Acidogenic biotreatment of wastewater containing 2 nitroaniline and copper

ACIDOGENIC BIOTREATMENT OF WASTEWATER
CONTAINING 2-NITROANILINE AND COPPER









KRISTHOMBU B. S. N. JINADASA










NATIONAL UNIVERSITY OF SINGAPORE
2003
ACIDOGENIC BIOTREATMENT OF WASTEWATER
CONTAINING 2-NITROANILINE AND COPPER

KRISTHOMBU B. S. N. JINADASA
(B.Sc.Eng. (Hons.) University of Peradeniya, Sri Lanka)







A THESIS SUBMITTED
FOR THE DEGREE OF MASTER OF ENGINEERING
DEPARTMENT OF CIVIL ENGINEERING
NATIONAL UNIVERSITY OF SINGAPORE
2003


ACKNOWLEDGEMENTS



I am deeply grateful to all the support and encouragement given to me
during the period of my research work at National University of Singapore
(NUS) by my supervisors, Prof. W.J. Ng and Assoc. Prof. M.A. Aziz.
Their guidance was crucial to the success of my research work at NUS.

I wish to thank all the staff of the Environmental Engineering Laboratory,
Department of Civil Engineering. Their friendly and helpful
attitudes made my research experience at NUS a very happy and
memorable one.

I would also like to express my appreciation to the National University of
Singapore for awarding a research scholarship which enabled me to
pursue a higher degree.










TABLE OF CONTENTS
Page

TABLE OF CONTENTS
ii


SUMMARY
vi

LIST OF FIGURES
viii


LIST OF TABLES
x


LIST OF PLATES
xii


LIST OF ABBREVIATIONS
xiii


CHAPTER ONE INTRODUCTION


1.1 Background

1
1.2 Objectives of the Study

3

1.3 Scope of the Study 4
1.4 Experimental Protocol 5

CHAPTER TWO LITERATURE REVIEW


2.1 Fundamentals of Anaerobic Wastewater Treatment

6
2.1.1 Phases of anaerobic biotreatment process

7
2.1.2 Substrate flow in anaerobic system

9
2.2 Inhibitory Substances

10
2.3 Persistent Organic Compounds

12
2.4 Nitroaromatic Compounds

12
2.4.1 Inhibitory characteristics of nitroaromatic compounds

13
2.5 2-Nitroaniline

14
2.6 Biotreatment of Nitroaromatic Compounds under Anaerobic Conditions

16

2.6.1 Feasibility of acidogenic pretreatment of wastewater


19

ii
2.6.2 Significance of acidogenic conditions in the reduction of
nitroaromatic compounds

20
2.6.3 Effect of other substitutes in aromatic ring in reduction of
nitroaromatic compounds

22
2.7 Heavy Metals

23
2.7.1 Sources

23
2.7.2 Copper

24
2.8 Effects of Heavy Metals on Microorganisms

26
2.9 Effect of Heavy Metals on Acidogenic Biotreatment

28
2.9.1 Activity factor

2.10 Need for Research

2.10.1 Biotransformation studies

28

29

30

CHAPTER THREE MATERIALS AND METHODS


3.1 Reactor Description

31
3.1.1 Reactor operation

32
3.1.2 Acidogenic reactor feed 33

3.2 Anaerobic Toxicity Assay (ATA) Test

35
3.2.1 Experimental setup

35
3.2.2 Operating protocol

36
3.2.3 Experimental procedure

37
3.3 Preparation and Analysis of Samples 38

3.3.1 Identification of metabolites

40
3.3.2 Quantification of metabolites 40

3.4 Track Runs with Acidogenic Sequencing Batch Reactor


41
3.5 ISO Inhibition Test (Aerobic)

42
3.5.1 Experimental setup 42

iii

3.5.2 Test procedure

43
3.5.3 Computation of inhibition

45
3.5.4 Determination of EC
50

value

45
3.5.5 Influent and effluent inhibitions 45

3.6 Microbiological Observations

46


CHAPTER FOUR RESULTS AND DISCUSSIONS


4.1 Acidogenic Biotreatment of Wastewater Containing 2-Nitroaniline

48
4.1.1 Biodegradation of 2-Nitroaniline

4.1.2 Biodegrdation pathway of 2-Nitroaniline

48

49
4.1.3 Effect of 2-Nitroaniline on biogas production

58
4.1.4 Effect of 2-Nitroaniline on Volatile Fatty Acids (VFAs)
production
62

4.1.5 Comparison of influent and effluent inhibitions


65
4.1.6 Anaerobic Sequencing Batch Reactor (anSBR) Track Run with
2-Nitroaniline

