Process Biochemistry 41 (2006) 22582263

www.elsevier.com/locate/procbio

Determination of optimum operating conditions of an acidification

reactor treating a chemical synthesis-based pharmaceutical wastewater
Yalcin Askin Oktem a, Orhan Ince b, Tom Donnelly c, Paul Sallis c, Bahar Kasapgil Ince d,*
b

a
Istanbul University, Department of Environmental Engineering, 34320 Avcilar, Istanbul, Turkey
Istanbul Technical University, Department of Environmental Engineering, 34469 Maslak, Istanbul, Turkey
c
University of Newcastle Upon Tyne, School of Civil Engineering & Geosciences, NE1 7RU, UK
d
Bogazici University, Institute of Environmental Science, 34342 Bebek, Istanbul, Turkey

Received 29 November 2005; received in revised form 28 April 2006; accepted 10 May 2006

Abstract
In this study, the optimum operating conditions of a lab-scale completely stirred acidogenic reactor treating a chemical synthesis-based
pharmaceutical wastewater were determined. Individual effects of hydraulic retention time (HRT), organic loading rate (OLR) and pH on the
degree of acidification were investigated at a mesophilic temperature (35  1 8C), for HRT in the range 824 h, OLR up to 14 kg COD/m3 d and pH
in the range of 5.06.3. A maximum acidification level of 44% and COD removal efficiency of 13%, were obtained at an OLR of 13 kg COD/m3 d
at pH of 5.5  0.1 with an HRT of 12 h. Under these conditions, acetic, propionic and n-butyric acids were found to be predominant volatile fatty
acids in the acidogenic reactor.
# 2006 Elsevier Ltd. All rights reserved.
Keywords: Anaerobic digestion; Degree of acidification; Chemical synthesis-based pharmaceutical wastewater; Optimum operating conditions; HRT; OLR

1. Introduction
Anaerobic processes have become a viable option for the
treatment of medium-high strength industrial wastewaters. The
pharmaceutical industry is a major source of industrial wastewaters and these comprise substrates which are difficult to treat in
biological treatment systems. Wastewaters from pharmaceutical
plants are characterized by a high COD and variable concentrations of salts [1,2]. Based on the production processes, the
pharmaceutical industry can be divided into five categories,
namely fermentation, natural product extraction, chemical
synthesis, formulation and research and development. Among
these processes, the most important one is chemical synthesis, in
which solvent-intensive processes are involved. The processes
generate wastewaters containing a variety of organic and
inorganic constituents including priority pollutants such as
methylene chloride, toluene, isopropyl alcohol, chloroform,
chlorobenzene, chloromethane, cyanide, phenol and benzene [3].
A substantial part of the COD may be made up of relatively
biodegradable solvents, but may also contain specific com-

* Corresponding author. Tel.: +90 212 359 70 16; fax: +90 212 257 50 33.
E-mail address: bahar.ince@boun.edu.tr (B.K. Ince).
1359-5113/$ see front matter # 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2006.05.016

pounds that are difficult to biodegrade and/or inhibit biological

treatment systems [4]. Physical and/or chemical processes
without a biological treatment are not generally considered to be
suitable for treating pharmaceutical wastewaters because of their
low efficiency for dissolved COD removal and high consumption
of chemicals [5]. The high COD content of pharmaceutical
wastewaters makes them particularly amenable to treatment
processes based on anaerobic technology. However, relatively
few studies on the anaerobic treatment of chemical synthesisbased pharmaceutical effluents have been reported in literature.
Although, the specific operational conditions for acetogenic/
methanogenic phases have been extensively studied, generally
relevant literature on the acidogenic phase is still scarce [6].
The acid-phase digestion products, such as volatile fatty acids
(VFA), CO2 and H2 can be greatly influenced by operational
and design parameters such as hydraulic retention time (HRT),
solid retention time (SRT), environmental factors such as pH,
temperature, oxidationreduction potential (ORP), as well as
reactor configuration, wastewater characteristics and availability of trace minerals [7]. To increase the efficiency of twophase digestion it is particularly important to optimize the
conditions within the acid-phase digester since hydrolysis and
liquefaction is often considered to be the rate limiting
fermentation step [8].

