PRODUCTION OF ENZYMES FOR INDUSTRIAL

WASTEWATER TREATMENT:
PROOF OF CONCEPT AND APPLICATION
TO THE TEXTILE DYE INDUSTRY

by

C Mutambanengwe, O Oyekola, C Togo and CG Whiteley

Department of Biochemistry, Microbiology & Biotechnology
Rhodes University, Grahamstown

Report to the Water Research Commission on the project

“The production of a cocktail of hydrolytic enzymes from an anaerobic sulphidogenic
bioreactor fed with sulphate reducing bacteria and municipal sludge for the treatment of
biological and industrial wastewater: proof of concept.”

WRC Report No 1541/1/08

ISBN 978-1-77005-748-7
OCTOBER 2008
 DISCLAIMER

This report has been reviewed by the Water Research Commission (WRC) and approved for
publication. Approval does not signify that the contents necessarily reflect the views and
policies of the WRC, nor does mention of trade names or commercial products constitute
endorsement or recommendation for use.

ii
EXECUTIVE SUMMARY

Background

A previous Water Research Commission Project (WRC Report No: 1170/1/04) into the
enzymology of solubilisation of municipal sewage sludge (Rhodes University BioSURE
Process®) identified the involvement of a plethora of hydrolase enzymes such as
phosphatases, sulphatases, proteases, lipases, endoglucanases and glucosidases (Pletschke et
al., 2004; Whiteley et al., 2002; Enongene, et al., 2003; Ngesi, et al., 2002) isolated from a
biosulphidogenic reactor. Furthermore it was found that these enzymes could be used, in situ,
to bioremediate effluents from acid mine drainage, tanneries and abattoirs. It is the intention
of the current research to exploit this idea further and undertake a thorough investigation to
show that hydrogenase enzymes, also found within the biosulphidogenic reactor, could be
used to bioremediate industrial waste effluent from the textile dye industry. Azo dyes are the
most commonly used colouring compounds (Pearce et al., 2003) and they were therefore used
in this study to investigate the ability of sulphate reducing bacteria (SRB) and associated
cytoplasmic hydrogenase enzymes to degrade them under anaerobic conditions. Several
advantages of using such a system are forthcoming:

1) Although the enzymes from the sulphidogenic bioreactors are produced under anaerobic
conditions, they are perfectly able to work within an aerobic environment.

2) Using enzymes in a sulphidogenic environment supports that the sulphide produced would
be strongly inhibitory to the survival and proliferation of pathogens.

3) Biological (enzymatic) processes have an added advantage over traditional

chemical/physical methods as they are regarded as "clean and green".

4) The high cost of industrial enzymes is prohibitively expensive and so the use of these
enzymes, from the BioSURE Process®, is a cheap alternative and a tool for bioremediation.

The novelty of our approach allows for the generation of the essential enzymes within a
sulphidogenic bioreactor with a simultaneous in situ ‘one-pot’ bioremediation of industrial
effluent from the textile industry.

Hypothesis and Objectives

Hypothesis

Enzymes generated within a sulphidogenic bioreactor were capable of totally decolouring and
degrading textile dyes, both from an authentic commercial source and from the influents and
effluents of a textile industry.

iii
Objectives

1. To set up a laboratory scale biosulphidogenic reactor to generate the enzymes under study.

2. To extract various enzymes (α-glucosidase, β-glucosidase, lipase, phosphatase, sulphatase,

protease, cellulase, endoglucanases, hydrogenase, azoreductase) from the reactor.

3. To assay the enzymes and monitor their percent distribution.

4. To test the enzyme mixture on industrial effluents from the textile dye industry.

5. To establish a resource base in the enzymology of and industrial waste water treatment
processes and to support cognate research areas in South Africa.

6. To promote student training and corporate technology collaboration to enhance wastewater

management in South Africa.

Outline of Approach

The overall initial objective of the present study was to develop a powdered enzyme extract
obtained from a biosulphidogenic reactor that would effectively bioremediate industrial
effluents such as paper and pulp, tanneries, olive mill, textile dye, petroleum, abattoir,
fishing, mining and wine distilling. Commercial enzymes are costly and so it would make this
approach fairly favourable and cost effective. Furthermore, ordinary municipal sewage
sludge, obtained through the Rhodes University Environmental Biotechnology Research Unit
(EBRU) BioSURE Process®, can be used as a prime source of carbon for the sulphate
reducing bacteria that exist in the reactor. The idea of a ‘cocktail of enzymes’ implies a
bacterial cell-free powdered preparation of crude enzymes. Though the production and
purification of several hydrolase enzymes (glucosidases, proteases, lipases) within this
‘cocktail of enzymes’ has met with considerable success with respect to the defouling of
membranes and the bioremediation of abattoir effluents, trials conducted using both the crude
and purified hydrogenase on textile dye industrial effluents extracts were not successful. In
the case of the hydrolases, the crude enzyme mixture is self sustaining and their respective
reactions can occur without any necessary cofactors. With the textile dye effluent, however, it
was necessary to reduce the azo –N=N-bond through the action of the hydrogenase enzyme.
Since this initial reduction had not taken place it was felt that either the redox potentials in the
reaction mixture were not favourable to facilitate azo bond cleavage or that specific and
essential co-factors were absent in the purified samples. This led us to examine the SRB cells
as a whole, or rather the SRB cells from within the BioSURE Process® sludge itself. We now
report that this work has resulted in a complete decolourisation – and degradation – of the azo
dyes from within the textile dye industrial effluent.

In view of the complex nature of both the industrial textile dye effluent and sewage sludge, it
was decided to follow a four-level protocol investigation with various control reactions:

iv
1) Use SRB that has been cultured on a ‘pure’ lactate medium;

2) Use SRB that have been cultured from BioSure Process® sludge;

3) Use five authentic dyes (orange II, amido black 10, reactive black 5, reactive red 120,
reactive blue 2) purchased from a commercial source;

4) Use various sources of influent and effluent from a textile dye industry either prior to the
dyeing process or after dyeing and before the effluent had passed to the Environmental
Treatment Plant.

Results

Following our success with a previous study that involved the enzymology of sludge
solubilisation using enzymes from a biosulphidogenic reactor, we decided to study a
possibility that hydrogenases and/or azoreductases also present in the reactor could decolour
and hence bioremediate industrial effluent from the textile dye industry. Consequently a batch
reactor to produce the enzymes was set up, seeded with an inoculum of sulphate reducing
bacteria (SRB), sulphate and a cheap source of carbon as sewage sludge from the Rhodes
University BioSURE Process®. After stabilising the reactor and ensuring that it was
operating maximally (by monitoring sulphate consumption, pH, COD levels, sulphide
production), methodology was established for optimum enzyme production and extraction. A
time course survey was conducted to determine optimal time at which highest enzyme
production occurred and this was found to be at six days. Sonication, of the SRB-Biomass, at
low amplitude (10 W) for four minutes proved to be the best disruptive method for releasing
the hydrogenases from the SRB.

Induction studies were then undertaken with four commercial azo dyes (Orange II, Reactive
Black 5, Amido Black 10 and Reactive Red 120) and one non-azo dye (Reactive Blue 2), to
confirm firstly that the growth of sulphate reducing bacteria are not compromised in any way,
and secondly that the yield of the enzymes could be increased in a shorter period of time. The
presence of the azo dyes in the bioreactor increased the relative hydrogenase activity by up to
140% in 24h except the non-azo anthraquinone dye Reactive Blue 2 that failed to induce
enzyme activity, even after 10 days of incubation. This was likely due to the structural moiety
of the dye which did not stimulate the production of more enzymes. It was shown that the
mono azo dye Orange II resulted in the highest enzyme production while the di-azo Reactive
Black 5, Amido Black 10 and Reactive Red 120 resulted in 80% increase in enzyme activity
in 24h. Corresponding decolourisation was also observed in the same order as for enzyme
induction with the decolourisation of the mono azo dye being higher than that of the di-azo
dyes. The threshold level of the dye concentration was determined to be in the range 100-500
mg.l-1; higher concentrations resulted in limited decolourisation of the dyes (decolourisation
was probably due to adsorption by the dead SRB cells). Lactate as a primary carbon source
resulted in higher rate of decolourisation when compared to sludge from the BioSURE
Process®.
v
Purification and characterisation of the hydrogenase enzymes using PEG to concentrate the
enzyme was achieved by chromatography on Sephacryl S-200 and analysis of the fraction by
a 10% SDS-PAGE showed a distinct band with a molecular size of 38.5 kDa which is in the
same magnitude as other hydrogenases purified from SRB. The hydrogenase operates
optimally at a pH of 7.5 and temperature of 40°C but has poor thermal stability with 50% loss
in activity in 32 minutes and 70% loss in activity within an hour under optimal conditions.
Kinetic parameters Km and Vmax for methyl viologen as the substrate were determined.

Absorbance spectra of the industrial textile reactive dye mixtures and their respective
effluents revealed several maximum absorbance peaks ranging from 215-625 nm, thereby
revealing the presence of different reactive dyes as specified by the textile company (Da
Gama Textiles, King William’s Town, South Africa).

After characterisation of the dyes, and effluents, they were then degraded by SRB under
anaerobic conditions. Four industrial textile dye samples were tested – two influents, that
consisted of a pre-dye mixture and another that is ‘fixed’ with caustic soda and silicates and
two effluents, consisting of a vat print rinse and a final effluent (after all of the dyeing
processes) just prior to passing into the Environmental Treatment Plant (ETP).
Decolourisation of 93% and 72% for the influent dye mixture and the dye mixture plus
silicate salts, respectively, were observed. While the primary function of these salts is to
facilitate dye-fabric interaction, their presence, downstream, inhibits the bio-catalytic action
of enzymes during effluent treatment and as such would need to be removed or diluted to
levels that don’t affect bioremediation of the effluent. Successful decolourisation of both
commercial dyes and industrial effluents with SRB-BioSURE Process® sludge was achieved
with decolourisations ranging from 96-49% over a five day period. The process of
decolourisation for each of the dyes can be monitored by a decrease in absorbance at the λmax
of the inherent chromophore. This is supported by a reduction of the azo link into two
colourless aromatic amine compounds. At the same time as there is a decrease in absorbance
of the dye in the visible region (480-610 nm) there is an increase in the absorbance at 280 nm,
reflecting an increase in concentration of single aromatic amines. With an extended period of
time, there was a subsequent decrease in the absorbance at 280 nm indicating that the
aromatic amines had been degraded further, perhaps by some other unknown factors, into
CO2, H2O and NH3. Both of the influent and effluent samples followed similar trends to the
authentic dyes in that:

1) There was decolourisation of the dye(s), monitored by a decrease of absorbance in the

visible region (480-610 nm).

