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Anaerobic granule formation and tetrachloroethylene (TCE) removal inan upflow anaerobic sludge blanket (UASB) reactor

Delia Teresa Sponza*

Dokuz Eylul University Engineering Faculty Environmental Engineering Department, Buca Kaynaklar Campus, I zmir, Turkey

Received 26 March 2001; received in revised form 11 June 2001; accepted 21 June 2001

Abstract

The granulation process was examined using synthetic wastewater containing tetrachloroethylene (TCE) in a 2 liters laboratory upflow

anaerobic sludge blanket (UASB) reactor. The anaerobic biotransformation of TCE was investigated during the granulation process by

reducing the HRT and increasing the chemical oxygen demand (COD) and TCE loadings. Anaerobic unacclimated sludge and glucose wereused as seed and primary substrate, respectively. Massive initial granules were developed after 1.5 months of start-up, which grew at an

accelerated pace for 7 months and then became fully grown. The effect of operational parameters such as influent TCE concentrations, COD

and TCE loading, food to microorganism (F/M) ratio and specific methanogenic activity (SMA) were also considered during granulation.

The granular sludge cultivated had a maximum diameter of 2.5 mm and SMA of 1.32 g COD (gTSS.day)1. COD and TCE removal

efficiencies of 92% and 88% were achieved when the reactor was operating at TCE and COD loading rates of 30 mg (l.day)1 and 10.5 g

(l.day)1, respectively. This corresponds to HRT of 0.40 day and F/M ratio of 1.28 gCOD (gTSS.day)1. Kinetic coefficients of k

(maximum specific substrate utilization rate), Ks

(half velocity coefficient), Y (growth yield coefficient) and b (decay coefficient) were

determined to be 2.38 mgCOD (mgTSS.day)1, 108 mgCOD l1, 0.17 mgTSS (mgCOD)1 and 0.015 day1, respectively for TCE

biotransformation together with glucose as carbon and energy source during granulation. © 2001 Elsevier Science Inc. All rights reserved.

Keywords: UASB reactor; Granulation; Granule; TCE treatment

1. Introduction

Anaerobic treatment of toxic and refractory industrial

wastewater has become a viable technology and has been

most commonly used. Since its introduction 15 years ago,

UASB reactors containing granular sludge have become

popular worldwide and have been commonly used for

wastewater from agricultural industries. Recent studies have

demonstrated that the UASB technology is applicable to

treating aromatic and aliphatic chlorinated chemicals such

as trichloro ethylene, carbon tetrachloride and chlorophenol.

Furthermore, the UASB granules exhibited higher resis-tance to the toxicity of chlorinated aromatic and aliphatics

than the flocculent digester sludge [1,2].

Granulation of methanogenic bacteria in UASB reactors

is important in the treatment of various industrial wastewa-

ter containing toxic substances due to their compact struc-

ture which protect the bacteria from inhibitory and toxic

pollutants. The ability of the granules to resist toxicity could

be attributed to their layered microstructure. However, these

granules occasionally disintegrate in industrial reactors

which results in loss of activity when they are developed in

carbohydrate containing wastewaters before being used as

seed [3]. This extends the granulation period which is gen-

erally known to be a very slow process.

The anaerobic granules developed in organic wastewa-

ters which do not contain chlorinated organic compounds

normally do not have significant dechlorinating activity.

Biomass in the reactor took a long time to become adaptedto the new substrate. Only after about 90 days of acclima-

tion did the biogas production rates and COD removal

efficiency begin to recover. Wu et al. determined that vol-

atile fatty acid (VFA) degrading granules did not exhibit

degrading activity for pentachlorophenol (PCP) [3]. During

an operational period of 225 days was spent on the devel-

opment of PCP degrading granules and one year after

start-up 80% PCP removal efficiency was obtained. PCP

degrading granules had a much higher tolerance to PCP* Fax: 90–232-453–1153.

E-mail address: [email protected] (D.T. Sponza).

www.elsevier.com/locate/enzmictecEnzyme and Microbial Technology 29 (2001) 417–427

0141-0229/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved.

PII: S0141-0229(01)00402-1

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concentrations than VFA degrading granules. The methane

production potential decreased 40% in VFA granules at a

PCP concentrations of 5 mg l1 while PCP degrading gran-

ules completely dechlorinated and mineralized [3]. On the

contrary, granules from industrial reactors, when fed with

carbohydrate and VFAs such as acetic and propionic acid,

changed their structure. Studies showed that granules from

the UASB reactor treating the pulp and paper mill waste-water when fed with VFA in continuous UASB reactors can

change their color, elemental composition, are not so

densely packed and may float [4]. Kosaric et al. observed

that three months after start-up granules appeared in the

ef fluents. Granules over 2 mm in diameter did not settle,

since a hollow core within the granules resulted in poor

settling [4].