66

4.2 Acidogenic Biotreatment of Wastewater Containing 2-Nitroaniline and
copper

69
4.2.1 Effect of copper on Volatile Fatty Acids (VFAs) production

4.2.2 Effect of copper on biogas production

69

72
4.2.3 Effect of copper on biodegradation of 2-Nitroaniline

75
4.2.4 Influent and effluent inhibitions 76

4.2.5 anSBR Track Run with 2-Nitroaniline and copper


77
4.3 Determination of EC

50
Values of 2-Nitroaniline and copper in
Aerobic System

80
4.4 SEM Observations of Microorganisms

81

iv


CHAPTER FIVE SUMMARY, CONCLUSIONS AND
RECOMMENDATIONS


5.1 Summary

84
5.2 Conclusions 85

5.3 Recommendations


REFERENCES
86


87

v

SUMMARY


Laboratory investigations were carried out to evaluate the feasibility of acidogenic
pretreatment of wastewater containing 2-nitroaniline and copper. The experiments
were conducted using the Anaerobic Toxicity Assay (ATA) Test, Anaerobic
Sequencing Batch Reactor (anSBR), and Aerobic Inhibition Test.

From the experimental results, it was found that acidogenic biotreatment process could
remove 2-nitroaniline effectively. Removal efficiency was around 95% when the
concentration of 2-nitroaniline was 12.5 mg/L. This suggested that the acidogenic
phase could remove 2-nitroaniline effectively at least in the short term. At 12.5 mg/L
2-nitroaniline, biogas production was found to be higher than that of the control. This
indicated the possibility of stimulatory effects by 2-nitroaniline on the acidogenic
process at low concentrations.

The inhibition potentials of the influent and effluent at varying feed concentrations of
2-nitraoniline were determined by the oxygen consumption rate measured in
accordance with International Standards Organisation method ISO 8192-1986-E. There
was a significant reduction in inhibition of the effluent compared to that of the influent.
This reduction in inhibition is presumed due to the conversion of the potentially

inhibitory organics in the wastewater into less inhibitory or non-inhibitory by-
products.

Metabolites were identified as 2-methylbenzimidazole and o-phenylenediamine using
gas chromatography mass spectrometry and quantified by high performance liquid

vi
chromatography. Around 80% of the 2-nitroaniline was recovered as metabolites and
other end products. 2-nitroaniline was transformed to its corresponding amine and then
further transformed to 2-methylbenzimidazole under acidogenic conditions. The
identification of o-phenylenediamine and 2-methylbenzimidazole was confirmed by
comparing their mass spectral fragmentation patterns against standards.


The effect of copper on the acidogenic process was studied using the Anaerobic
Toxicity Assay (ATA) Test and an Anaerobic Sequencing Batch Reactor (anSBR).
Copper inhibition on bacterial activity was estimated by considering volatile fatty
acids (VFAs) production. Propanoic acid was most severely affected followed by
pentanoic acid, butanoic acid and ethanoic acid with increasing concentrations of
copper. The removal of 2-nitroaniline was observed to be around 90 % and 78 % when
the copper concentrations were 0 mg/L and 25 mg/L respectively with 2-nitroaniline
acclimated biomass in the anSBR track runs. The acidogenic process could, therefore,
be an attractive alternative for upgrading present aerobic treatment processes which are
not effective at removing nitroaromatic compounds such as nitrobenzenes,
nitroanilines and nitrophenols from industrial wastewaters. These compounds could be
in a wastewater containing heavy metals such as copper.

vii
LIST OF FIGURES

Title

Page
Figure 1.1 Schematic diagram of experimental protocol

5
Figure 2.1 Three phases of the anaerobic biotreatment process

8
Figure 2.2 Flow chart illustrating the mechanism of anaerobic
biotreatment system

9
Figure 2.3 Typical effect of an inhibitory compound on biotreatment
processes

11

Figure 2.4 Reduction pathway of nitrobenzene to aniline under anaerobic
conditions