2) There was an increase in absorbance at 280 nm due to an increase in aromatic amines.

3) There was subsequent decrease in absorbance at 280 nm due to a total breakdown of these
aromatic compounds.

vi
As a control measure, the effect of a ‘pure’ culture of SRB (using lactate medium as a carbon
source) on both authentic dyes and on the various influents and effluents from the textile
industry was studied. The time taken to degrade the dyes using SRB from BioSURE
Process® sludge in the sulphidogenic bioreactor was much longer than if the SRB were used
from a ‘pure’ culture.

It is interesting to reiterate that with ‘pure’ SRB from a culture on lactate medium there was
very little breakdown of the single aromatic compounds as the absorbance at 280 nm
remained fairly significant. This was evident with both authentic dyes and industrial samples.
With SRB from the BioSURE Process® sludge there was complete degradation and a
subsequent removal of the aromatic compounds absorbing at 280 nm. It supports other
factors, within the sulphidogenic reactor, that may be responsible for complete degradation. It
is hypothesised that an anaerobic degradation of the dyes into their constituent aromatic
amines followed by an aerobic degradation into CO2, H2O and NH3. With the ‘pure’ SRB
system this doesn’t happen.

Though each bioreactor is different on any particular day and consequently the yield and
activity of the hydrogenase enzyme varies, the amount of sludge required to completely
decolour a specific volume of textile effluent can be estimated. For example if the enzyme
activity per ml of sulphidogenic sludge is estimated at 2200 µmol.min-1 then 1 kg of sludge
(1000 ml) would decolour 2.2 mols of azo dye (770 grams Orange II) in one minute.

Recommendation

Purified hydrolase enzymes, extracted from a sulphidogenic bioreactor can be concentrated

into a dried powdered cocktail preparation, using established concentration techniques.
Though this powdered extract was suitable to bioremediate certain abattoir effluents and acid
mine drainage they failed to decolour and degrade dyes from the textile industry. It is the
recommendation from this project that in order to completely decolour and degrade the azo
dyes from an industrial waste effluent a dried powdered extract of SRB-BioSURE Process®
sludge from a biosulphidogenic reactor, including all of the necessary enzymes and cofactors
in situ be used.

vii
viii
ACKNOWLEDGEMENTS

The research in this report emanated from a project funded by the Water Research
Commission and entitled:

“The production of a cocktail of hydrolytic enzymes from an anaerobic sulphidogenic

bioreactor fed with sulphate reducing bacteria and municipal sludge for the treatment of
biological and industrial wastewater: Proof of concept”

The reference Group responsible for this project consisted of the following persons:

Mr HM Du Plessis Water Research Commission (Chairman)

Prof PD Rose Rhodes University
Prof M Wentzel University of Cape Town
Prof E Chirwa University of Pretoria
Mr A Wurster Golder Associates Africa

The financing of the project by the Water Research Commission and the contribution of the
members is gratefully acknowledged.

The authors also wish to record their sincere thanks to the following:

The Technical staff, Department of Biochemistry, Microbiology and Biotechnology.

The Dean of Research office and Department of Finance, Rhodes University.

Dr CA Togo for extensive proof-reading and formatting of the manuscript.

ix
ABBREVIATIONS

AB 10 Amido Black 10
CMC Carboxymethylcellulose
DEAE Diethylaminoethyl
ETP Environmental treatment plant
FAD Flavine adenine dinucleotide
FMN Flavine mononucleotide
MV Methyl viologen
NAD(H) Nicotinamide adenine dinucleotide (reduced)
OII Orange II
PEG Polyethyleneglycol
PSS Primary sewage sludge
RB2 Reactive Blue 2
RB5 Reactive Black 5
RR 120 Reactive Red 120
SDS-PAGE Sodium dodecylsulphate-polyacrylamide gel electrophoresis
TRIS Tri(hydroxymethyl)aminomethane
U Units of enzyme activity
p-NP- α-G p-Nitrophenol – α-glucopyranoside
p-NP- β-G p-Nitrophenol – β-glucopyranoside
p-NPP p-Nitrophenolphosphate
p-NPS p-Nitrophenolsulphate

x
TABLE OF CONTENTS

EXECUTIVE SUMMARY III

BACKGROUND III
HYPOTHESIS AND OBJECTIVES III
Hypothesis iii
Objectives iv
OUTLINE OF APPROACH IV
RESULTS V
RECOMMENDATION VII

ACKNOWLEDGEMENTS IX
ABBREVIATIONS X
TABLE OF CONTENTS XI
LIST OF FIGURES XIII
LIST OF TABLES XV
1 INTRODUCTION 1
2 POTENTIAL USE OF ENZYMES WITH INDUSTRIAL EFFLUENTS 3
2.1 PAPER AND PULP 3
2.2 OLIVE MILL 4
2.3 MINING 4
2.4 TEXTILE 4
3 MATERIALS AND METHODS 6
3.1 SOURCE OF SLUDGE 6
3.2 REACTOR DESIGN AND OPERATION 6
3.3 PREPARATION OF SEWAGE SLUDGE AND EXTRACTION OF ENZYME MIXTURE 6
3.4 ANALYTICAL PROCEDURES 7
3.5 ENZYME ASSAYS 7
3.5.1 Lipases 7
3.5.2 Proteases 8
3.5.3 Glucosidases 8
3.5.4 Sulphatases and phosphatases 8
3.5.5 Endoglucanases 8
3.5.6 Azo-reductases 9
3.5.7 Hydrogenases 9
3.6 ENZYME PURIFICATION 9
3.6.1 Glucosidase 9
3.6.2 Hydrogenase/azoreductase 9
3.6.3 Endoglucanase 10
3.7 ENZYME PROPERTIES 10
3.7.1 pH 10
3.7.2 Temperature 10
3.7.3 Stability 10
3.7.4 Kinetic parameters: Km and Vmax. 10
3.8 ENZYME SCREENING 10
3.8.1 Textile dyes 10
3.9 INDUCTION STUDIES 11
3.9.1 Textile dyes 11

xi
 3.10 INDUSTRIAL EFFLUENT ANALYSIS 11
3.10.1 Textile industry 11
3.11 FIELD TESTING OF THE PRODUCT 11
3.11.1 Textile industry 11
4 RESULTS 13
4.1 REACTOR OPERATION 13
4.2 ENZYME EXTRACTION 14
4.3 ENZYME DISTRIBUTION 14
4.4 ENZYME PURIFICATION 14
4.4.1 Glucosidase 14
4.4.2 Hydrogenase/azoreductase 15
4.4.3 Endoglucanase 16
4.5 ENZYME PROPERTIES 16
4.6 ENZYME SCREENING 17
4.6.1 Textile dyes 17
4.7 INDUCTION STUDIES 18
4.7.1 Textile dyes 18
4.8 INDUSTRIAL EFFLUENT ANALYSIS 20
4.8.1 Textile industry 20
4.9 FIELD TESTING 22
4.9.1 Textile industry 22
5 DISCUSSION 33
6 CONCLUSIONS AND RECOMMENDATIONS 36
6.1 CONCLUSIONS 36
6.2 RECOMMENDATIONS 36
7 FUTURE RESEARCH 37
8 REFERENCES 38
9 CAPACITY BUILDING 41
9.1 STUDENTS 41
9.1.1 MSc Students 41
9.1.2 Post doctoral fellow 41
9.2 PUBLICATIONS 41
9.2.1 Papers 41
9.2.2 Conference presentaions 42

xii
LIST OF FIGURES

Page
Figure 2.1 Mechanism to show the reduction of orange II by a hydrogenase enzyme
effluent wastewater containing these dyes is ongoing with an increasing
number of studies being reported. 5
Figure 3.1 The design of the laboratory-scale biosulphidogenic reactor. 7
Figure 4.1 Time survey to show typical sulphate (■) and sulphide (▲) levels in the
bioreactor 13
Figure 4.2 Purification of β-glucosidase with Sepharose 4B gel exclusion. 15
Figure 4.3 Sephacryl S-200 ion exchange chromatography for hydrogenase after PEG
concentration. 15
Figure 4.4 DEAE-Cellulose ion exchange chromatography for endoglucanase. 16
Figure 4.5 Sulphate reducing bacteria degrading Orange II azo dye in agar medium. 17
Figure 4.6 Time course survey of hydrogenase activity produced by SRB. 18
Figure 4.7 Time course survey of hydrogenase activity produced by SRB after been
induced in the presence of orange II reactive dye. 19
Figure 4.8 Induction of hydrogenase in the presence of commercial dyes. 19
Figure 4.9 UV/Vis scan between 200-700 nm of a mixture of reactive dyes (D1) used
for dyeing textile fibres at Da Gama Textiles; reactive dyes with silicates
and caustic alkali (D2); vat print rinse (D3) and waste produced (D4) after
dyeing before the Environmental Treatment Plant. 20
Figure 4.10 Concentrations of the dyes and the effluents measured by determining the
chemical oxygen demand (COD). 21
Figure 4.11 Sulphate reducing bacteria was incubated with single commercial dyes as
well as mixtures ranging from 100 mg.l-1 to 200 mg.l-1 for each dye over a
period of 24h. 23
Figure 4.12 Effect of dye concentration on dye decolourisation efficiency over 24h. 23
Figure 4.13 Anaerobic degradation of D1 by SRB 24
Figure 4.14 Anaerobic degradation of D2 by SRB 24
Figure 4.15 Anaerobic degradation of D3 by SRB 25
Figure 4.16 Anaerobic degradation of D4 by SRB 25
Figure 4.17 Degradation of Orange II and dissapperance of aromatic amines by SRB
cultured in BioSURE Process® sludge. 26
Figure 4.18 Decolourisation of orange II by SRB from BioSURE Process® 26
Figure 4.19 Anaerobic degradation of Amido Black 10 by SRB from the BioSURE
Process®. 27
Figure 4.20 Anaerobic degradation of Amido Black 10 with SRB from BioSURE
Process® 27
Figure 4.21 Anaerobic degradation of Reactive Black 5 by SRB from the BioSURE
Process®. 28
xiii
Figure 4.22 Anaerobic degradation of Reactive Black 5 with SRB from BioSURE
Process®. 28
Figure 4.23 Anaerobic degradation of Reactive Red 120 by SRB from BioSURE
Process®. 29
Figure 4.24 Anaerobic degradation of Reactive Red 120 with SRB from BioSURE
Process ®. 29
Figure 4.25 Anaerobic degradation of Reactive Blue 2 by SRB from the BioSURE
Process®. 30
Figure 4.26 Anaerobic degradation of Reactive Blue 2 with SRB from BioSURE
Process®. 30
Figure 4.27 Progressive removal of Reactive Red 120 by SRB from the BioSURE
Process®. 31
Figure 4.28 Progressive removal of Reactive Blue 2 by SRB from the BioSURE
Process®. 31
Figure 4.29 Decolourisation rates of Da Gama dyes and effluents by SRB from pure
lactate media after 24h and monitored at 590 nm. 32
Figure 5.1 Anaerobic – aerobic bacterial azo dye degradation. 35

xiv
LIST OF TABLES

Page
Table 3.1 A factorial matrix of experiments of the incubation of the enzyme from
various sources with both authentic and industrial effluent dyes. 11
Table 4.1 Typical enzyme activity and extracted from a biosulphidogenic reactor. 14
Table 4.2 Properties of enzymes extracted from the biosulphidogenic reactor. 17

xv
xvi
1 INTRODUCTION

According to Aitken (1993) enzymes were first proposed for the treatment of industrial waste
in the 1930’s but it wasn’t until recent that enzyme technology received much attention
(Whiteley and Lee, 2006) for the improvement in biological remediation for industrial
effluents.