Higher loading rates and upflow velocities of wastewater

containing toxic compounds results in mortalities in gran-

ules during granulation process in UASB reactors. In a

study performed by Wu et al. PCP degrading granules were

used as seed material to start-up a (pentachloroethylene)PCE dechlorinating UASB reactor [4]. Lower PCE removal

ef ficiencies were observed at loading rates as high as 79 mg

(l day)1. Ninety-five percent PCE removal ef ficiency was

achieved at an HRT 4 h and a PCE loading rate of 36 mg (l

day)1. Fang et al. observed that benzoate acclimated gran-

ules showed better resistance to chlorinated aromatics than

starch degrading and distillery granules since they did not

have prior exposure to aromatic chemicals during granula-

tion in UASB reactors [5]. For instance, acclimated and

benzoate-degrading granules exhibited better resistance to

cresol, phenol, cathetol and PCE toxicity. The inhibition of

methanogenic activity via these substances was not severe

for UASB reactors containing benzoate-degrading granules

[5]. Hydrophobic functional group such as aromatic and

aliphatic chemicals were very toxic and showed a higher

inhibition effect on methanogenesis [6]. Anaerobic granules

with special dechlorinating activities can be developed by

using microbial consortia which are able to form granular

structures together with additional dechlorinating organisms[7]. On the other hand, the actual values of ef ficiency may

vary when slowly or rapidly degrading primary substrates

are used together with acclimated culture. When rapidly

degrading substrates such as lactate, methanol, sugar and

acetate are used the dechlorination fraction of substrate may

increase during acclimation and granulation. Anaerobic

sludge should be acclimated to toxic organics at the begin-

ning of granulation or during these processes [8].

Tetrachloroethylene (C2Cl4) is widely used as a soil and

grain fumigant, a industrial solvent and a dry-cleaning or

degreasing fluid. Biotransformation of TCE has been stud-

ied several times by different researchers. TCE was trans-formed by reductive dehalogenation to trichloroethylene

and dichloroethylene [9], to ethylene [10], to ethane [11,12],

and vinyl chloride [13] under anaerobic conditions. In ad-

dition, TCE was at least partially mineralized to CO2 [14].

In a study performed by Distefano et al. high concentrations

of TCE such as 550 mM were routinely dechlorinated to

80% ethylene and 20% vinyl chloride in 160 ml serum

bottles [15]. High concentrations of TCE (550 mM) in-

hibited the methanogenic transformations. Eighty percent

and 85% TCE and COD removal ef ficiencies were obtained

up to 39 mg (l.day)1 of TCE loading rate in a UASB

reactor at a HRT of 4.5 h and influent TCE concentrations

varied between 6.5–9.3 mgl1 by using butyrate and glu-cose as carbon source. The intermediates of TCE were

found to be trichloroethylene, dichloroethylene, vinyl chlo-

ride and ethylene [3]. Christiansen et al. , studied the reduc-

tive dechlorination of TCE in a UASB reactor operating in

batch mode [16]. It was found that TCE was reductively

dechlorinated to dichloroethylene (DCE). When the TCE

concentration was increased from 4.6 mM to 27 mM, the

transformation rate decreased [16]. 80 and 75% removal

ef ficiencies for TCE were obtained in CSTR reactor at

loading rates varying between 35 and 110 mg TCE (l.day)1

at a HRT of 10 days and average biomass concentration of

2300 mg l1

when acetic acid and propionic acid were usedas primary substrate, respectively. Various substrates in-

cluding acetate and glucose can serve as electron donors for

dechlorination of TCE [3]. The effects of different types of

supplemented substrates such as acetate, glucose, lactate

and methanol on the biotransformation of TCE were dem-

onstrated [17,18]. TCE degradation in wastewater was stud-

ied by Tatsumoto et al. in reactors containing an anaerobic

sludge and granular biologic activated carbon [19]. It was

found that TCE was degraded as adsorption and biotrans-

formation, the microbial activity of which signified an 80%

decrease of TCE. Prakash and Gupta studied the biodegra-

dation of TCE in an UASB reactor containing 5–50 mg l1

Abbreviations

Lr specific substrate utilization rate (day)1

X concentration of microorganisms in UASB reac-

tor (mg l1)

dS/dt microbial substrate utilization rate [mg (l.day)1

]k maximum specific substrate utilization rate

[mgCOD (mgTSS.day)1]

Ks half velocity coef ficient (mgCOD l1)

S TCE or COD concentrations surrounding the

microorganisms (mg l1)

Q influent flow rate (l day1)

So influent TCE or COD concentration (mg l1)

V reactor volume (l)

c solid retention time (SRT) (day)

Y growth yield coef ficient [mgTSS(mgCOD)1]

b bacterial decay coef ficient (day1)

Qw waste flow rate (l day1)

Xw waste microbial concentration (mg l1)

Xe ef fluent microbial concentration (mg l1)

418 D.T. Sponza / Enzyme and Microbial Technology 29 (2001) 417 – 427

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of TCE to develop granular sludge [20]. 92% TCE and 94%

COD removal ef ficiencies were obtained and trichloroethyl-

ene, 1,2-dichloroethylene, vinyl chloride and ethylene were

formed on dehalogenation of TCE. Granules of 2.3 mm sizes

were bioaugmented 82 days following the start-up period.

In spite of the studies mentioned above, granule forma-

tion and the effectiveness of operational factors for UASB

reactor containing TCE has not been fully investigated. The

granulation process was not extensively examined using

TCE containing wastewaters. The studies showed that gran-

ules with TCE proceed more slowly than in a UASB reactorfed with readily biodegraded carbohydrates. This can be

attributed to the use of granules developed in carbonaceous

wastewater for wastewater treatment. Furthermore, studies

involving granule formation in UASB reactors containing

TCE are limited. Investigations concerning the anaerobic

granulation process in TCE containing wastewater, influ-

ence of operational parameters such as HRT, F/M ratio and

organic loading rate have not been adequately researched in

recent studies.