16
Figure 2.5 Significant pathways in acidogenic conditions relate to the
reduction of nitroaromatic compounds

21
Figure 3.1 Schematic diagram showing the experimental set-up of
laboratory anSBR system

31
Figure 3.2 Procedure for preparation and analysis of samples

38
Figure 3.3 Schematic diagram of ISO inhibition test apparatus assembly

43
Figure 4.1 Removal efficiency of 2-nitroaniline at concentrations of
12.5~100 mg/L

49
Figure 4.2 Mass spectra identification of o-phenylenediamine
(Reactor sample)

50
Figure 4.3 Mass spectra identification of 2-methylbenzimidazole
(Reactor sample)

51

Figure 4.4 Mass spectra identification of o-phenylenediamine
(Using standard chemicals)

52
Figure 4.5 Mass spectra identification of 2-methylbenzimidazole
(Using standard chemicals)

53
Figure 4.6 Degradation of 2-nitroaniline and possible metabolic products
in acidogenic biotreatment

54
Figure 4.7 Possible biotransformation pathway for 2-nitroaniline to
o-phenylenediamine



54

viii
Figure 4.8 Possible transformation pathway for 2-nitroaniline to
2-methylbenzimidazole in presence of aliphatic carboxylic
acids

55
Figure 4.9 Quantification of metabolic products

57
Figure 4.10 Variation of cumulative total gas production with varying
concentrations of 2-nitroaniline

59
Figure 4.11 Effect of increasing concentration of 2-nitroaniline on carbon
dioxide production

60
Figure 4.12 Effect of increasing concentration of 2-nitroaniline on methane
production

60
Figure 4.13 Variation of biogas composition with time (control)

61
Figure 4.14 VFA production at various 2-nitroaniline concentrations

63
Figure 4.15 Relationship between VFA production activity and
2-nitroaniline concentration

64
Figure 4.16 Biodegradation of 2-nitroaniline during REACT in anSBR
cycle
67
Figure 4.17 Variation of MLVSS and MLSS during REACT in anSBR
cycle

67
Figure 4.18 VFA production with varying copper concentrations

70

Figure 4.19

Relationship between VFA production activity and copper
concentration

71
Figure 4.20 Effects of copper on cumulative total gas production

73
Figure 4.21 Relative gasification activities with time

74
Figure 4.22 Effect of increasing concentration of copper on carbon dioxide
production

75
Figure 4.23 Biodegradation of 2-nitroaniline with varying copper
concentrations
76
Figure 4.24

Biodegradation of 2-nitroaniline during REACT in anSBR
cycle

78
Figure 4.25 Variation of MLVSS and MLSS during
REACT in anSBR cycle

79
Figure 4.26 Inhibition at different concentrations of 2-nitroaniline and

copper
81


ix
LIST OF TABLES

Title Page

Table 2.1 Components in a carbohydrate-fed anaerobic biotreatment
system

10
Table 2.2 Molecular weight and IC
50
values of some aromatics to
methanogenic activity

14
Table 2.3 Physical and chemical properties of 2-nitroaniline

15
Table 2.4 Typical metals concentration in raw sewage

23
Table 2.5 Limitation of metal concentration in the effluent for general
metal industry

24
Table 3.1 Reactor operating parameters and sequences

33
Table 3.2 Composition of synthetic wastewater

34
Table 3.3 Constituents of inorganic nutrients supplement

34
Table 3.4 Constituents of trace elements supplement

34
Table 3.5 Concentrations of 2-nitroaniline used for ATA test

36
Table 3.6 Concentrations of 2-nitroaniline and Cu used for ATA test

36
Table 3.7 HPLC conditions for 2-nitroaniline and
2-methylbenzimidazole

41
Table 3.8 HPLC conditions for o- phenylenediamine

41
Table 3.9 Constituents of the synthetic medium

43
Table 3.10 Mixture for preliminary test materials

44

Table 4.1 Comparison of influent and effluent inhibitions at different
2-nitroaniline concentrations

66
Table 4.2 Reduction in dissolved COD in the acidogenic reactor

68
Table 4.3 Inhibition test results (2-nitroaniline = 100 mg/L) 68

Table 4.4 Variation of Activity Factor with increasing
2-nitroaniline concentration