The heterogeneous complexity of industrial waste has created gross uncertainty and deviation
in predictions of suitable models for its bioremediation. Microorganisms can express specific
xenobiotic metabolising enzymes that would degrade even the most recalcitrant industrial
waste but the limiting capacity for bioremediation emphasises the fact that micro-organisms,
by themselves, are insufficient as they lead to the generation of a considerable amount of
biomass and have a very slow rate of degradation. Intrinsic bioremediation is the removal,
transformation or detoxification of any contaminant to a less toxic form by any natural
process and it is possible to enzymatically attack this complex industrial waste to recover
valuable resources, remove toxic materials and recover the water.

A recent Water Research Commission Project (WRC Report No: K5/1170/0/1) into the
enzymology of solubilisation of municipal sewage sludge using a sulphidogenic bioreactor
(Rhodes University BioSURE Process®) identified the involvement of a plethora of
hydrolase enzymes such as phosphatases, sulphatases, proteases, lipases, endoglucanases and
glucosidases. (Enongene et al., 2003; Ngesi et al., 2002; Pletschke et al., 2004; Whiteley et
al., 2002). In view of the fact that municipal sewage sludge acts as the carbon source for the
sulphate reducing bacteria (SRB) and that the generation of hydrogen sulphide can precipitate
heavy metals as their metal sulphide, the sulphidogenic bioreactor has met with considerable
success in the bioremediation of acid mine drainage (Rose et al., 1998). Furthermore it was
found, in our laboratories, that these enzymes, within the bioreactor, could be used, in situ, to
defoul membranes used in the bioremediation of abattoir effluents (Melamane et al., 2003)

Several advantages of using such a system are forthcoming:

1) Although the enzymes from the sulphidogenic bioreactors are produced under
anaerobic conditions, they are perfectly able to work within an aerobic environment.

2) Using enzymes in a sulphidogenic environment supports that the sulphide produced

would be strongly inhibitory to the survival and proliferation of pathogens.

3) Biological (enzymatic) processes have an added advantage over traditional

chemical/physical methods as they are regarded as "clean and green".

4) The high cost of industrial enzymes is prohibitively expensive and so the use of these
enzymes, from the BioSURE Process®, is a cheap alternative and a tool for
bioremediation.
1
It is the intention of the current research to exploit this idea further and undertake a thorough
investigation to show that these enzymes could be used to bioremediate industrial waste
effluent from various industries such as paper and pulp, olive mill, petroleum, textile dye,
mining and fishing. The novelty of our approach allows for the generation of the essential
enzymes within a sulphidogenic bioreactor with a simultaneous in situ ‘one-pot’
bioremediation of the industrial effluent.

2
2 POTENTIAL USE OF ENZYMES WITH INDUSTRIAL EFFLUENTS

Industrial waste effluents contain a variable mixture of both organic and inorganic substances
though the actually nature and concentrations will depend on the source. Much of the organic
matter is contained within fatty acids, carbohydrates, proteins that can arise from the
agricultural sector (crops, fruit vegetables, pesticides, herbicides, fertilizers, nitrates,
phosphates); the fishing industry (oils, cleaning fluids); animal production processes (manure,
nitrogen, phosphorous, microorganisms); paper and pulp industries (lignin, celluloses,
colouring dyes, inks, sulphites, dioxins); abattoir effluents; wine distilling and brewing
industries; meat canning and processing; dairy industries; petroleum industry (phenols,
toluenes, benzenes, xylenes, sulphides) and in the tannery pond effluents. The inorganics
contain predominantly the heavy metals, brine salts, sulphites and sulphides.

This heterogeneity in industrial wastewaters has warranted extensive investigations and

different strategies for their total bioremediation and water recovery. Though microorganisms
can express enzymes that can degrade the most recalcitrant pollutants they are wrought with
problems. Any approach to use microorganisms alone for the bioremediation and
biodegradation of industrial wastewaters has limited potential since individual bacteria,
capable of remediating a given pollutant, maybe inhibited by the presence of other pollutants.
Moreover bacteria exercise a very slow rate of degradation of polluted sites limiting their
overall practical use for this purpose.

Consequently enzymology may be regarded as a key stone in the context of environmental

biotechnology and biological remediation whether they are aerobic or anaerobic processes.
The role of novel enzymes for use in bioremediation of industrial effluents remains a safe,
economic and ‘clean and green’ alternative to redundant chemical processes.

2.1 Paper and Pulp

Enzymes can be used in the biodeinking of newspaper before recycling which, in general,
gives a better result as compared to any chemical method (Pelach et al., 2003). Wastepaper is
a major constituent of solid waste and any enzymatic bioconversion of cellulose-rich waste
into fermentable sugars (glucose) may limit environmental pollution and encourage reuse and
sustainability (van Wyk and Mohulatsi, 2003). Such effluents also contain significant
amounts of coloured material, generated from quinone type products during the pulp
bleaching process and a major challenge towards bioremediation is its ultimate removal.
Biopulping is the enzymatic treatment of lignocellulosic material just before
thermomechanical pulping takes place and is believed to increase the strength of the product
while at the same time decrease environmental impact and chemical energy consumption.
The enzymatic degradation of cellulose by cellulases has been the focus of several studies for
their use in the bioconversions of agricultural wastes, in the improvement of the manufacture
of recycled paper (Stork et al., 1995), in the production of food and fuel (Clark, 1997),
biopolishing of textiles (Cavaco-Paulo, 1998), additives in washing powder and animal feed,

3
pulping, processing of fruit juices and beverages, baking and in bioethanol production (Tolan
and Foody, 1999; Zaldivar et al., 2001). Endoglucanases cleave the internal glycosidic bonds
of cellulosic chains and act synergistically with exoglucanases and E-D-glucosidases during
the solubilisation of crystalline cellulose (Bhat and Bhat, 1997). The current state of
knowledge invites new and improved endoglucanases with varying pH and temperature
optima, stability and substrate specificities for increased efficiency and economics of various
biotechnological and bioremediation processes. Since endoglucanase and glucosidase
enzymes have been found in the sulphidogenic bioreactor in our laboratories (Oyekola et al.,
2007) their role in the bioremediation of industrial waste was to be considered.

2.2 Olive Mill

These industrial wastes are usually dark red to black coloured effluents containing COD
values greater than 200 g.l-1. Along with sugars, tannins, lipids and pectin the colours
predominate from the phenolics including polyalcohols and polyphenols. Consequently the
decolourisation of this waste material is of paramount importance.

2.3 Mining

Microorganisms offer a potentially large gene pool to choose from when searching for
enzymes that may be potentially useful for the treatment, and recovery, of metals from
contaminated wastewaters. In particular enzymes from SRB have received much attention
due to their ability to enzymatically aid reductive precipitation of metal salts like mercury,
palladium, chromium, technetium, arsenic (Lloyd et al., 1998; 2001). Reports are
forthcoming from our laboratories on the enzymatic recovery/reduction of two platinum
group metals (PGM) – rhodium (Ngwenya and Whiteley, 2006) and platinum (Rashamuse
and Whiteley, 2007).

2.4 Textile

Commercial dyes are classified as azo, anthraquinone, phthalocyanine, triphenylmethane and

heterocyclic with azo dyes, characterized by the functional azo group (-N=N-), representing
one of the largest class used in the printing and textile processing industries. The decrease in
colour, for orange II, is monitored at 490 nm and is due to a reduction of the azo group –
N=N- first to the bis-amine then to two separate amines (Figure 2.1). These dyes and their
degradative aromatic amine products (Brown and Hamburger, 1987) are toxic and mutagenic
to living systems and consequently are undesirable if released into the environment. The
decolourisation of industrial

4
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NaSO3 N
CH3

Figure 2.1 Mechanism to show the reduction of orange II by a hydrogenase

enzyme effluent wastewater containing these dyes is ongoing with an
increasing number of studies being reported. These brightly coloured
dyes are water-soluble and are extremely resistant to microbial and
physico-chemical degradation, including conventional processes of
wastewater treatment.

Enzymes from both anaerobic and aerobic systems have been reported to be effective in the
decolourisation of the dyes with the majority of results forthcoming from white rot fungi
Phanaerochaete and Trametes. These organisms secrete laccases and manganese/lignin
peroxidases that are capable of oxidative free radical cleavage of the azo bond. Non specific
cytoplasmic reductases and hydrogenages from bacteria (Pseudomonas, Clostridium), also
degrade azo dyes. These examples, however, are wrought with limitations in that they have a
limited substrate range or have been reported with model chromophoric compounds. Due to
the variability in industrial effluent composition as well as the structural diversity of the dye
itself the biodegradation of dyes found in the textile and/or printing sectors are more
recalcitrant. Since hydrogenase enzymes have been found within the sulphidogenic bioreactor
in our laboratories (Ngwenya and Whiteley, 2006) it was decided to investigate their role in
the bioremediation of industrial textile dye effluents.