In this study, anaerobic granular sludge was developed

for the treatment of TCE in a UASB reactor. The perfor-

mance of anaerobic granules bio-augmented for the removal of

TCE was monitored with SMA, COD, and TCE treatment

ef ficiencies during granulation period. The effect of opera-

tional parameters such as F/M ratio, HRT and organic loading

on treatment ef ficiency were examined during granulation.

2. Materials and methods

2.1. Seed

The flocculent anaerobic microorganisms used in this

study were obtained from an anaerobic CSTR reactor con-

taining acidogenic and methanogenic phase biomass of a

Yeast Beaker Factory in Izmir-Turkey. This seed was not

granulated or acclimated to TCE before the start-up of the

UASB reactor.

Anaerobic sludge was acclimated and granulated to-

gether during the start-up period in a continuous operation

of the UASB reactor by increasing the influent concentra-

tion and loadings of TCE gradually due to the low growth

and long start-up period of anaerobic microorganisms.

The properties of anaerobic sludge was as follows: Sus-

pended solid (TSS) concentrations of 33 g l1, volatile

suspended solid concentrations (VSS) of 21 g l1 and

sludge volume index (SVI) of 90 ml g1 TSS. The specific

methanogenic activity (SMA) and median bioparticle diam-

eter of the sludge were 0.07 g CH4-COD (g TSS.day)1 and

0.02 mm, respectively.

2.2. UASB reactor and experimental methodology

Fig. 1 illustrates the 2 liter stainless-steel UASB reactor

used in this study, which had an internal diameter of 90 mm

and a height of 1000 mm. Five evenly distributed sampling

ports were installed over the top of the reactor. The studies

were conducted at 35 2°C by means of a temperature

controlled heater in the reactor. The reactor was filled with

350 ml of settled anaerobic sludge resulting in a seed con-

centration of 8 g l1 of TSS fed in continuous mode with

glucose and TCE containing Vanderbilt Mineral Medium.

The operational conditions including influent concentra-

tions of TCE, COD, TSS concentrations were measured in

the reactor and upflow rates applied throughout 230 days of

operation are depicted in Table 1. During the operation

period, pH and gas production were measured daily, COD,

TCE and VFA concentrations in ef fluents were monitored

Fig. 1. Schematic configuration of UASB reactor.


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weekly. TSS concentrations in the reactor and ef fluent sam-

ples were monitored biweekly.

2.3. Substrate and mineral medium

The feed solution of the UASB reactor consisted of

glucose as a primary carbon source and TCE dissolved in

Vanderbilt mineral medium. During the fist two months, for

rapid granulation S/Fe2 and Cl/Fe2 ratios were main-

tained between 1.6 –1.8 and 0.3– 0.5, respectively, by using

a suitable amount of Na2S, FeCl2 and chlorinated salts as

given in the composition of Vanderbilt Mineral Medium.

800 mg l1 of NH4-N, 100 mg l1 of Ca2 and 100 mg l1

of PO4-P concentrations were adjusted by adding 3000 mg

l1 of NH4Cl, 370 mg l1 of CaCl2. 2H2O and 415 mg l1

of (NH4)2 HPO4, respectively, to the feed medium duringthe start-up of granulation.

S/P and Ca/Fe2 ratios were maintained between 0.4 –

0.6 and 2.1–2.3, respectively, during the first two months of

continuous operation of the UASB [3,21]. COD/N/P ratios

were adjusted between 105–108, 2.6 –3.2 and 0.8 –1.2, re-

spectively. Furthermore, every day two milliliters of stock

trace metal solution was added to the reactor feed in con-

tinuous operation for growth stimulatory purpose. The in-

gredients of this solution consisted of: (gram per liter)

FeCl2.6H2O (28.5), NiCl2.6H2O (8), CoCl2.6H2O (8). 0.01 g

l1 of sodium thioglicollate was added to reduce the redox

potential and sustain the anaerobic conditions. NaHCO3

ranging between 3000 and 6000 mg l1 was used to main-

tain the neutral pH throughout the continuous feed.

The composition of the Vanderbilt mineral medium that

was used as the basal medium in all batches (anaerobic

toxicity assay-ATA and specific methanogenic activity test-

SMA) and continuous experiments was as follows (in mil-

ligrams per liter of basal medium): NH4Cl (1200), MgSO4

(400), KCl (400), Na2S.9H2O (300), CaCl2.2H2O (50),

(NH4)2HPO4 (80), FeCl2. 4H2O (40), CoCl2.6H2O (10), Kl

(10), (NaPO3)6 (10), MnCl2.4H2O (0.5), NH4VO3 (0.5),

CuCl2.2H2O (0.5), ZnCl2 (0.5), AlCl3.6H2O (0.5),

NaMoO4.2H2O (0.5), H3BO3 (0.5), NiCl2.6H2O (0.5),

NaWO4.2H2O (0.5), Na2SeO3 (0.5), Cystein (10), NaHCO3

(6000). The medium was made up in demineralized water.

2.4. Analytical procedures

2.4.1. Routine analyses

Cumulative gas and methane gas were measured byliquid displacement method. Volatile fatty acids(VFA),

were measured by a two stage titration method developed

by Anderson and Yang [22]. Biomass was measured as total

suspended solids (TSS) by following Standard Methods

[23]. COD was measured by closed Reflux colorimetric

spectrophotometric method numbered 5220 D [23].