72
Table 4.5 Variation of Activity Factor with increasing
Copper concentration
72

x
Table 4.6 Comparison of influent and effluent inhibitions at different
copper concentrations
77

Table 4.7

Reduction in dissolved COD in the acidogenic reactor
(2-nitroaniline = 100 mg/L, Cu 25 mg/L)


80
Table 4.8 Inhibition test results for Track Run 2

(2-nitroaniline = 100 mg/L, Cu = 25 mg/L)


80

xi
LIST OF PLATES

Title Page

Plate 3.1 Laboratory-scale acidogenic-anaerobic SBR

32
Plate 3.2 Anaerobic toxicity assay setup

35
Plate 4.1 SEM photo showing microorganisms in acidogenic
enriched culture (magnification= 14,000)

82

Plate 4.2

SEM photograph showing microbial populations exposed to
2-nitroaniline concentration 100 mg/L
(magnification = 14,000)

82
Plate 4.3

SEM photograph showing microbial populations exposed to
copper concentration 25 mg/L
(magnification = 14,000)

83
Plate 4.4

SEM photograph showing microbial populations exposed to
2-nitroaniline 100 mg/L and
copper concentration 25 mg/L (magnification = 14,000)


83

xii
LIST OF ABBREVIATIONS

2-NA 2-nitroaniline

AAS Atomic Absorption Spectroscopy

anSBR Anaerobic Sequencing Batch Reactor

ATA Anaerobic Toxicity Assay

COD Chemical Oxygen Demand

A
v
VFAs production activity

Cu Copper

d day

DO Dissolved Oxygen

EC
50
Effective concentration of toxicant causing 50 % reduction in
oxygen uptake rate

FID Flame Ionization Detector

F/M Food/Microorganism

g gram

GCMS Gas Chromatography Mass Spectrometry

h hour

HPLC High Performance Liquid Chromatography

HRT Hydraulic Retention Time

IPCS International Programme on Chemical Safety and the European
Commission

L Litre

mg milligram

min minute

mL milli-Litre

MLSS Mixed Liquor Suspended Solids


xiii
MLVSS Mixed Liquor Volatile Suspended Solids

N Nitrogen

NADH Nicotinamide Adenine Dinucleotide Hydrogen

NIST

NB

National Institute of Standards and Technology

Nitrobenzene

P Phosphorus

OSHA Occupational Safety & Health Administration

pH Reciprocal of logarithm of hydrogen-ion concentration

SBR Sequencing Batch Reactor

SEM Scanning Electron Microscope

SRT Solid Retention Time

TCD Thermal Conductivity Detector

US-EPA

United States Environmental Protection Agency

VFA Volatile Fatty Acid

VFAs Volatile Fatty Acids

xiv
Chapter 1

1
CHAPTER ONE


INTRODUCTION


1.1 Background


Wastewaters generated from industrial activities such as textile dyeing, oil refining,
petrochemical processes, electroplating and semi-conductor, and pharmaceuticals
manufacturing may contain heavy metals such as copper and persistent organic
compounds such as 2-nitroaniline. These wastewaters when discharged into

waterbodies can create severe water pollution problems resulting in human health
hazards.

The anaerobic biotreatment process is widely used for treating strong industrial
wastewaters. However, anaerobic degradation of some persistent organics generally
requires a long period due to their inhibitory and recalcitrant nature (Razo-Flores et al.,
1999). In an anaerobic reactor, different groups of microorganisms (acidogens,
methnogens, etc.) work together to degrade organic matter. Acidogens have been
found to be more resistant to potentially inhibitory substances than methanogens (Lin,
1993). Therefore, use of the first phase of the anaerobic process, the acidogenic stage,
as a pretreatment process to partially convert persistent organic compounds into readily
biodegradable substances which could be efficiently removed by the subsequent
aerobic biotreatment process could be a viable treatment approach (Aziz et al., 1994;
Ng et al., 1999).