5
3 MATERIALS AND METHODS

Methodology focused on the production of an enzyme extractive for use in an industrial and
biological wastewater treatment process and testing the results. Enzymes under consideration
are endoglucanases, proteases, glucosidases, phosphatases, sulphatases, lipases, cellulases,
hydrogenases and azoreductases. Computer software packages (Statistica 7, StatSoft)
(SigmaPlot 6) were used for statistical analyses of the data, for graphical determinations of all
the kinetic parameters and for determining the relevant mechanisms and models.

3.1 Source of Sludge

Sludge used for these experimentations was from two different sources. It was either
collected from the anaerobic digesters from a local sewage treatment plant or from a
batch/continuously fed, laboratory scale, sulphidogenic bioreactor.

3.2 Reactor Design and Operation

An experimental sulphidogenic bioreactor (10000 ml), containing modified Postgate medium

B (Postgate, 1984), was seeded with an inoculum (750 ml) of a mixed consortium of sulphate
reducing bacteria obtained from the Environmental Biotechnological Research Unit (EBRU)
BioSURE Process® (Grahamstown, South Africa). The bioreactor was covered with
aluminium foil to minimise exposure to light and was kept anaerobic by purging regularly
with N2: CO2 (80:20%) (Afrox, South Africa) for 15 minutes followed by H2 (99%), (Afrox)
for 5 minutes at room temperature. It was sub-cultured every 3 weeks with new Postgate
medium. A 15% (v/v), (750 ml) inoculum was taken from this stock bioreactor and used as a
starter for a smaller 5000 ml bioreactor (Figure 3.1) which was used for all experiments
carried out during the research. This was connected to a reservoir containing a solution of
sulphate (2000 mg.l-1) to represent the final electron-acceptor. The carbon source for the
reactor was fresh raw primary sewage sludge (PSS) obtained from the primary clarifiers of
the Grahamstown sewage plant. In order that the sulphate was not limiting within the reactor
the influent sulphate and COD concentrations were both 1000 mg.l-1 resulting in a COD:SO4
ratio of 1:1 (m/m). The mean CODSoluble of the feed was 261 mg.l-1 with a corresponding
CODParticulate of 738 mg.l-1 and the mean pH of the reactor was 6.31. The reactor was operated
at ambient temperatures (20-22°C). A gas trap containing a solution of zinc acetate was
installed to absorb any released hydrogen sulphide.

3.3 Preparation of Sewage Sludge and Extraction of Enzyme Mixture

Anaerobic sludge (100 ml) from the biosulphidogenic bioreactor was centrifuged (10000 x g,
30 min, 4ºC), the pellet washed twice with distilled water and the supernatants combined
(S1). The pellet (P1) was then suspended in phosphate buffer (pH 6, 50 mM) and sonicated
(40 W, 30 second bursts) with varying times involving 2-10 min intervals in order to establish
optimal sonication protocol. The suspended pellet was centrifuged (10000 x g, 10 min, 4 ºC)

6
to afford a cell-free supernatant (S2) and a pellet (P2). All of the enzymes in S2 and P2 were
assayed as described below. The soluble cell-free extract was subjected to dialysis overnight
and then freeze-dried to afford a crude dark brown-black amorphous solid.

Figure 3.1 The design of the laboratory-scale biosulphidogenic reactor. (A = Zinc acetate
trap; B = rubber stopper; C = 5 litre glass reactor vessel; D = sampling port; E
= Metal clamp; F/G = magnetic stirrer; H/H1 = Tygon tubing (impermeable to
oxygen).

3.4 Analytical Procedures

All analyses were carried out in triplicate and values reported as the means with standard
deviations. All reagents and standards used were of analytical grade from Sigma-Aldrich
(Inc. USA) or Merck Chemicals (Pty) Ltd. Samples used for sulphate, sulphide, alkalinity and
CODSoluble were filtered through glass micro fibre filters (GF/A Circles 25 mmØ – Whatman®
Int Ltd England) and the filtrate refiltered through 0.45 micron (Millipore AcetatePlus, #
A04SP002500, Osmonics Inc). Total chemical oxygen demand (CODTotal), soluble chemical
oxygen demand (CODSoluble) and particulate chemical oxygen demand (CODParticulate) were
determined using the Merck Spectroquant reagent kit as per manufacturer’s instructions. Prior
to COD determination, all samples were acidified with concentrated HCl to pH 2 in order to
remove any dissolved sulphide followed by nitrogen purge to fully remove traces of hydrogen
sulphide. Sulphide was determined with a Merck Spectroquant reagent kit and absorbance
read at 665 nm. Protein concentration was determined by the Bradford method (Bradford,
1976) using bovine serum albumin as standard. Carbohydrate (Yemm and Willis, 1954) and
lipid concentrations (Folch et al., 1957) were determined using published procedures.

3.5 Enzyme Assays

3.5.1 Lipases

Lipase activity was measured by the enzymatic cleavage of glycerol from a commercial lipid.
(Korn, 1954). The supernatant from the sonicated sludge (1.0 ml) was incubated at 25°C for
10 min in 0.1 M sodium phosphate buffer, pH 7.5 with triacetin (1%w/v, 1.0 ml). The
reaction was stopped by the addition of sulphuric acid (5 M, 50 Pl) and sodium periodate
7
(NaIO4) (0.1 M, 250 Pl). The sample was mixed, NaHSO3 (250 Pl) added, then transferred to
chromotropic acid reagent (2.5 ml) and heated (80°C, 60 min). After cooling the glycerol
released was measured by reading the absorbance at 570 nm and concentrations determined
from a standard curve.

3.5.2 Proteases

Protease activity was measured according to the published procedures using azocasein as
substrate (Goel et al., 1998; Pin et al., 1995). The soluble cell-free extract (1.0 ml) was
incubated (37°C, 1 h) in suitable buffer at the optimum pH with 2% (w/v) azocasein (1.0 ml).
Ice-cold 10% (w/v) trichloroacetic acid (TCA) (1.0 ml) was added to stop the reaction and the
tubes left at –20°C to facilitate precipitation. The precipitated protein was removed by
centrifugation (4 000 x g, 10 min) and the A440nm of TCA-soluble peptides was measured.

3.5.3 Glucosidases

D- and E-glucosidase activity was measured by a modification of the published procedure

using the corresponding anomeric methylumbelliferyl-glucopyranoside as substrate and
liberating the fluorogenic methylumbelliferone product which is measured fluorometrically at
455 nm. (Hattenberger et al., 2001). The soluble cell-free extract (1 ml) was incubated at
50°C for 5 min in glycine buffer (0.4 M, pH 10.8) with methylumbelliferyl-glucopyranoside
(1.5 mM, 1 ml). Ice-cold ethanol (2.5 ml; 95%) was added to stop the reaction and the tubes
were centrifuged (2 000 x g, 5 min) and the fluorescence measured in a spectrofluorimeter at
an excitation wavelength of 365 nm and an emission wavelength of 455 nm. Glucosidase
activities were calculated from a standard curve.

3.5.4 Sulphatases and phosphatases

The activity of these enzymes was measured by the enzymatic cleavage of U-

nitrophenolsulphate (or nitrophenolphosphate) liberating U-nitrophenol that generates an ion
in alkaline solution that can be measured at 405 nm. The soluble cell-free extract was
incubated at 25°C for 20 min in acetate buffer (10 mM) at the relevant pH with U-
nitrophenolsulphate (or nitrophenolphosphate) (30 Pl; 60 PM). The reaction was stopped with
the addition of NaOH (2.0 ml, 0.5 M) and the absorbance of the yellow colour measured in a
spectrophotometer at 405 nm. One unit of enzyme activity is the amount of enzyme required
to convert 1 PM of U-nitrophenolsulphate (nitrophenolphosphate) to U-nitrophenol in 1 min at
25°C at 405 nm. (Bergmeyer, 1986).

3.5.5 Endoglucanases

Endoglucanase activity was measured from the release of reducing sugar (glucose) from
carboxymethylcellulose (CMC) using the published method (Colowick and Kaplan, 1988).
The enzyme sample (1.0 ml) was treated with carboxymethylcellulose (1.0 ml, 1% (w/v),
50ºC, 30 min). Dinitrosalicylic acid reagent (3.0 ml) was added and the whole boiled for 5
minutes then diluted with distilled water (20 ml) and A540nm determined. One unit of

8
enzymatic activity is the amount of enzyme that releases 1 Pmol of reducing sugar per minute
under the assay conditions.

3.5.6 Azo-reductases

In an assay volume of 2.0 ml was placed Tris HCl, pH 7.4, 25 mM; NADH 0.21 mM; FAD
20 µM; Orange II or Reactive Black 5, 25 µM and soluble enzyme extract, 50 µl. The
reaction was monitored at 490 and 595 nm respectively with 1U (unit) of enzyme
representing the reduction of 1 µmol of dye per minute (Liger et al., 2004).

3.5.7 Hydrogenases

In a total assay volume of 3.105 ml was placed Tris HCl, 20 mM, pH 7.8, methyl viologen 1
mM; sodium dithionite, 100 mM and soluble enzyme extract 100 µl. The reaction was kept in
an atmosphere of nitrogen and the activity monitored by reduction of methyl viologen at 604
nm. 1 U(unit) of enzyme activity is the reduction of 1 µmol of methyl viologen per minute
(Bennet et al., 2003; DeLacy et al., 2000).

3.6 Enzyme Purification

3.6.1 Glucosidase

The crude cell-free extract (50 ml) was treated with cold (<10 ºC) acetone to a final
concentration of 60% and allowed to stand at this temperature overnight. The suspension was
centrifuged (15300 x g, 20 min, 4ºC) and the supernatant treated further with cold acetone
(5h) followed by a final centrifugation. The combined precipitates were resuspended in Tris
HCl buffer (50 mM, pH 7.7, 25 ml) and then loaded on to a Whatman cellulose (CC31)
affinity column (2 x 15 cm) equilibrated with Tris HCl buffer (50 mM, pH 7.7). The column
was washed with the same buffer until the absorbance at 280 nm (A280nm) of the eluate
reached base line. β-Glucosidase was eluted from the column at a flow rate of 3.5 ml.min-1.
Fractions containing β-glucosidase activity were pooled and then loaded on to a Sepharose
4B gel exclusion column (0.9 x 18 cm) equilibrated with the same buffer. At a flow rate of
7.0 ml min-1, fractions (5 ml) were collected and analysed for glucosidase activity (Oyekola
et al., 2007).