GC-MS Purge–Trap capillary column method (6210 D)

was used in the determination of TCE concentrations in

water [23]. HP 6890 GC system (Hewlett Packard, Avon-

dale, Pa) with micro-cell electron capture (ECD) detector

and a Column DB-624 (25 m*0.32 mm*0.52 m, Supelco

Inc. Bellefonte, PA) were used. Helium was the carrier gas

with a flow rate of 1 ml (min)1. Column temperature waskept at 35°C for 5 min then programmed at 6°C (min)1 to

160°C and at 20°C (min)1 to 220°C. Mass range was

maintained between 45 and 180 amu. Injector and detector

temperature were adjusted to 200 and 280°C, respectively.

2.4.2. Determination of granule diameter

Sludge samples taken from the reactor were prepared on

a slide for size measurement in a light microscope (Prior,

England). Granule diameter was measured by stage and

ocular micrometers. Approximately six granule diameters

were measured and the average granule size was calculated

for every sampling by determining how many units of theocular micrometer superimposed a known magnitude on the

stage micrometer on overall magnification 1500.

2.4.3. Anaerobic toxicity assay (ATA)

ATAs were performed at 35°C using serum bottles with

a capacity of 150 ml as described by Owen [24]. Serum

bottles were filled with 35 ml of Vanderbilt mineral medium

containing glucose. The liquid volume of the serum bottles

was 50 ml and these were seeded with 15 ml of anaerobic

granules which were enriched in yeast industry wastewater

and unacclimated to TCE. The glucose COD in the serum

bottles was stoichiometrically restored to 3500 mg l1 daily

Table 1

Operational parameters in UASB reactor throughout 230 days of

operation

Days COD

influent

(mg 11)

TCE

influent

(mg 11)

Upflow rate

(1 day1)

TSS

inside reactor

(g 11)

0–10 3500 2 1 810–20 3500 2 1 6.5

20–30 3500 2 1 6

30–40 3500 5 2 7

40–50 3500 5 2 9.5

50–60 3500 10 3 10

60–70 3500 10 3 10.5

70–80 3500 14 4 11

80–90 3500 14 4 12

90–100 3500 25 2 12.5

100–110 3500 14 5 13

110–120 3500 20 5 14

120–130 3500 14 2 16

130–140 3500 14 2 17

140–150 3500 25 5 18.5

150–160 3500 25 6 19160–170 3500 30 6 20

170–180 3500 30 5 21

180–190 3500 40 7 22.5

190–200 3500 30 5 23.2

200–210 3500 40 8 23.3

210–220 3500 40 8 23.5

220–230 3500 40 8 24


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based on the volume of the methane gas produced the

previous day. The glucose COD level of 3500 mg l1

ensured the presence of non limiting substrate conditions in

the serum bottles. The measured bicarbonate alkalinity in

the test bottles ranged between 3500 and 4000 mg l1 as

CaCO3. Gas production was measured daily using a glass

gas displacement device which was filled with a salt satu-rated 5% sulfuric acid and water solution colored with

methyl orange [23]. The total gas produced passed through

a 2 N NaOH solution to absorb the CO2, the remaining CH4

gas was collected and measured daily. Before the toxicity

experiments, serum bottles were operated until the variation

in daily gas production was less than 15% for at least 7

consecutive days. All the control samples and the serum

bottles were run in triplicate. After observing the steady-

state conditions, TCE concentrations of 5– 40 mg l1 were

administered to serum bottles as slug-doses from concen-

trated stock solutions of this chemical. This chemical was

reagent grade and was obtained from Merck Chemical Co.,Inc. The effect of TCE on glucose utilization was compared

to the control samples. Inhibition was defined as a decrease

in cumulative methane compared to the control sample.

2.4.4. Speci fic methanogenic activity (SMA)

The anaerobic granulated sludge samples taken from the

bottom sampling port of the UASB reactor were measured

during granulation for SMA. The SMA test was conducted

in 150 ml serum bottles at 35°C under anaerobic conditions.

Wastewater samples taken from feed during operations of

the UASB reactor containing glucose, minerals and suitable

TCE doses were added to the serum bottles in the same

manner as those used in the ATA.

2.5. Start-up

Anaerobic bacterial biomass was acclimated and granu-

lated with gradually increasing concentrations of TCE in the

influent in a step-wise manner with decreasing the HRT.

Unless there was a considerable transformation and a stable

level of COD and TCE concentrations in ef fluent, the or-

ganic loadings were not raised. To accelerate the acclima-

tion and shorten the granulation period, TCE doses, COD

loadings and upflow rates were gradually increased depend-

ing on COD and TCE removal ef ficiencies. The HRT in thisstudy was initially 2 days but was decreased to 0.25 days

after 230 days of operation. The corresponding loading rates

for COD and applied HRT are illustrated in Fig. 2 while

TCE loadings are given in Fig. 3 for 230 day of operation

period. The COD in influent was kept at constant concen-

trations of 3500 mg l1 by adding glucose to the feed as a

primary substrate. Synthetic wastewater comprising glucose

and increased concentrations of TCE plus balanced miner-

als, nutrients and alkalinity were added to the feed of the

reactor using a peristaltic pump. NaHCO3 was added con-

sistently at 5000 500 mg l1 to maintain proper pH and

buffering capacity. Addition of 0.01 g l1 of sodium thio-

glicollate ensured the anaerobic condition and reduced the

redox potential in the reactor.