Chapter 1

2
Nitroaromatic compounds are priority pollutants which are found in wastewaters
originating from industries such as textile dying, pesticides, explosives and
pharmaceuticals manufacturing (Gurevich et al., 1993). Anaerobic biotreatment of
nitroaromatics has only been marginally successful due to incomplete metabolism and
production of unidentified intermediates. Prediction of the fate of these compounds in
the environment and the development of effective biotreatment systems is hindered by
the lack of information regarding some fundamental aspects such as detoxification
mechanism, identification and quantification of metabolites, and end-products
(Gorontzy et al., 1993). The first part of this study investigated the detoxification of a
wastewater containing 2-nitroaniline, and identifying and quantifying the metabolites.

Some inhibitory substances such as heavy metals are stimulatory at lower
concentrations. When present at higher concentrations, they can have inhibitory effects
resulting in an impairment of bacterial activities (Speece, 1996) and at still higher
concentrations, toxic effects leading to process failure (Bhattacharya et al., 1996).
While studies have been carried out to investigate the possible inhibitory effects of
persistent organic compounds and heavy metals on the methanogenic phase and
anaerobic process (Jin et al., 1996), little has been reported on the acidogenic phase in
relation to effect of persistent organic compounds and heavy metals. The second part
of this study was, therefore, carried out to investigate the possible inhibitory effects of
copper on acidogenic biotreatment of a wastewater containing 2-nitroaniline.




Chapter 1

3

1.2 Objectives of the study

The main objective of this study is to investigate the feasibility of acidogenic
pre-treatment of wastewaters containing 2-nitroaniline and copper.

The following are the sub-objectives of this study:

1. To study the feasibility of treating wastewater containing 2-nitroaniline using
the acidogenic process.

2. To examine the biodegradation pathway of 2-nitroaniline in the acidogenic
process identifying and quantifying the metabolites.

3. To study the inhibitory effect of copper on acidogenic biotreatment of a
wastewater containing 2-nitroaniline.









Chapter 1

4

1.3 Scope of the Study

The following is the scope of the study:

1. Monitoring of the variation of the following parameters under varying
concentrations of 2-nitroaniline and copper to determine process performance.
• production of VFAs (ethanoic, butanoic, propanoic and pentanoic acids)
• generation of gases (methane, carbon dioxide and nitrogen)
• pH
• dissolved COD
• MLSS and MLVSS

2. Conducting track runs to evaluate the performance profile of the anSBR during
a cycle.

3. Determination of EC
50
values of 2-nitroaniline and copper in aerobic system to
assess inhibitory effects.

4. Microscopic observations of microbial populations under varying
concentrations of 2-nitroaniline and copper to determine morphology.

Chapter 1
5
1.4 Experimental Protocol

A schematic diagram of study sequences is shown in Figure 1.1

Figure 1.1 Schematic diagram of the study sequences
Biotreatment of wastewater containing 2-nitroaniline
ISO Inhibition Test (aerobic) for 2-nitroaniline
Biodegradation pathway for
acidogenic pretreatment of
2-nitroaniline

Anaerobic Toxicity Assay Test
(2-nitroaniline and copper)

Biotreatment of wastewater containing
2-nitroaniline and copper

Acclimation and track runs with shock loads of 2-nitroaniline and copper in
Anaerobic Sequencing Batch Reactor

Anaerobic Toxicity Assay Test (2-nitroaniline)
Inhibition on acido
g
enisis b
y
2-nitroaniline

Inhibitory effect of copper on
acidogenic biotreatment
Chapter 2
CHAPTER TWO

LITERATURE REVIEW

2.1 Fundamentals of Anaerobic Wastewater Treatment

Anaerobic biotreatment process is widely used for treating organic wastewaters
(Stronach et al., 1986). Principally, in this process, the organic matter is converted to
methane (CH
4
) and carbon dioxide (CO
2
). Methane is a useful end-product since it is
an energy source. Therefore, this process has become increasingly popular for treating
both municipal sludges and industrial wastewaters. Anaerobic biotreatment has
significant advantages compared to the aerobic process. These include lower excess
sludge production, lower nutrients requirement, ability to accommodate high organic
loadings, and generation of useful end-products such as methane. However, there are
disadvantages such as low microbial growth rate, odour production and high buffer
requirement for pH control (Rittman and McCarty, 2000).

Industries that are currently served with full-scale anaerobic treatment facilities include
breweries and distilleries, chemical manufacturing, dairy product processing, textile
manufacturing, food processing, fish processing and pharmaceuticals manufacturing
(Rajeshwari et al., 2000). Wastewaters generated from these industries contain highly
persistent organic compounds such as nitroaromatic compounds and heavy metals such
as copper, which make the biotreatment of these wastewaters potentially difficult.