3.6.2 Hydrogenase/azoreductase

The cell free extract (100 ml) was concentrated using PEG 20000 followed by dialysis
against 10 mM Tris-HCl pH 7.6 to remove any salts. Enzyme activity and protein
concentration were determined at each stage. The concentrated cell free enzyme extract was
loaded onto Sephacryl 200 size exclusion – ion exchange column (2x30 cm) equilibrated with
50mM Tris-HCl pH 7.6 buffer. Fractions were collected at a rate of 2.2 ml.min-1. Protein
determination and enzyme assays were determined and fractions exhibiting enzyme activity
were pooled together and freeze dried. The freeze dried cell free extract was resuspended in
ddH2O. SDS-PAGE was performed to verify the proteins exhibiting enzyme activity.

9
3.6.3 Endoglucanase

The cell-free extract (200 ml) was concentrated to 35 ml against PEG 20000 (3 h), removed
from the dialysis tubing, centrifuged (10000 x g, 10 min, 4°C) to remove any debris and
enzyme activity and protein analysis determined as before. The concentrated cell-free enzyme
extract (6 ml) was loaded on to a DEAE-cellulose ion exchanger column (1 x 25 cm)
equilibrated with sodium phosphate buffer (0.05 M, pH 6.0). The column was washed with
the same buffer until the absorbance at 280 nm (A280nm) of the eluate reached base line.
Bound endoglucanases were eluted from the column by a stepwise increase in NaCl (0-1 M)
in sodium phosphate buffer (0.05 M, pH 6.0) at a flow rate of 1 ml.min-1. Fractions
containing endoglucanase activity were pooled, concentrated and subjected to SDS-PAGE.

3.7 Enzyme Properties

3.7.1 pH

The pH profiles for the enzymes were performed, at their respective optimum temperature
between pH 4 and 11. The following buffers were used: 0.2 M sodium acetate buffer,
pH 4-5.5; 0.1 M sodium phosphate buffer, pH 6 and pH 6.5; 0.1 M Tris-HCl buffer, pH 7.0,
7.5, 8.0, 8.5, 9.0 and 0.1M carbonate/bicarbonate buffer, pH 9.5-11.

3.7.2 Temperature

The temperature profiles for the enzymes were performed, at their respective optimum pH
between 20-70°C.

3.7.3 Stability

The stabilities of all of the individual enzymes were investigated at respective optimum
temperatures and pH over incubation times of up to 5h and recorded as the percent decrease
in activity per hour. The stability of the enzyme mixture as a whole was not determined.

3.7.4 Kinetic parameters: Km and Vmax.

Ten different concentrations each of the respective substrates were chosen to give measurable
reaction rates and the reactions were performed in triplicate. The affinity constant, Km and the
rate of reaction, Vmax were determined, at the optimum pH and temperature, by linear
regression plots. (Lineweaver and Burk, 1934).

3.8 Enzyme Screening

3.8.1 Textile dyes

In order to establish whether the SRB would be viable and survive in the presence of the dyes
agar plates with azo dye concentrations ranging from 10-100 mg.l-1 were inoculated with SRB
mixed consortia and incubated at 20°C for 48h.

10
3.9 Induction Studies

3.9.1 Textile dyes

To induce and consequently increase enzyme activity (azoreductase/hydrogenase) the

inoculum of SRB in lactate media/agar was, initially, incubated with the azo dyes in
concentration of 100-400 mg.l-1. A time course survey was performed in order to determine
the greatest production of hydrogenase and/or azoreductase. Bioreactors were started with
five different dyes (orange II, reactive black 5, amido black 10, reactive red 120 and reactive
blue 2 (non-azo)).

3.10 Industrial Effluent Analysis

3.10.1 Textile industry

To test the principle of the process an industrial effluent from a textile industry was selected.
A visit was made to the Da Gama Textile Company, King William’s Town, Eastern Cape and
contact made with Mr Donald Tarr and Mr John Durrands. After a brief tour of the facilities
including the dyeing and printing processes and the Effluent Treatment Plant (ETP) samples
of dye mixtures were obtained at various stages: soluble dye mixture before being ‘fixed’
(D1); dye mixture after ‘fixing’ (D2); vat print rinse (D3); effluent dye mix (raw feed) before
ETP (D4).

3.11 Field Testing of the Product

3.11.1 Textile industry

To test the enzyme mixture for specificity in the bioremediation and decolourisation of the
effluent from the textile industry (Da Gama Textiles) a factorial matrix of 3 x 2 experiments
was set up (Table 3.1)

Table 3.1 A factorial matrix of experiments of the incubation of the enzyme from
various sources with both authentic and industrial effluent dyes.

Soluble extract or Enzymes from SRB from Enzymes from SRB

pure hydrogenase enzyme ‘pure’ lactate medium. BioSURE Process®
Authentic Dyes from Authentic Dyes from Authentic Dyes from
dyes industry dyes industry dyes industry

3.11.1.1 Soluble enzyme extract

Sulphate reducing bacteria cell-free soluble extract (1.0 ml) was incubated with either
industrial textile dye influent or effluent (D1, D2, D3 or D4) (5.0 ml) or an authentic dye
(orange II, reactive black 5, amido black 10, reactive red 120, reactive blue) (100 mg.l-1, 1.0
ml) for 24h. Duplicate samples were removed at timed intervals and monitored for dye
decolourisation at the respective wavelength.

11
3.11.1.2 Purified hydrogenase
Purified hydrogenase (1.0 ml) was added to a sealed flask containing either industrial effluent
(D1, D2, D3 or D4) (1.0 ml) or authentic dye (100 mg.l-1, 1.0 ml) for 24h. Duplicate samples
were removed at timed intervals and monitored for dye decolourisation at the respective
wavelength.

3.11.1.3 Whole SRB cells from a ‘pure’ lactate medium culture

SRB cells (0.5 g.l-1) in Tris-HCl buffer (20 mM, pH 7.6, 1.0 ml) previously cultured on a
lactate medium were incubated with either industrial textile dye influent/effluent (D1, D2, D3
or D4) (1.0 ml) or authentic dye (100 mg.l-1, 1.0 ml) for 24h. Duplicate samples were
removed at timed intervals and monitored for dye decolourisation at the respective
wavelength.

3.11.1.4 Whole SRB cells from a biosulphidogenic BioSURE Process® reactor

SRB cells (0.5 g.l-1) in Tris-HCl buffer (20 mM, pH 7.6, 1.0 ml) previously cultured on a
lactate medium were incubated with either industrial textile dye effluent (D1, D2, D3 or D4)
(1.0 ml) or authentic dye (100 mg.l-1, 1.0 ml) for 24h. Duplicate samples were removed at
timed intervals and monitored for dye decolourisation at the respective wavelength.

12
4 RESULTS

4.1 Reactor Operation

After a 15-day acclimation period the reactor performance was routinely assessed by the
determination of sulphate removal and a corresponding sulphide production. As expected the
sulphate levels dropped over the first 5 days from 1000 mg.l-1 to 400 mg.l-1 with a
concomitant increase in sulphide (Figure 4.1). Final concentrations of sulphide reached 160
mg.l-1 and there was a fair assumption that sulphate reduction was taking place, endorsed by a
corresponding and concomitant low CODSoluble that indicates a rapid utilisation of the soluble
fraction within the reactor. Within the reactor there were fluctuating values of CODTotal and
CODParticulate of between 1000 mg.l-1 and 1500 mg.l-1 and these indicate that some of the
organic matter within the reactor was non-biodegradable. The pH within the reactor
fluctuated slightly between 7.2 and 7.4.

1200 200
1000
Sulphate (mg/l)

Sulphide (mg/l)
150
800
600 100
400
50
200
0 0
0 5 10 15
Time (days)

Figure 4.1 Time survey to show typical sulphate (■) and sulphide (▲) levels in the
bioreactor

The facts that sulphate reduction was taking place, that the pH had remained fairly constant
and the COD levels, especially the soluble form, were low indicated that, after 15 days, the
reactor was under optimum conditions for the production of enzymes. After this time the
enzymes were extracted from the bioreactor and their specific activity and distribution
determined (Table 4.1).

It was interesting to note the yield (specific activity) of crude enzyme per ml sludge. From
this volume the amount of sludge required to completely decolour a specific volume of textile
effluent can be estimated. Table 4.1 reports typical enzyme activity per ml of sulphidogenic
sludge. Units of activity (U) are µmol.min-1 and since the original volume of anaerobic
sulphidogenic sludge was 100 ml (Section 3.3) the specific activity of the respective enzymes

13
can be estimated as µmol.min-1.ml sludge-1. In other words, for the case of the hydrogenase
enzyme, 1000 ml of sulphidogenic sludge (1 kg) would decolour 2.2 mols of azo dye (770
grams Orange II) in one minute.

Table 4.1 Typical enzyme activity and extracted from a biosulphidogenic reactor.

Enzyme Substrate Specific Activity

Endoglucanase CMC 22
Hydrogenase MV; OII 2200
Azoreductase OII; RB5 45
Lipase Triacetin 24
Protease Azocasein 1850
α - Glucosidase p-NP- α-G 1500
β - Glucosidase p-NP- β-G 300
Sulphatase p-NPS 1200
Phosphatase p-NPP 3000

4.2 Enzyme Extraction

Enzyme activity (protease, sulphatase, phosphatase, glucosidase, hydrogenase, azoreductase)

was found predominantly in the supernatants though there was slight activity of some
enzymes remaining in the pellet and these could not be removed.

4.3 Enzyme Distribution

It should be noted that the distribution values of the enzymes obtained from a
biosulphidogenic reactor are not consistent and vary from reactor to reactor.

4.4 Enzyme Purification

4.4.1 Glucosidase

The enzyme was purified to homogeneity on SDS-PAGE by acetone precipitation followed

by affinity (Whatman cellulose CC31) and gel exclusion (Sepharose 4B) (Figure 4.2) column
chromatography. Fractionation of the resuspended pellet after acetone precipitation on a
Whatman affinity cellulose CC-31 column yielded one major peak with specific activity of
108.4 U.mg-1, 1.8 fold purification and a yield of 34.6%. This pooled fraction was further
purified on Sepharose 4B gel exclusion column to afford a β-glucosidase in a 5 fold
purification and a 15% yield. One prominent protein band was observed by SDS-PAGE with
a molecular mass of 66 kDa with a second minor band being observed at 54 kDa. These
bands agreed with polypeptides from the cellulosome of other organisms and reported
elsewhere (Bayer et al., 1998).

14
 1.4 2
1.8

Absorbance @ 280 nm
1.2
1.6

Activity (nmol/min)
1 1.4
0.8 1.2
1
0.6 0.8
0.4 0.6
0.4
0.2
0.2
0 0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29
Fraction No.
protein activity
Figure 4.2 Purification of β-glucosidase with Sepharose 4B gel exclusion.