Y.A. Oktem et al. / Process Biochemistry 41 (2006) 22582263

Studies have been conducted into the effect of temperature

on acidification of a synthetic substrate, mainly glucose, over
the range 2060 8C, with an HRT of 810 h and a pH 5.8 [9] and
showed that the optimum temperature for maximum acidification was at 37 8C for mesophilic and 52 8C for thermophilic
processes. Although a slightly higher degree of acidification
occurred in the thermophilic ranges, it was pointed out that
mesophilic ranges are more robust due to the advantages of
having greater stability. Acetic, propionic and n-butyric acids
were found to be the main VFA. Similar studies investigated the
effects of temperature and pH on the acidification of dairy
wastewater at a constant HRT (12 h) [8]. It was reported that the
optimum operating conditions were found within the temperature range of 3436 8C and pH in a range of 5.75.8. It was also
stated that acetic acid was the predominant VFA. In a study
investigating effect of HRT on mesophilic acid-phase digestion
of a dairy wastewater [10], it was observed that the degree of
acidification increased from 28 to 54% with increases in HRT
from 4 to 12 h; however, further increases in HRT from 16 to
24 h only increased acidification slightly from 56 to 59%. In
contrast, other studies showed the degree of acidification
increased from 6 to 56% as the HRT was decreased from 24 to
12 h [11]. Effect of pH on acidification of various wastewaters
has been investigated [12,13] and the optimum pH range for

2259

acidification of gelatin-rich wastewater was found to be 72% at

pH 6.5 [14].
However, despite these studies on the acidification of
industrial wastewaters, there is still lack of specific information
on acidification of chemical synthesis-based pharmaceutical
wastewaters. Therefore, the aim of this study was to determine
the optimum design and operating conditions of a laboratoryscale completely mixed acidogenic reactor treating a chemical
synthesis-based pharmaceutical wastewater.
2. Materials and methods
2.1. Acidogenic reactor
The acidogenic reactor was constructed from acrylic with an active volume
of 5 l (Fig. 1). The reactor included a sampling port, a gas collection unit, an
influent and effluent line and was operated continuously over a period of
approximately 280 days. Mixing was provided by a magnetic stirrer. The reactor
was placed in a water bath where the temperature was maintained between
35  1 8C using temperature controller. The pH value was controlled by
automatic addition of 1 N NaOH and/or 1 N HCL.

2.2. Wastewater characteristics

The wastewater was taken from a local chemical synthesis-based pharmaceutical manufacturing plant. The main production at the site was bacampicil-

Fig. 1. Schematic diagram of the experimental system.

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Y.A. Oktem et al. / Process Biochemistry 41 (2006) 22582263

Table 1
Characteristics of the chemical synthesis-based pharmaceutical wastewater

Table 3
Conversion factors for each VFA

Parameter

Concentration (mg/l)

VFA

Carbon (%)

COD equivalent

COD
TKN
PO4-P
Volatile suspended solids/suspended solids (VSS/SS)
Alkalinity (as CaCO3)
pH a

4000060000
800900
36
0.60.7
9001000
78

Acetic
Propionic
Butyric
Valeric
Caproic

40.00
48.64
54.53
58.80
62.04

1.066
1.512
1.816
2.036
2.204

Unitless.

line and sultampicilline toshylate. The major chemical contaminants arising

from production activities were n-butyl acetate, dimethyl formamide, isopropyl
alcohol, ethyl acetate and methylene chloride. Although the site had a solvent
recovery system in place, the final combined effluent from the manufacturing
process still contained a significant quantity of solvents due to the malfunction
of the solvent extraction unit. The general characteristics of the wastewater are
given in Table 1.

This stepped feeding strategy was used in order to allow microorganisms to

acclimatize gradually to the pharmaceutical wastewater from the glucose feed
which was totally biodegradable substrate under anaerobic conditions [16,17].
OLR was then increased to 13 kg COD/m3 d as 100% pharmaceutical wastewater to determine performance at high organic loading to the acidogenic
reactor. In addition, the optimum operating conditions for HRT and pH were
also determined in Stage 3 (Table 2).

2.3. Seed sludge

2.6. Calculation for degree of acidification

The acidogenic reactor was inoculated with sludge taken from a full-scale
up-flow anaerobic sludge blanket (UASB) reactor treating an alcohol industry
effluent. Due to the granular characteristics of the sludge, filtration could not be
accomplished, therefore, total solids (TS) and total volatile solids (TVS)
parameters were used; TS concentration of the seed sludge was approximately
105 g/l of which 96% of TVS and after inoculation the reactor had a sludge
concentration of 10 g/l TS.

As VFA concentration is the most widely used parameter to assess acidification [18], the combined COD equivalent of each individual VFAwas used to
express the total COD of VFA in the effluent (COD of VFAeff) and this was
compared to the soluble COD of the influent (Soluble CODinf) and the degree of
acidification calculated by Eq. (1). The COD equivalents and percentage of
organic carbon for individual VFA are given in Table 3.
Degree of acidification%

2.4. Analytical methods

During the operation of the acidogenic reactor, routine analyses such as
COD, TS and TVS, pH, total Kjeldahl nitrogen (TKN), ammoniacal nitrogen
(NH3-N), orthophosphate phosphorus (PO4-P) and alkalinity were carried out in
accordance with standard methods [15]. VFA were measured using a HP Model
5890 Series II Gas Chromatograph (GC) (HP FFAP Column,
10 m  530 mm  1 mm) with nitrogen as a carrier gas.