3. Results and discussion

3.1. ATA tests

The purpose of the batch ATA experiments was to obtain

information on the toxicity of TCE in order to develop a

spike pattern strategy of this compound in the UASB oper-

ation. In this study, inhibition is defined as a reduction in the

activity, in terms of gas production, of a batch reactor

relative to its activity before the addition of the TCE. The

initial biomass concentration of anaerobic unacclimated

sludge and daily gas production were measured to be 2300mg l1 and 60 ml (day)1, respectively. The average cu-

mulative methane production was not significantly affected

by 5 mg l1 of TCE as compared to the control samples.

The 10 mg l1 of TCE spike demonstrated an inhibitory

effect compared to the control sample from day 7 to 10.

TCE concentrations of 10 mg l1 and above resulted in

inhibition (Fig. 4). The concentrations of TCE resulting in

50% inhibition of the rate of production (IC50) of methane

gas in a 2-day contact time period were found to be 18 mg

l1 by extrapolation of the results in Fig. 5. This result was

lower than the findings of Blum and Speece1 (IC50 22 mg

Fig. 2. COD loading rates and HRT changes in TCE fed UASB reactor.

Fig. 3. TCE loading rates in TCE fed UASB reactor.


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l1) [25], Chiu (IC50 28 mg l1) [14], and Freedman and

Gossett (IC50 20 mg l1) [10].

3.2. Performance of UASB experiments

In the beginning of start-up and acclimation during gran-

ulation, F/M ratios were continuously increased based on

the SMA measured, with the F/M to SMA ratio maintained

consistently at about 0.75, on average. The effect of F/M

ratio applied to SMA is depicted in Fig. 6. High substrate

ef ficiency has been proved appropriate to fast granulation

[26,27,28,29,30,31]. Herbert et al. reported that a high or-ganic loading may be applied when there is dif ficulty in

developing granulation [32]. Lettinga et al. suggested that

the if sludge granulation at the beginning stage of the

reactor were well developed, a very high organic loading

could be applied to the UASB reactor [33]. For this reason,

the COD and TCE loading rates were slowly increased

when the reactor removed over 75% of soluble COD and

TCE from the wastewater. The stepwise increase of the F/M

ratios was achieved through the decrease of hydraulic re-

tention time (HRT) coupled with the increase of COD and

TCE loading rates. See Figs. 2, 3 and 6.

The smooth operation was interrupted four times on days

75, 110, 160 and 180. The interruptions were due to the

deterioration of the reactor performance as reflected by the

lowering of SMA and the COD removal ef ficiency on each

of these occasions, the TCE loading rates being reduced

from 28, 50, 90, 40 mg (l.day)1 to 10, 25, 75, 75 mg(l.day)1, respectively. This corresponded to reduced COD

loading rates from 7, 8.75, 10.5, 12.25 g (l.day)1 to 3.5,

3.5, 8.75, 8.75 g (l.day)1 and was kept at that level until

COD removal ef ficiency and SMA were fully recovered

before the loading rates were increased once more. The

substrate removal was not apparent during the first month of

operation and thereafter until day 80. During this period, the

ef fluent COD and TCE removal ef ficiencies in the ef fluent

were between 40%–70% and 35%– 85% respectively. Figs.

7 and 8 show the COD and TCE concentrations in influent

and ef fluent samples.

As can be seen from Figs. 2, 3 and 8, until day 30, theef fluent TCE concentration was 1 mg l1 corresponding to

TCE conversion of around 50% at TCE loading rates rang-

ing between 1–5 mg TCE (l.day)1 and corresponding

HRTs varied between 1–2 days. After day 30, the ef fluent

TCE concentration started to decrease and went down to 1

mg l1 from 10 mg l1 between days 50 and 60 at a loading

rate of 15 mg TCE(l.day)1 a HRT of 0.66 days. The COD

removal ef ficiency was measured at between 80 and 95%. In

this period the ef fluent total volatile fatty acid (TVFA)

Fig. 4. Toxicity effects of TCE on anaerobic unacclimated sludge.

Fig. 5. IC50 value for TCE (IC50 18 mg/liter).

Fig. 6. F/M and SMA changes in TCE fed UASB.

Fig. 7. Influent and ef fluent COD concentrations in TCE fed UASB reactor.


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concentrations varied between 25 and 42 mg l1 (Fig. 9).

Although the influent characteristics were altered (increas-

ing in TCE, COD loading and decreasing in HRT) this

change in the ef fluent quality was attributed to acclimation

of anaerobic sludge to TCE. On day 80, after observing 30%ef fluent quality with an influent TCE loading of 28 mg TCE

(l.day)1, the influent TCE loading was decreased to 10 mg

(l.day)1 by day 90. On the same date, TVFA concentra-

tions in ef fluent were measured to be 670 mg l1, corre-

sponding to about 40% removal of COD. As can be seen

from Figs. 7 and 8, the ef fluent COD and TCE concentra-

tions were 2000 and 7 mg l1, respectively, on the same

days. The TCE and COD loadings increased any further

after observing a steady state ef fluent quality. On day 100,

the ef fluent TCE concentrations dropped to less than 2 mg

l1. The percentage transformation of TCE vas found to be

98%, while the percentage of COD removal varied between35 and 42%. This low COD removal was due to the forma-

tion and dif ficulty in biotransformation of intermediates of

glucose, namely acetic and propionic acids which were

measured as TVFA(890 mg l1).