6
Chapter 2
2.1.1 Phases of anaerobic biotreatment process

Anaerobic bioconversion of the organic matter present in wastewaters generally occurs
in three steps. The first step involves the enzyme-mediated transformation (hydrolysis)
of organic matter of higher molecular mass into simple compounds suitable for use as
a carbon and energy source. The second step (acidogenesis) involves bacterial
conversion of the organic compounds resulting from the first step into identifiable
lower molecular-mass intermediate compounds-mainly ethanoic acid, propanoic acid,
and other volatile fatty acids. Lastly, the third step (methanogenesis) involves the
bacterial conversion of the intermediate compounds into simpler end-products
principally methane and carbon dioxide (Malina et al, 1992; Sawyer et al., 1994).

In an anaerobic reactor, different groups of microorganisms work together to degrade
the organic matter. One group is responsible for hydrolysing organic polymers and
lipids to basic structural building blocks such as monosaccharides, amino acids, and
related compounds. Another group ferments these products to simpler organic acids.
This group consists of both facultative and obligate anaerobic microorganisms. These
microorganisms are often identified as acidogens or acid formers.

The third group of microorganisms converts the hydrogen and acetic acid formed by
the acidogens to methane and carbon dioxide. The microorganisms responsible for this
bioconversion are strict anaerobes and are known as methanogens or methane formers.

Among the three groups, the most important is the methanogens which utilize
hydrogen and acetic acid. Hydrogen (H

2
) reacts as an electron donor and carbon

7
Chapter 2
dioxide (CO
2
) reacts as electron acceptor to form methane. Figure 2.1 shows the flow
diagram for the biotransformation pathway of organics present in wastewaters.

Complex
organics
Intermediary
products
Organic acids +
H
2

CH
4
+ CO
2


Hydrolysis Acidogenesis Methanogenesis

Figure 2.1 Three phases of the anaerobic biotreatment process (Rittman and
McCarty, 2000)

Temperature is one of the most important factors that can affect anaerobic performance

(Leighton et al., 1997). Growth rates generally roughly double for each 10°C rise in
temperature within the usual operational range from 10°C to 35°C. The mesophilic
range (25°C - 40°C) is most often used because thermophilic anaerobic reactions need
greater energy thus resulting in a higher operating cost. The optimum temperature for
the anaerobic system is usually around 35°C (Rittman and McCarty, 2000). A buffer
solution is added to control the reactor pH. System performance is checked regularly
by measuring pH and organic acids concentration. Acidogenesis occurs best at around
pH 5 to 6 while methanogenesis does so at pH 6.6-7.6.

In addition to the fundamental requirements such as carbon, nutrients (nitrogen,
phosphorus and trace elements) are required for microbial growth. Municipal
wastewaters and sludges could comply with these requirements. However industrial
wastewaters may not contain adequate nutrients to facilitate microbial growth. As a
result it may be necessary to add supplemental nutrients to support biotreatment.
Generally, a BOD
5
: N: P ratio of 100:5:1 is required in the feed composition to sustain
desirable microbial growth (Toe, 2000).


8
Chapter 2
2.1.2 Substrate flow in anaerobic system
Figure 2.2 shows the flow diagram illustrating the mechanism of anaerobic
biotreatment system. Various components in this carbohydrate-fed anaerobic
biotreatment system is given in Table 2.1. This explains flow of intermediate
molecules in an anaerobic biotreatment system that starts with carbohydrate, forms
intermediate organic acids and hydrogen and ultimately generates methane and carbon
dioxide (Bagley and Brodkorb, 1999).

.












(g
lucose
)
X
P
X
B
X
A
X
F
X
F
X
L

X
X
S
S
I
S
S
S
C
X
I
S
F
S
B
S
L
S
A
S
H
S
CO2
S
H
S
CO2
S
p
S

H
S
A
S
A
S
CO2
S
M
S
CO2
X
H
X
L
Cell l
y
sis
H
y
drol
y
sis
Acido
g
enesis
Aceto
g
enesis
Methano

g
enesis
X
F
Figure 2.2 Flow diagram illustrating the mechanism of anaerobic biotreatment
system





9