COD of VFA mg=leff

 100
soluble COD mg=linf

(1)

3. Results and discussion

3.1. Acclimation to pharmaceutical wastewater
During the initial study (Stage 1), the acidogenic reactor was
gradually loaded with increasing levels of glucose in the feed,
OLR of 16 kg COD/m3 d, at a HRT of 24 h and pH in a range
of 5.5  0.1. The degree of acidification achieved was
approximately 47% at OLR of 6 kg COD/m3 d (Stage 1). It
has been recommended that pre-acidification should be partial
(2040%) in achieving higher organic loading rates [19].
Similarly, other researchers have stated that degree of
acidification should be in a range of 4050% to maintain
stability of process [20]. Following the initial study (day 41),
the glucose containing feed was replaced with increasing
proportion of the pharmaceutical wastewater, until at the end of

2.5. Reactor operation

After inoculation the reactor was flushed with nitrogen gas for a period of
15 min in order to maintain anaerobic conditions inside the acidogenic reactor.
Temperature inside the acidogenic reactor was increased gradually over 24 h
from room temperature to the operational temperature of 35  1 8C which was
maintained throughout the study. The acidogenic reactor was initially fed with
glucose solution of 1000 mg COD/l. During the initial study employing glucose
feeding, a HRT of 24 h was applied and the OLR was gradually increased to
6 kg COD/m3 d. Then, the glucose containing feed was replaced with increasing
proportion of the pharmaceutical wastewater from 10 (w/v), 30 (w/v), 70 (w/v),
to 100% (w/v) at above conditions. Overall feeding regime is given in Table 2.

Table 2
Summary of the operating schedule with feeding strategy applied to the acidogenic reactor
Stage

Operation

Initial studies

Steady-state

4153

Steady-state

5379

Steady-state
Steady-state

Time (d)
141

Feeding strategy
Glucose

OLR (kg COD/m3 d)

HRT(h)

Period

16

2412

Acidification with glucose

12

12

7993

10% wastewater
90% glucose
30% wastewater
70% glucose
100% wastewater

12

98285

100% wastewater

614

812

Acclimation to the wastewater

Determination of maximum
organic loading and operating conditions

Y.A. Oktem et al. / Process Biochemistry 41 (2006) 22582263

2261

Fig. 2. Effect of organic loading rate (OLR) on the degree of acidification (*) and COD removal efficiency (*) during different stages of the investigation.

Fig. 3. Composition and concentration of VFA produced within the acidogenic reactor during initial study with glucose (Stage 1), acclimation to the pharmaceutical
), acetate (*), butyrate (^), propionate ().
wastewater (Stage 2) and determination of maximum organic loading rate (Stage 3). Total volatile fatty acids (

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Y.A. Oktem et al. / Process Biochemistry 41 (2006) 22582263

Table 4
Summary of related studies
Wastewater

Reactor
type

Dairy
Dairy
Gelatinaceous
Gelatinaceous -rich
Synthetic
This study

Lab-scale
Lab-scale
Lab-scale
Lab-scale
Lab-scale
Lab-scale

CSTR
UAR
UAR
UAR
UAR
CSTR

pH
range

HRT
(h)

Acidification
degree (%)

Loading
(kg COD/m3 d)

Temperature
(8C)

Reference

No control
5.5
5.5
47
5.5
5.5

0.5
0.51
0.161
0.5

56
5459
8490
3267
5927
44

3.1
4
230
4
424
13

35  1
37
37
37
3755
35  1

[11]
[10]
[30]
[14]
[31]
[32]

0.5

UAR: upflow anaerobic reactor; CSTR: continuously mixed stirred tank reactor.