On day 110, the TCE and COD loading rates were raised

to 50 mg (l.day)1 and 8.75 g COD (l.day)1, respectively,

after reaching 90% treatment ef ficiencies in the ef fluent

TCE and COD from earlier days. Under these conditions the

percentage COD and TCE removals decreased to 50 and

60%. The TVFA concentrations also increased to 850 mg

l1 on the same day. This can be explained by the accumu-

lation of both glucose intermediates such as propionic and

acetic acid and TCE intermediates even though these were

not measured. This indicated some dif ficulties in the bio-

transformation of both intermediates due to high COD load-

ing and TCE toxicity to biomass. Under these conditions theCOD and TCE loadings were dropped to 3.5 g (l.day)1 and

14 mg (l.day)1, respectively, on day 120. Loading rates

were gradually increased when COD and TCE removal

ef ficiencies reached 80% in the ef fluent. The TCE and COD

loading rates still increased up to 80 mg (l.day)1 9.5 g

(l.day)1, respectively, until day 160. On day 150, 89%

TCE removal ef ficiency was obtained for influent TCE

concentrations of 25 mg l1 at TCE loading rates of 75 mg

(l.day)1. This result is significantly better than those ob-

tained by Wu et al. [8] in an acetic acid fed UASB reactor.

They reported treatment ef ficiencies of about 69% and 90%

for influent TCE concentrations of 5 and 20 mg l

1

at TCEloading rates of 42 and 49 mg (l.day)1, respectively, [8].

When TCE loading rate of 90 mg (l.day)1 was main-

tained in the influent which corresponded to COD loading

of 10.5 g COD (l.day)1, the ef fluent TCE concentration

was raised to 17 mg l1 from 3 mg l1 on day 160. The

ef fluent TVFA concentrations were increased to 580 mg l1

after the introduction of 90 mg (l.day)1 of TCE loading.

On day 180, a HRT of 0.28 day was maintained in the

UASB reactor with the TCE and COD loading rate in the

feed kept at 140 mg (l.day)1 and 14 g (l.day)1, respec-

tively. The ef fluent COD and TCE concentrations were

increased to 1500 and 20 mg l1, respectively. Even though

the percentage of removal ef ficiency (50%) was not rela-tively good, the ef fluent TVFA concentrations were not

significantly increased (180 mg l1) This suggested that

there was still residual toxicity damage from TCE to the

anaerobic biomass from the previous TCE feed or new COD

loading shocks to microorganisms. Under these operating

conditions, the TCE loading rate was dropped to 75 mg

(l.day)1 by day 190. This change was reflected immedi-

ately in the ef fluent quality of the system. TVFA concen-

trations in the ef fluent dropped to 20 mg l1 and the per-

centage of TCE and COD removal increased to around 96

and 94%, respectively.

After day 190, even though the loadings were raised theef fluent TCE and COD concentrations started to decrease

and went down to 300 and 5 mg l1 between days 210 and

230, respectively. In this period the ef fluent TVFA concen-

trations were measured to be 30 mg l1. Because none of

the operating parameters or influent characteristics were not

altered during the 40 days, this change in the ef fluent quality

was attributed to the acclimation of granular sludge to TCE

concentrations of 40 mg l1 in influent at TCE, COD load-

ings of 160 mg (l.day)1 and 14 g (l.day)1, respectively.

Approximately 90% TCE and 94% COD mean removal

ef ficiencies were obtained. The results obtained in this study

were significantly better than the study of Christiansen et al.

Fig. 8. Influent and ef fluent TCE concentrations.

Fig. 9. TVFA concentrations in ef fluent.


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where an UASB reactor was fed with methanol at maximum

TCE loading rate of 35 mg (l.day)1 with 90% TCE bio-

transformation performance in ef fluent [34].

3.2.1. Relationships between SMA and F/M ratio during

granulation process

The SMA, bioparticle size and TSS concentrations in thereactor were used to characterize granulation. The SMA is

an indicator of the methanogenic activity of the biomass

forming granules. The SMA started increasing almost im-

mediately after start-up as an increasing F/M ratio was

applied. After just 1.5 months of operation, the SMA had

increased from 0.29 g COD (g TSS.day)1 initially to 0.52 g

COD (g TSS.day)1 as the F/M ratio was increased to

0.44 g COD (g TSS.day)1 from 0.2 g COD (g TSS.day)1.

This can be attributed to the high primary substrate suf fi-

ciency applied and biodegradability of the TCE with the

appropriate minerals and nutrients maintained, all of which

led to the rapid subsequent development of bacterial accli-

mation. The SMA sharply decreased 4 times during the

operation period as a resulting of high TCE and COD

loadings applied on days 75, 110, 160 and 180. Until day

90, increasing the F/M ratio had a pronounced effect on the

increase in SMA. After this day the SMA ratio was not

influenced by F/M ratio (Fig. 6). After day 110, the F/M

ratio in the reactor remained stable around 0.64 or slightly

decreased due to high TSS concentrations measured in the

reactor(14 –1 6 g l1), although TCE and COD loadings

were increased. In this case, increased substrate concentra-

tions might have had a greater impact than reduced F/M on

the increase in SMA.