4.4.2 Hydrogenase/azoreductase

0.90 1.6
Protein concentration

0.80 0.3 M 1.4

0.70 1.2

Enzyme activity
0.60
(mg/ml)

(umol/min)
0.50
0.8
0.40
0.6
0.30
0.20 0.4
0.10 0.2
0.00 0
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77
Fraction No.
Protein Hydrogenase activity

Figure 4.3 Sephacryl S-200 ion exchange chromatography for hydrogenase after PEG
concentration. Column dimensions: 2 × 30 cm. Equilibrated with Tris HCl (pH
7.6, 50 mM); flow rate: 2.2 ml.min-1. Hydrogenase was eluted, with 0-0.3 M
NaCl gradient.

Concentrating the enzyme extract with PEG 20000 followed by chromatography on

Sephacryl 200 (size exclusion/ion exchange) gave one protein (identified by SDS-PAGE)
eluting at 0.08 M NaCl, with molecular mass of 54 kDa and with hydrogenase activity
(Figure 4.3).

15
Though there was over a 3-fold purification after this chromatographic step the yield was
only 32% and consequently this placed in doubt, from a commercial industrial point of view,
on the need to actually purify the enzymes. A reason why there was a level of uncertainty
about whether the enzyme was a hydrogenase or an azoreductase was because under the
conditions of the assay, though there was hydrogenase activity present (using hydrogen as
electron donor), no azo-reductase activity was found. This can be related to large difference
in redox potentials of specific mediators and/or the fact that NADH cofactor was the
hydrogen donor. It is believed that a hydrogenase enzyme had been purified and was capable
of reducing the azo –N=N-bond.

4.4.3 Endoglucanase

During the purification of the endoglucanase(s) it was realised that ammonium sulphate
fractionation led to total denaturation of the enzymes and consequently the enzyme was
concentrated with PEG 20000. Chromatography on DEAE – Cellulose gave five active
fractions (Figure 4.4) indicating that several enzymes were present. Fractions in the second
peak (between fractions 55 and 60) eluted in 0.5 M salt and the third peak (between fractions
100 and 110) eluted in 1 M salt had 88% of enzyme activity. These components were purified
13 and 25 fold with respective yields of 2.4 and 1.3% and specific activity of 6.5 and 12.7
U.mg-1 protein. The extract containing these endoglucanases arose from a complex
sulphidogenic bioreactor and so it was not surprising that several enzymes with varying
molecular mass and ionic charge existed.
1M
1 0.18
0.9 0.16
Absorbance @ 280 nm

0.8

Activity (umol/min/ml)
0.14
0.7
0.5 M 0.12
0.6
0.1
0.5
0.08
0.4
0.06
0.3
0.2 0.04

0.1 0.02
0 0
0 20 0.2540
M 60 80 100 120 140 160 180
Fractions (5 ml/tube)
Protein Activity

Figure 4.4 DEAE-Cellulose ion exchange chromatography for endoglucanase. Column

dimensions: 1 × 25 cm. Flow rate: 1 ml.min-1. Endoglucanases were eluted,
stepwise, with 0-1 M NaCl in sodium phosphate buffer (50 mM, pH 6).

4.5 Enzyme Properties

The optimum pH and temperature, the stability and kinetic properties of all of the enzymes
from the biosulphidogenic reactor are shown in Table 4.2.

16
Table 4.2 Properties of enzymes extracted from the biosulphidogenic reactor.

Enzyme Opt Opt Stability Vmax Km Substrate

pH T°C (% loss/hr) (µmol /min/ml) (µM)
Endoglucanase 6.0 50 0 0.3 4 CMC
Cellulase 5.4-7 55-60 95 ND ND CMC
Hydrogenase 7.5 40 60-70 1100 5.2 MV
Lipase 6-8 50-60 40-80 2.09 0.22 Triacetin
Protease 5-10 50-70 10-25 2.31 0.1 Azocasein
α – Glucosidase 6-7 55 70 0.85 0.16 p-NP- α-G
β – Glucosidase 5-7 60 70 0.49 0.19 p-NP- β-G
Sulphatase 6 50-60 40-65 119 23 p-NPS
Phosphatase 4.5 60 50 248 16 p-NPP
ND = Not determined

It was somewhat misleading that for the majority of enzymes greater than 50% of activity
was lost if the isolated enzyme was kept at its optimum temperature for an hour. This was not
the case if the enzyme remained in situ in the sulphidogenic bioreactor. The maximum
hydrogenase activity was observed at 40°C and at pH 7.5. A loss of hydrogenase activity of
25% and 80%, respectively, was noted with 10°C and 20°C change from optimum.

4.6 Enzyme Screening

4.6.1 Textile dyes

Control Test

Figure 4.5 Sulphate reducing bacteria degrading Orange II azo dye in agar
medium.

A control and test experiment with SRB on agar plates after having been incubated with
orange II dye is shown (Figure 4.5). With scrutiny of the test plate it can see that the SRB are
capable of not only surviving and growing in the presence of the dye (in this case orange II)
but there was also decolourisation of the dye as well. The first catabolic step in the reduction
of azo dyes, accompanied by decolourisation, is the reduction of the azo (-N=N-) bridge
(Figure 2.1, page 5).

17
Once the ability of SRB to grow in the presence of an azo dye had been demonstrated it was
necessary to then identify the enzymes and electron acceptors responsible for the reductive
cleavage of the azo bond. Lignin modifying enzymes (laccase, lignin and manganese
peroxidase) reputed to aerobically degrade azo dyes were, understandably, not found in the
anaerobic sulphate reducing environment of the bioreactor. During this exploratory study an
interesting result was forthcoming when the green azino bis ethane sulphonic acid (ABTS)
which, under normal circumstances, acts an indicator for laccase type enzymes was used as
the electron acceptor. Normally this dye rapidly turns a darker green in the presence of
laccase, a suitable substrate and oxygen. In our case, using SRB whole cells from the
biosulphidogenic reactor this dye was decoloured within a few minutes with an activity of
1.81 nmol.min-1. The possibility of this being a chemical reaction, due to the presence of
sulphide in the soluble extracts, was ruled out since the decolorisation still persisted
(measured activity of 1.67 nmol.min-1) even after the sulphide had been removed with zinc
acetate. The fact that the SRB whole cells may contain either a hydrogenase or an azo
reductase to ‘reduce’ ABTS is supported since after the extract had been boiled (to destroy
the enzyme) the enzyme activity decreased to 0.31 nmol.min-1.

4.7 Induction Studies

4.7.1 Textile dyes

4
Specific activity (U/mg)

0
0 2 4 6 8 10 12
Time (days)

Figure 4.6 Time course survey of hydrogenase activity produced by SRB.

18
Without any induction of the enzymes the highest hydrogenase activity (specific activity 3.0
U.mg-1) was observed after 6 days (Figure 4.6) while in the presence of orange II the greatest
yield of enzyme was found after 24h with specific activity of 4.7 U.mg-1 (Figure 4.7). The
enzyme activity decreases after an initial 24h due to the fact that product metabolites
(aromatic amines from the azo dyes) may act as potential inhibitors of the hydrogenase
enzymes.

5
Specific activity (U/mg)

0
1 2 3 4 5 6 7
Time (days)

Figure 4.7 Time course survey of hydrogenase activity produced by SRB after been
induced in the presence of orange II reactive dye.

180 7
160 6
Relative Actvity (%)

140
5 Time (days)
120
100 4
80 3
60
2
40
20 1
0 0
C CD 1 O II AB 10 Mix RB 5 RR RB 2
120
Relative Activity (% ) Time (days)

Figure 4.8 Induction of hydrogenase in the presence of commercial dyes. Relative

activity (100%) is 3.0 U.mg-1 achieved at day 6. C represents the maximum
activity of hydrogenase in the absence of dye and CD 1 (control (day 1))
represents hydrogenase activity after one day in the absence of dye. O II
(orange II); AB 10 (amido black 10); Mix (mixture of all dyes); RB 5 (reactive
black 5); RR 120 (reactive red 120); RB 2 (reactive blue 2).

19
It is interesting to note that, in the control at day one there was only 25% of the activity
whilst, with the induced sample, the activity was at 160% over the same time period (Figure
4.8). The same applied for Amido Black 10 and the mixture of the dyes. Although, the overall
yield in hydrogenase activity was not as high as the control for reactive black 5 and reactive
red 120, it is still evident that there was increased enzyme activity (78 and 45% respectively)
after only one day of incubation. It can therefore be deduced that the presence of azo dyes
induced the production or activation of hydrogenase activity. Induction with reactive blue 2,
which is an anthraquinone dye, yields the highest activity of 0.8 U.mg-1 after 6 days and
consequently hydrogenase is not induced by this dye as the specific activity levels were lower
than the control.

4.8 Industrial Effluent Analysis

4.8.1 Textile industry

In the textile industry the diversity and magnitude of dyes and colours is extremely vast and
so analysis and characterisation of the industrial samples from Da Gama Textiles, King
William’s Town was performed to gain an insight into their constituents. A total UV/Vis scan
between 200-700 nm of D1 dye mixture (Figure 4.9) revealed the presence of various
components with absorbance maxima at 270, 290, 400, 450, 490, 515, 600 nm. Since Da
Gama Textiles could only specify a ‘code’ used by the dye manufacturers the concentrations
of the dye was represented as COD values and was 178260 mg.l-1. The pH was 5.28.
Absorbance peaks observed in the range 215-260 nm are likely to be representative of
aromatic chromophores

1.4
1.2
1
Absorbance

0.8
0.6
0.4
0.2
0
200 250 300 350 400 450 500 550 600 650 700
Wavelength (nm)
D1 D2 D3 D4

Figure 4.9 UV/Vis scan between 200-700 nm of a mixture of reactive dyes (D1) used for
dyeing textile fibres at Da Gama Textiles; reactive dyes with silicates and
caustic alkali (D2); vat print rinse (D3) and waste produced (D4) after dyeing
before the Environmental Treatment Plant.

within the dye. Peaks in the range 400-490 nm are representative of green, yellow, orange
and red dyes and those in the range of 500-600 nm are indicative of purple, violet and blue
20
dyes. This dye mixture was ‘fixed’ with caustic soda to increase the pH to 11.14 and sodium
silicates to optimise the fixing of the dyes on to the fabric (D2) (Figure 4.9). There was a
slight dilution of the dye on addition of silicates and caustic soda as the COD levels dropped
to 131427 mg.l-1. The absorbance peaks recorded electronically were 215, 260, 290, 400, 450,
490, 515, 600, 625 nm. A mixture of insoluble vat dyes and soluble reactive dyes that did not
absorb to the fabric during the dye process (D3) is also illustrated (Figure 4.9). The scan
shows absorbance peaks at 230, 240, 250, 310 and 510 nm and the concentration of this
effluent was 1378 mg.l-1 and the pH was 4.35. The raw dyes and materials remaining after the
whole textile dyeing process before it goes to the Environmental Treatment Plant (ETP) (D4)
is shown (Figure 4.9). It includes dyes that have not been adsorbed onto the fibre, fabric
material, silicates, caustic soda and rinse water from all of the different stages of the process.
It was very dilute with a COD of 2520.5 mg.l-1 and a pH of 11.14. A scan of this effluent
shows peaks at 215, 230, 275, 290, 385, 600 nm.