the acclimation period (day 93) the reactor received 100%

pharmaceutical wastewater and achieved a 37% degree of
acidification in Stage 2 (Fig. 2).
3.2. Determination of maximum organic loading rate and
optimum operating conditions
In order to determine the maximum organic loading
applicable to the acidogenic reactor, OLR was gradually
increased during Stage 3 from 7 to 14 kg COD/m3 d and a 39%
degree of acidification and a 17% COD removal efficiency were
achieved at an OLR of 7 kg COD/m3 d. Changes in COD
removal and degree of acidification throughout this part of the
study were observed (Fig. 2). VFA in the acidogenic reactor
mainly comprised acetic (41%), propionic (20%) and n-butyric
(29%) acids. Every step increase in OLR was followed by an
increase in VFA production until OLR of 13 kg COD/m3 d at
which point 43% acidification degree was obtained and VFA
composition did not change (48%) acetic, (18%) n-butyric and
(13%) propionic. Further increase in OLR to 14 kg COD/m3 d
(day 136) resulted in a dramatic decrease in the degree of
acidification to 19%. Moreover, total VFA concentration
decreased from 3410 to 1370 mg/l as acetate, which can be
explained by the stress imposed on the acidogenic bacteria.
Therefore, OLR was reduced to 13 kg COD/m3 d and the
acidogenic reactor recovered within 15 days. Fig. 3 shows the
composition and concentration of VFA produced within the
acidogenic reactor to determine maximum OLR.
After the determination of the most applicable OLR, the
effects of HRT and pH on the acid-phase reactions were
investigated by varying these parameters (Fig. 2). Optimum
HRT was investigated by gradually decreasing from 24 to 18,
12, then 10 and finally 8 h. The degree of acidification was
determined to be approximately 40, 43, 44, 35 and 30% at HRT
of 24, 18, 12, 10 and 8 h, respectively. Therefore, an HRT of
12 h was considered to be optimum for acidification of the
pharmaceutical wastewater under pre-determined conditions of
OLR of 13 kg COD/m3 d, pH of 5.5  0.1 and temperature of
35  1 8C and acetic, propionic and butyric acids were still the
main VFA products under these conditions. Previously
published studies do not agree on the role of HRT in
determining the degree of acidification, one group concluding
that product distribution (i.e. VFA composition) is not
influenced to any substantial degree by HRT [21,22], whereas,
the other group maintains that VFA composition in acidification

of biological sludge, primary sludge and dairy wastewater was

strongly affected by HRT [7,23,24]. A summary of related
studies is presented in Table 4. Results from this study indicated
that the pharmaceutical wastewater was acidified at HRT as low
as 8 h and the VFA composition was not influenced to any
substantial degree by HRT (Fig. 4).
During the final part of the Stage 3 investigation, the effect of
pH on the degree of acidification was determined by varying pH
under the above pre-determined conditions (OLR 13 kg COD/
m3 d and HRT 13 h). The degree of acidification increased from
30% at pH 5.0 to 44% at pH 5.5, but reduced to 35% at pH 6.3,
therefore, a pH of 5.5  0.1 was considered to be optimum for
acidification of the pharmaceutical wastewater. The predominant VFA products were acetic acid, butyric acids and
propionic acids at pH 5.05.5, whereas acetic and propionic
acids became the main products at pH 6.3. The VFA
distribution was therefore sensitive to pH changes during the
acidification of the pharmaceutical wastewater, a result that
agrees with the results of other researchers who also found that
the VFA composition was dependent on pH [21,25,26].
In another study, carried out to determine the influence of
dairy wastewater strength (230 g COD/l) on acidification at pH
5.5 and temperature of 37 8C [7], it was reported that the degree
of acidification decreased from 57 to 33% with increases in
wastewater COD from 2 to 20 g/l and further increases in COD to
30 g/l decreased degree of acidification to 29%. Acetic, butyric
and propionic acids were main VFA products. It was also stated
that COD had a considerable effect on the distribution of VFA.
However, some researchers have claimed that acidification

Fig. 4. Effect of HRT on the degree of acidification. (OLR of 13 kg COD/m3 d,

pH of 5.5  0.1 and temperature of 35  1 8C).

Y.A. Oktem et al. / Process Biochemistry 41 (2006) 22582263

products were dependent on pH [26,27] and whereas other

researchers stated have that HRT was more effective than pH in
determining the acidification products [25,28,29]. However, it is
difficult to make direct comparisons between these different
investigations since they all used different wastewaters, reactor
configurations and operating conditions.
4. Conclusions
Based on above results, an HRT of 12 h, pH of 5.5  0.1 and
OLR of 13 kg COD/m3 d were found to be the best combination
of conditions, within the ranges studied, for efficient acidification
of a chemical synthesis-based pharmaceutical wastewater. Under
these conditions, a maximum acidogenic conversion of 44% of
the influent soluble COD was achieved. Throughout the
investigation, COD removal was in a range of 1025% and
the main VFA were found to be acetic, propionic and n-butyric
acids. Acetic acid was produced in higher concentrations than
other VFA in the acidogenic reactor at all HRT and pH ranges
investigated. HRT had only a minor effect on the composition of
VFA produced, however, VFA distribution was found to be
highly sensitive to the operating pH within the reactor.
Acknowledgements
O. Ince would like to acknowledge the project coded 1536 of
the Institute of Science and Technology of Istanbul Technical
University. Author Y. Oktem also acknowledges Institute of
Environmental Sciences, Bogazici University and Engineering
Faculty of Istanbul University.
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