The SMA increased from 0.44 g COD (g TSS.day)1 to

0.68 g COD (g TSS.day)1 70 days following start-up and

acclimation period with the change in the F/M ratio applied.

After day 70, the F/M ratio was increased from 0.27 gCOD

(g TSS.day)1 to 0.65 gCOD (g TSS.day)1. On day 80,

when the F/M ratio was raised to 0.65 gCOD (g TSS.day)1

the SMA decreased to 0.28 g COD (g TSS.day)1 from

earlier levels of 0.68 g COD (g TSS.day)1. This corre-

sponded to high TCE loading of 28 mg TCE (l.day)1

maintained in the influent on the same day. The ef fluent

COD and TCE concentrations also increased to 2000 and 7

mg l1 from earlier levels of 150 and 2 mg l1, respectively.

This indicates accumulation of some intermediates since the

VFA concentrations were measured to be 810 mg l1.

The SMA decreased on days 160 and 180 depending on

high TCE and COD loadings such as 90 mg (l.day)1,

10.5 g (l.day)1, and 140 mg (l.day)1 and 12.25 g

(l.day)1, respectively. After day 200, SMA remained sta-

ble as a maximum value of 1.32 g COD (g TSS.day)1. This

result is comparable with those obtained by Yan and Tay in

an UASB reactor treating only glucose, peptone and meat

extract without any chlorinated toxicant (1.72 g COD (g

TSS.day)1 [28].

3.2.2. Granule size

Divalent cations (Ca2, Fe2) are reported to bridge

negatively charged bacteria together faster for initiated

granulation [28]. Furthermore, S/Fe and Cl/Fe ratio influ-

ences the composition of granules and have been shown to

be beneficial for granule formation [3]. Supporting these

statements the Ca2, Fe2, S2 and Cl1 amounts were

adjusted as mentioned in the Materials and Methods section

for rapid granulation together with reducing HRTs and

increasing organic loadings.

The bioparticle size increased slowly during the initial

month of operation. At around 1–1.5 months, massive initial

granules were being formed in the lower part of the UASBreactor and began to grow during acclimation and pre-

granulation. Fig. 10 shows the variations of granule size and

TSS concentrations during granulation. The mean granule

diameter was measured to be 1.0 mm at the end of 1 months

of operation. The initiated granules continued to grow rap-

idly for 3 months until large granules were formed with

mean diameters of 1.5 mm. The granule size reached 1.8

mm after 100 days of operation period. After 220 days the

maximum granule diameter was measured to be 2.5 mm.

The bioparticles had grown to about 100 times their initial

original size of 0.02 mm.

3.2.3. TSS concentration in and in the ef fl uent of the

UASB reactor

The profiles of TSS concentrations in and in the ef fluent

of the reactor showed a gradual and progressive change

during start-up and differed significantly before and after

granulation. As mentioned above, an increase in TSS con-

centration during granulation was as a result of biomass

synthesis in the UASB reactor (Fig. 10). Initially, the bio-

mass synthesis was low due to the low F/M ratio applied.

On the other hand, the washout was higher due to the poor

settleability of seed sludge until day 40. The sludge con-

centration in the reactor slightly decreased with the mini-

Fig. 10. Granule diameter and TSS concentrations in TCE fed UASB

reactor.


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mum of 7 g l1 achieved after about 1 month of operation.

By day 20, with the progressive improvement of granule

initiation, the TSS concentration gradually increased due to

higher granulated biomass and lower washout from the

ef fluent. After 200 days of operation the biomass concen-

tration in the reactor reached 24 g l1. The TSS concentra-

tions in the ef fluent increased to 900 mg l1 during the

start-up of the pre-granulation period due to the disintegra-

tion of microbial aggregates and washout. On days 130 and

200, TSS concentrations in ef fluent were measured to be

400 and 250 mg l1, respectively (data were given else-

where). This can be explained by high upflow rates applied

and consequently high gas production even though the data

were not given. The gas produced inside in the granules gets

more dif ficult to release by increasing upflow rates. This

result causes rising gas bubbles and disintegration of the

granule and can split the large granules.

3.2.4. Pro files of TSS concentrations and granule size at

different reactor heights

The sludge stayed loose initially and expanded easily

like a blanket. As the granulation process proceeded, the

sludge bioparticles were progressively stratified with the

larger ones settling down in the lower part of the reaction

zone and the smaller ones expanded or suspended in the

upper part of the reaction zone. Fig. 11 illustrates the max-

imum granule diameter at different reactor heights on dif-

ferent days. The larger particles settled in the lower part of

the sludge bed. The smaller ones were also suspended dueto mixing and increasing of upflow rates and the release of

more gas bubbles during anaerobic treatment. The maxi-

mum sludge diameter at the bottom part of the reactor was

found to be 1 mm and 2.05 mm at the end of 30 and 180

days of operation periods, respectively. During the 230 days

of full granulation, the TSS concentrations were monitored

two times along the reactor height (Fig. 12). As can be seen

from this figure on day 30, the maximum TSS concentra-

tions were measured to be 5 and 10 g l1 at reactor heights

of 40 and 5 cm, respectively. On day 180, the measured TSS

concentrations varied between 18 and 20 g l1 at the same

heights.

