower Higher

1 1.5�106 4.9�105 3.9�106

3 1.5�106 4.7�105 3.5�106

5 7.0�105 2.0�105 2.2�106

7 2.4�105 7.7�104 7.2�105

10 4.3�105 1.1�105 1.4�106

12 8.6�105 2.6�105 2.0�106

18 1.1�107 4.4�106 4.0�107

31 3.0�108 9.9�107 7.8�108

35 1.7�107 5.8�106 4.9�107

40 1.8�107 6.0�106 5.1�107

Ts

T

(w

1

1

1

3

3

4

506

fold, could be explained by the attachment of some of

these species to the media and wash out in the ef¯uent

from the up¯ow anaerobic ®lter. The medium rods

were the dominant group in the samples from the drain

in week 1, however there were changes during the 34

weeks of operation and Methanococcus species became

the dominant group followed by short rods, medium

rods, long rods, Methanosarcina and ®laments.

The dominant methanogenic species in samples

taken from the port of the up¯ow anaerobic ®lter

changed during the study. The dominant group was

medium rods in week 1 followed by ®laments,

Methanococcus, short rods and equally least dominant

groups of Methanosarcina and long rods. The numbers

of ®lamentous species were reduced 4-fold during the

®rst 7 weeks of operation, after which they remained

fairly constant and after week 19 started to increase

but were still in low numbers compared with the other

species. This can be explained by the same mechanism

which might have caused the decreases in the numbers

of methanogenic species in the drain. Noticeable

changes in the numbers of methanogenic species

occurred throughout the operation, resulting in the

changes in the dominant methanogenic species.

Methanococcus species were found to be the dominant


4

Figure 10. Changes in ratio of totalmethanogens to viable methanogens(MPN) in two-stage anaerobicdigestion system.


group in the samples taken from the port of the up¯ow

anaerobic ®lter at the end of the study followed by

short rods, medium rods, long rods, Methanosarcinaand ®laments.

Slight changes in the numbers of methanogens

occurred in the ef¯uent from the up¯ow anaerobic

®lter throughout the operation resulting in changes in

the dominant methanogenic species. Medium rods

and Methanococcus species were the ®rst equally

dominant groups in week 1 followed by short rods,

long rods and the least dominant groups of Methano-sarcina species and ®laments whereas Methanococcusspecies became the dominant group at the end of the

operation followed by medium rods, short rods, long

rods, ®laments and the least dominant Methanosarcinaspecies. The numbers of methanogenic species, except

®laments in the drain and the port, were higher than

were found in the ef¯uent. This could imply that most

of the ®laments on the media in the upper part of the

up¯ow anaerobic ®lter because detached and were

washed out.

The proportion of methanogens in total anaerobic

bacterial population was shown to vary from 1 to

10%.1 In this study, the maximum ratio of methano-

gens to total bacteria was found to be 13% in the drain

of the up¯ow anaerobic ®lter. The numbers of

methanogens counted using direct microscopic tech-

niques however, do not completely re¯ect the metha-

nogenic activity in the reactor. Another disadvantage

of this technique is that the viable and non-viable

methanogens cannot be distinguished from each

other. The MPN technique was, therefore, used to

count viable methanogens. Compared with the micro-

scopic count, the MPN technique gives up to 3-fold

fever numbers18 because only three different metha-

nogenic groups are identi®ed by this technique,

namely Methanosarcina, Methanobacterium formicicumand Methanococcus species.

In this study, the ratio of the number of methano-

gens obtained by microscopic count to the number of

viable methanogens was initially 20, 40, 250 and 20 in


the acid reactor, the drain, the port and the ef¯uent

respectively, becoming 700 in the acid reactor, 2 in the

drain, 5 in the port and 80 in the ef¯uent of the up¯ow

anaerobic ®lter at the end of the study. The reason for

the observed increase in the ratio of total auto¯uor-

escent methanogens to viable methanogens in the acid

reactor was that the numbers of auto¯uorescent

methanogens increased after an acclimatization period

of approximately 10 weeks operation and a general

decrease in the numbers of viable methanogens

throughout the study. The number of viable methano-

gens doubled in the port at the end of the study and

decreased in the ef¯uent from the up¯ow anaerobic

®lter during the ®rst 17 weeks after which the number

remained low, indicating successful bacterial attach-

ment. The up¯ow anaerobic ®lter performed well at an

OLR of 7kg COD mÿ3 dayÿ1 and achieved 90% COD

removal ef®ciency and a methane yield of 0.33m3

CH kgÿ1 COD removed.

5 CONCLUSIONSThis study showed that changes in the numbers and

the composition of the microbial populations occurred

both in the acid and methane reactors during 34 weeks

of operation. The medium rods and Methanococcusspecies were found to be the dominant groups in the

acid reactor and in the up¯ow anaerobic ®lter

respectively at the end of study. The ratio of total

auto¯uorescent methanogens to total bacteria re-

mained between 0.01±3% in the acid reactor and 2±

13% in the up¯ow anaerobic ®lter. The number of

viable methanogens decreased 590-fold in the acid

reactor and 10-fold in the ef¯uent whereas they

increased 10-fold in the port and remained fairly

constant in the drain of the up¯ow anaerobic ®lter.

This resulted in successful bacterial attachment in the

anaerobic ®lter resulting in COD removal ef®ciency of

90% and methane yield of 0.33m3 CH4kgÿ1 COD

removed.

507

BK Ince et al

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changes to bacterial community make-up in a two-phase anaerobic digestion system

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