200 14
178.26
180
12
160
COD (mg/l) [x 1000]

140 131.427 10
120 8

pH
100
80 6
60 4
40
2
20 1.378 2.52
0 0
Dye - No Alkali Dye- Vat Print Raw Feed to
Silicate/NaOH Rinse ETP

COD pH
Figure 4.10 Concentrations of the dyes and the effluents measured by determining the
chemical oxygen demand (COD). The pH of the dyes and effluents was also
measured.

The dye mixture without alkali had the highest concentration (COD) of 178260 mg.l-1 (Figure
4.10). Addition of silicate salts and sodium hydroxide into the dye mixture are necessary to
enhance both the solubility of the dyes and their fixation onto the fabric. While it is apparent
that an increased pH is necessary for dye application, biological treatment demands mild
alkaline conditions for optimal bioreactor performance, leading to slight neutralisation of the
reactive mixtures. It remains to be answered what consequences these additives have on
downstream bioremediation of the effluent.

21
4.9 Field Testing

Both the industrial textile dye effluent and sewage sludge are dark complex mixtures of many
materials and substances. Consequently it was necessary to follow protocols that included
various control reactions in order to answer the following questions:

1) Can purified hydrogenase and/or SRB cell-free extract degrade and decolour both
authentic dyes (orange II, amido black 10, reactive black 2, reactive black 5, reactive
red 120) and dyes from the textile industry?

2) Can SRB from a ‘pure’ culture degrade the same authentic commercial dyes?

3) Can this ‘pure’ culture decolour and degrade typical industrial textile dye effluent?

4) Can SRB, which had been isolated from the Biosure sewage sludge, bioremediate the
same authentic commercial textile dyes as well as the dyes from the industrial
effluents?

4.9.1 Textile industry

4.9.1.1 Purified hydrogenase and/or cell-free extract with authentic dyes

Over the incubation period of 24h there was absolutely no decolourisation of the authentic
dye with either the purified hydrogenase enzyme or the SRB soluble extract. In view of this
and the fact that the industrial effluents (D1, D2, D3 and D4) were extremely dark in colour
and non-transparent it must be assumed that both the purified enzyme and the cell-free extract
had failed to decolour this industrial effluent as well. Consequently attention turned to whole
SRB cells.

4.9.1.2 SRB whole cells from a ‘pure’ lactate medium with authentic dyes
Over a period of 24h and using dye concentrations of 100 mg.l-1 decolourisation of 92%
(orange II), 89% (reactive black 5), 88% (amido black 10), 64% (reactive red 120) and only
15% (reactive blue 2) were observed (Figures 4.11 and 4.12). When a mixture of dyes at 100
mg.l-1 each (i.e. total of 500 mg.l-1) was incubated with SRB the decolourisation decreased
slightly to 88%, 82%, 56%, 42% and 12% respectively. With dye concentrations of 200 mg.l-
1
each (i.e. total of 1000 mg.l-1) these decolourisation decreased to 52% (orange II), 44%
(reactive black 5), 38% (amido black 10), 37% (reactive red 120), and 10% (reactive blue 2).
It was observed that the decolourisation rates decreased with increase in dye concentration.
The highest decolorisation occurred within 24h for all dyes except Reactive Blue 2 which
remained undegraded during the experiment.

22
 100
90
Decolorisation rates (%)
80
70
60
50
40
30
20
10
0
Orange II Reactive Amido Reactive Reactive
Black 5 Black 10 Red 120 Blue 2

single-100mg/l mix 1-100mg/l mix 3-200mg/l

Figure 4.11 Sulphate reducing bacteria was incubated with single commercial dyes as well
as mixtures ranging from 100 mg.l-1 to 200 mg.l-1 for each dye over a period
of 24h.

100
90
Decolourisation rates (%)

80
70
60
50
40
30
20
10
0
100 500 1000
Dye Concentration (mg/l)
Orange II Amido Black 10 Reactive Black 5
Reactive Red 120 Reactive Blue 2

Figure 4.12 Effect of dye concentration on dye decolourisation efficiency over 24h.

23
4.9.1.3 SRB from the BioSURE Process® and Da Gama textiles dyes and effluents.

0.8

0.6
Absorbance

0.4

0.2

0
200 250 300 350 400 450 500 550 600 650 700
Wavelength (nm)
0 hrs 8 hrs 16 hrs

Figure 4.13 Anaerobic degradation of D1 by SRB with respect to time.

The decolourisation, monitored at 550 nm, of the Da Gama Textile soluble dye mixture (D1)
by SRB from the BioSURE process was 92% over a 16h period (Figure 4.13) and 86% for
the dye mixture containing silicates (D2) (Figure 4.14). Furthermore evidence for dye
degradation was reflected in the increase in concentration of aromatic compounds absorbing
at 280 nm (indicative of a break in the conjugation of the azo bond) by 234% over 8-6h.

1.2

1
Absorbance

0.8

0.6

0.4

0.2

0
200 250 300 350 400 450 500 550 600 650 700
Wavelength (nm)
0 hrs 8 hrs 16 hrs
Figure 4.14 Anaerobic degradation of D2 by SRB with respect to time.

There appeared to be total breakdown of all compounds with respect to time across the whole
range of the UV-Vis spectrum. With D3 (Figure 4.15) there was a 51% decolourisation over
two days, monitored at 530 nm, and a 26% increase, over 6 days, in the concentration of the
aromatic compounds absorbing at 280 nm while with D4 (Figure 4.16) there was 37%
decolourisation (at 530 nm) over 2 days and a 26% increase, in 4 days, in the concentration of
aromatic compounds absorbing at 280 nm. It was interesting to note that the absorbance due

24
to these aromatic amines decreased in concentration reflecting that there were other unrelated
factors within the BioSURE Process® sludge that were responsible for their degradation.

1.6
Absorbance

1.2

0.8

0.4

0
200 250 300 350 400 450 500 550 600 650 700
Wavelength (nm)
0 days 2 days 4 days 6 days

Figure 4.15 Anaerobic degradation of D3 by SRB with respect to time.

1.6
1.4
1.2
Absorbance

1
0.8
0.6
0.4
0.2
0
200 250 300 350 400 450 500 550 600 650 700
Wavelength (nm)
0 days 2 days 4 days 6 days

Figure 4.16 Anaerobic degradation of D4 by SRB with respect to time.

4.9.1.4 SRB from the BioSURE Process® and authentic dyes

When the SRB from the BioSURE Process® was treated with Orange II a 60%
decolourisation (monitored between 490-530 nm) was realised after 6-8 days of incubation
(Figures 4.17 and 4.18). After 2 days of incubation there was a sharp increase in absorbance
at 280 nm which indicated an accumulation of aromatic compounds as a result of azo dye

25
degradation into single aromatic amines. This peak at 280 nm decreased after 6 days of
incubation.

1.6
Absorbance

1.2

0.8

0.4

0
200 250 300 350 400 450 500 550 600 650 700
Wavelength (nm)
0 days 2 days 4 days 6 days

Figure 4.17 Degradation of Orange II and disappearance of aromatic amines by SRB

cultured in BioSURE Process® sludge.

0.9
0.8
0.7
Absorbance 490nm

0.6
0.5
0.4
0.3
0.2
0.1
0
0 2 4 6 8 10
Time (days)
Sludge Biomass lactate

Figure 4.18 Decolourisation of orange II by SRB from BioSURE Process® with respect to
time. A comparison is made with the respective decolourisation using SRB
from a pure lactate medium.

26
 1.2

1
Absorbance
0.8

0.6

0.4

0.2

0
200 250 300 350 400 450 500 550 600 650 700
Wavelength (nm)
0 days 2 days 4 days 6 days

Figure 4.19 Anaerobic degradation of Amido Black 10 by SRB from the BioSURE
Process®.

1.6
Absorbance 620nm

1.2

0.8

0.4

0
0 2 4 6 8 10
Time (days)
sludge lactate
Figure 4.20 Anaerobic degradation of Amido Black 10 with SRB from BioSURE
Process® with respect to time. A comparison is made using SRB from a pure
lactate medium.

With Amido Black 10, 50% anaerobic decolourisation (monitored at 620 nm) occurred within
48h with a loss of a further 42% colour after a further 48h (Figures 4.19 and 4.20).

Reactive Black 5 was also decoloured with 75% colour removal, at 595 nm, after 48h
incubation (Figures 4.21 and 4.22).

27
 2

Absorbance 1.6

1.2

0.8

0.4

0
200 250 300 350 400 450 500 550 600 650 700
Wavelength (nm)
0 days 2 days 4 days 6 days

Figure 4.21 Anaerobic degradation of Reactive Black 5 by SRB from the BioSURE
Process®.

1.2

1
Absorbance 595nm

0.8

0.6

0.4

0.2

0
0 2 4 6 8 10
Time (days)
Sludge lactate

Figure 4..22 Anaerobic degradation of Reactive Black 5 with SRB from BioSURE
Process®. A comparison is made using SRB from a pure lactate medium.

28
With Reactive Red 120 there was an initial slow decolourisation, monitored at 525 nm, of
48% by day 2/4 which then increased to 68% by day 6 and then to 78% by day 8 after which
there is no further degradation (Figures 4.23 and 4.24).

1.6

1.2
Absorbance

0.8

0.4

0
200 250 300 350 400 450 500 550 600 650 700
Wavelength (nm)
0 days 2 days 4 days 6 days

Figure 4.23 Anaerobic degradation of Reactive Red 120 by SRB from BioSURE
Process®.