3.3. Determination of kinetic coef ficients

The rate of substrate utilization is directly related to the

concentration of microorganisms in the granules and con-

centration of the growth limiting substrate surrounding thegranule microorganisms. This relationship can be described

by an expression similar to Monod’s equation which is

widely used to describe the relationship between bacterial

growth rate and the concentration of growth limiting sub-

strate:

Lr 1/X (dS/dt) kS/(Ks S) (1)

At quasi steady-state conditions, Lr may be expressed as:

Lr Q(So S)/VX (2)

Microbial substrate utilization rate is related to solids reten-

tion time(SRT) as follows:

1/ c YLr b (3)

By definition, c is the total quantity of active biomass in the

reactor divided by the total quantity of active biomass with-

drawn daily and was determined as follows;

c VX/[QwXw (Q Qw)Xe] (4)

The term Qw Xw only makes sense if there is a waste

sludge stream, and there is no mention here of sludge

wastage. Therefore c can be expressed as follows,

c VX/(QeXe) (5)

The kinetic constant of Y, b, k and Ks were determined

by using the experimental data obtained during granulation

on days 40, 50, 70, 100, 140, 150, 170 and 200 when it had

reached a steady-state condition. The kinetic coef ficients

mentioned above were calculated based on the COD since

the glucose was the primary substrate and energy source

used by the anaerobic microorganisms for growth. The y

intercept and slope of the line from the graph plotted be-

tween Lr and 1/ c (1/SRT) gave the values of b(decay

coef ficient) and Y(growth yield coef ficient), respectively. Y

was determined to be 0.17 mg TSS (mg COD)1 while b

was found to be 0.015 day1 (1/SRT 0.2523* Lr

Fig. 11. Granule size through reactor height.Fig. 12. TSS concentration through reactor height.


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0.0163, R2 0.78). From the plot between 1/S versus

1/literr, k (maximum specific substrate utilization rate) and

Ks (half velocity coef ficient) were determined to be 2.38 mg

COD (mg TSS.day1) and 108 mg COD l1 (1/literr

5.571*1/S 0.3161, R2 0.98). The maximum COD

utilization rate (k), b and Y values determined in this study

were compared with similar coef ficients as reported byother researchers. k is significantly higher than the findings

of Guiot et al. [35] Unal [36] and slightly lower than Sponza

[37,38] in continuous UASB reactors containing only glu-

cose as a carbon source which did not contain chlorinated

toxic organic substrate (1.1, 0.91, 3.11 and 2.99 mgCOD

(mg TSS.day)1, respectively). In some other studies per-

formed by de Bruin et al., [11] and Horber et al. [39], the

TCE dechlorination rate (k) of 0.4 mgTCE (mg TSS.day)1

and 0.6 mgTCE (mg TSS.day)1, respectively were ob-

tained during continuous operation of UASB reactor fed

with sucrose, format and acetate. These values are about

60% of the maximum substrate utilization rate observed inour study. The b value found in this study appears to be on

the lower side as compared to suspended type sludge, while

the Y value seems to be significantly higher even if TCE

was treated with glucose in the case of granular sludge. This

can be attributed to high COD utilization by dense biomass

in granules. Lower Ks values indicate the af finity of gran-

ular sludge to substrate and shows that the TCE and glucose

were not accumulated in the reactor.

4. Conclusions

TCE was inhibitory to unacclimated anaerobic bacteria

in the batch reactor at concentrations as low as 5 and 10

mg/liter. TCE in synthetic wastewater was effectively de-

graded in continuous operation of a laboratory scale UASB

reactor at loading rates ranging between 28 and 75 mg TCE

(l.day)1 during TCE degrading granule formation from

unacclimated anaerobic culture. Over 87% TCE was re-

moved at 37°C and an HRT of 0.28 day. This corresponds

to 92% of COD removal ef ficiency at COD loading rates

varying between 7 and 10.5 g (l.day)1. Thereafter, at TCE

loading rates ranging between 80 and 160 mgTCE (l.day)1

the TCE removal ef ficiency decreased to 80%.When the loading rates were lowered; the COD, and

TCE removal ef ficiencies recovered gradually. At high

loading rates the COD, TC and TCE removal ef ficiencies

dropped to 50% on average but they readily recovered to

88% in about 1 or 2 weeks by reducing the loading rates and

maintaining the F/M ratio at 75% of SMA during start-up

and maturation period. Under these conditions, biomass in

the reactor took a short time to become adapted to the new

loading rate. The granulation process was examined through

TCE removal in UASB reactor by measurements of granu-

lar size and SMA. TCE degrading granules were developed

having a maximum diameter of 2.5 mm and SMA of 1.32 g

COD (g TSS.day)1 at the end of acclimation and granula-

tion.

The results of this study showed that TCE degrading

granules can be ef ficiently developed by appropriate HRT,

TCE, COD loading rates and F/M ratios even at the begin-

ning of granulation if the start-up period is well operated.

The development of granules was influenced by the waste-water itself, HRT, organic loadings and the F/M ratio ap-

plied, particularly for toxic chemicals containing wastewa-

ters.

Acknowledgments

The author would like to express her thanks to

TUBITAK (The Turkish Scientific and Technical Research

Council) for financial support under Grant YDABCAG-485

and to Dokuz Eylul University Support Foundation, Grant

no 0908. 97.02.03.

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