0.9
0.8
0.7
Absorbance 530nm

0.6
0.5
0.4
0.3
0.2
0.1
0
0 2 4 6 8 10
Time (days)
Sludge lactate

Figure 4..24 Anaerobic degradation of Reactive Red 120 with SRB from BioSURE
Process ®. A comparison is made using SRB from a pure lactate medium.

The slower rates of decolourisation generally observed with Amido Black 10, Reactive Black
5 and Reactive Red 120 are probably as a result of the double azo linkages that make up the
structure and consequently require more reducing equivalents to effect complete bond
cleavage and subsequent colour removal. Orange II has only one azo link and was reduced
and decoloured relatively quickly. The absorbance at 280 nm, which corresponds to the single

29
aromatic molecules, initially increased then gradually decreased at day 6. Once again it is
suspected that there may be a degradation of the dyes by unknown factors that exist within
the BioSURE Process®.

1.5

1.2
Absorbance

0.9

0.6

0.3

0
200 250 300 350 400 450 500 550 600 650 700
Wavelength (nm)
0 days 2 days 4 days 6 days 8 days

Figure 4.25 Anaerobic degradation of Reactive Blue 2 by SRB from the BioSURE
Process®

1.2

1
Absorbance 610nm

0.8

0.6

0.4

0.2

0
0 2 4 6 8 10
Time (days)
sludge lactate
Figure 4.26 Anaerobic degradation of Reactive Blue 2 with SRB from BioSURE
Process®. A comparison is made using SRB from a pure lactate medium.

With Reactive Blue 2 there was negligible decolourisation of this non-azo anthraquinone dye
up to day 6. This was followed by over 96% decolourisation by day 8 and it is inferred that
this was due to the induction of enzymes that degrade these types of dyes (Figures 4.25 and

30
4.26). Furthermore there was a sudden decrease in absorbance at 280 nm which could imply
that the simple aromatic degradation compounds acted as electron donors for the SRB.
Interestingly it was noticed that in all cases there was a decrease in A280 concomitant with a
decrease in colour reflecting the total loss of aromatic compounds.

It was pleasing to note that the red colour of Reactive Red 120, and the blue colour of
Reactive Blue 2, had been completely removed at the end of 10 days (Figure 4.27 and 4.28).

Figure 4.27 Progressive removal of Reactive Red 120 by SRB from the BioSURE
Process®. The numbers represent days of incubation.

Figure 4.28 Progressive removal of Reactive Blue 2 by SRB from the BioSURE Process®.
The numbers represent days of incubation.

31
4.9.1.5 SRB from ‘pure’ lactate medium with Da Gama textiles dyes and effluents
As a final control experiment experiments were conducted with SRB that had been cultured
from a ‘pure’ lactate medium and the industrial textile dye influents/effluents from Da Gama
Textiles. A decolourisation rate of 93% was achieved in 24h for the mixture of the reactive
dyes (Figure 4.29) (D1) which is a similar result to the authentic dyes reported earlier. The
addition of salts and alkali did, however, affect the rate of decolourisation to decrease to 72%
(D2). Since the presence of the alkali was not significant as the pH had been neutralised
before starting the bioreactors, the presence of the silicate salts must have contributed to the
decreased efficiency. Perhaps these silicates affected the structural moiety of the
hydrogenases and since their concentration was considered dilute, in order to meet the
requirements for bacterial incubation, there was not complete inhibition of enzyme action.
Decolourisation, at 490 nm, of the effluents (vat print rinse (D3) and raw feed to the ETP
(D4)) were low at 52% and 41% respectively. Unfortunately no determination of the
absorbance maximum at 280 nm was determined and so the levels of single aromatic
compounds was not determined.

100
% Decolourisation

80

60

40

20

0
D1 D2 D3 D4
Da Gama textiles influent/effluent

Figure 4.29 Decolourisation rates of Da Gama dyes and effluents by SRB from pure
lactate media after 24h and monitored at 590 nm.

32
5 DISCUSSION

The initial idea behind this project was to develop a powdered cocktail of enzymes, extracted
from a sulphidogenic bioreactor, that could be used, in situ, to bioremediate industrial waste-
water. Furthermore the exorbitant cost of commercial enzymes makes this approach fairly
favourable and cost effective. In conjunction with this idea was the fact that ordinary
municipal sewage sludge, obtained through the Rhodes BioSURE Process® can be used as a
prime source of carbon for the sulphate reducing bacteria that exist in the reactor. These SRB,
along with the ubiquitous methanogenic bacteria, are responsible for the production of these
so called “cocktail of enzymes”. There is an added advantage with this system since we have
found, in our laboratories, that the activities of all of these enzymes are enhanced in a
sulphide environment as would be the case in the sulphidogenic reactor.

Industrial effluents that were intended targets included paper and pulp, tanneries, olive mill,
textile dye, petroleum, abattoir, fishing, mining and wine distilling. Though the production of
several hydrolase enzymes (glucosidases, proteases, lipases) within this ‘cocktail of enzymes’
has met with considerable success with respect to the defouling of membranes associated
with the bioremediation of abattoir effluents there was virtually no success when it came to
water recovery from the textile industry. The idea of a ‘cocktail of enzymes’ implies a
bacterial cell-free powdered preparation of crude enzymes. In the case of the hydrolases the
crude enzyme mixture is self sustaining and their respective reactions can occur without any
necessary cofactors. In view of the fact that many of the coloured dyes used in the textile
industry contain the ‘azo’ bond (-N=N-) we have established that one of the enzymes
responsible for the decolourisation is indeed either an azoreductase or a hydrogenase. Both of
these enzymes require specific cofactors for their operation. Though the cell-free extract gave
a positive assay for hydrogenase (since the cofactor is a constituent of the assay) it failed to
decolour any of the authentic dyes. Furthermore these cofactors would not have been
extracted into the purified powdered cocktail of enzymes. This led us to examine the SRB
cells as a whole or rather the SRB cells from within the BioSURE Process® sludge itself.
Indeed we found that, with the authentic dyes (Orange II, Reactive Black 5, Amido Black 10
and Reactive Red 120) there was a fairly rapid decolourisation when the dyes were added to
the sulphidogenic reactor containing SRB from the BioSURE Process® sludge. The non azo
dye (Reactive Blue 2) took considerably longer to decolour since it was suspected that ‘other’
unknown enzymes had to be induced for the initial degradation of the aromatic carbon
skeleton.

The process of decolourisation for each of the dyes can be monitored by a decrease in
absorbance at the λmax of the inherent chromophore. This is supported by a reduction of the
azo link into two colourless aromatic amine compounds. At the same time as there is a
decrease in absorbance of the dye in the visible region (480-610 nm) there is an increase in
the absorbance at 280 nm reflecting an increase in concentration of single aromatic amines.
With an extended period of time there was a subsequent decrease in the absorbance at 280 nm
indicating that the aromatic amines had been degraded further, perhaps by some other

33
unknown factor, into CO2, H2O and NH3. This observation was especially noticeable with
authentic dyes in the sulphidogenic bioreactor containing SRB from the BioSURE Process®
sludge.

When this system and set up was tested with dyes from an industrial enterprise (Da Gama
Textiles, King William’s Town) a similar result was forthcoming. Four industrial textile dye
samples were tested in the biosulphidogenic reactor containing SRB from the BioSURE
Process® sludge. Two, regarded as influent, that consisted of a pre-dye mixture and another
that is ‘fixed’ with caustic soda and silicates and two, regarded as effluents, consisting of a
vat print rinse and a final effluent (after all of the dyeing processes) just prior to passing into
the Environmental Treatment Plant (ETP).

All four of the industrial samples followed similar trends to the authentic dyes in that:

1) there was decolourisation of the dye(s), monitored by a decrease of absorbance in the

visible region (480-610 nm).

2) there was an increase in absorbance at 280 nm due to an increase in aromatic compounds.

3) there was subsequent decrease in absorbance at 280 nm due to a total breakdown of these
aromatic compounds.

As a control measure the effect of a ‘pure’ culture of SRB (using lactate medium as a carbon
source) on both authentic dyes and on the various influents and effluents from the textile
industry was studied. In all cases, the time taken to degrade the dyes using SRB from
BioSURE Process® sludge in the sulphidogenic bioreactor was considerably longer than if
the SRB were used from a ‘pure’ culture. It is interesting to reiterate that with ‘pure’ SRB
from a culture on lactate medium there was very little breakdown of the single aromatic
compounds as the absorbance at 280 nm remained fairly significant. This was evident with
both authentic dyes and industrial samples. With SRB from the BioSURE Process® sludge
there was complete degradation and a subsequent removal of the aromatic compounds
absorbing at 280 nm. It supports other factors, within the sulphidogenic reactor, that may be
responsible for complete degradation. It is hypothesised that an anaerobic degradation of the
dyes into their constituent aromatic amines followed by an aerobic degradation into CO2,
H2O and NH3 (Figure 5.1). With the ‘pure’ SRB system this is not the case.

34
 R N N R* Azo dye

Anaerobic Aerobic

R NH2 + H2N R* Aromatic amines

O2 Autoxidation

Anaerobic Aerobic
CO2 + H2O + NH3

Figure 5.1 Anaerobic – aerobic bacterial azo dye degradation.

35
6 CONCLUSIONS AND RECOMMENDATIONS

6.1 Conclusions

Successful decolourisation of both commercial dyes and industrial effluents with

SRB-BioSURE Process® sludge was achieved with decolourisations ranging from 96-49%
over a 6-8 day period. At the same time as there is a decrease in absorbance of the dye in the
visible region (480-610 nm) there is an increase in the absorbance at 280 nm reflecting an
increase in concentration of single aromatic amines. With an extended period of time there
was a subsequent decrease in the absorbance at 280 nm indicating that the aromatic amines
had been degraded further.

1) There was decolourisation of the dye(s), monitored by a decrease of absorbance in the

visible region (480-620 nm).

2) There was an increase in absorbance at 280 nm due to an increase in aromatic amines.

3) There was subsequent decrease in absorbance at 280 nm due to a total breakdown of these
aromatic compounds.

With ‘pure’ SRB, from a culture that had been grown on a lactate medium, there was
complete decolourisation of both authentic dyes and industrial influents and effluents as
monitored by the decrease of absorbance in the visible region (480-620 nm). There was,
however, very little breakdown of the single aromatic compounds as the absorbance at 280
nm remained fairly significant. It supports that, within the sulphidogenic reactor, there may
be other factors that are responsible for complete degradation.

6.2 Recommendations

Purified hydrolase enzymes, extracted from a sulphidogenic bioreactor can be concentrated

into a dried powdered cocktail preparation, using established concentration techniques.
Though this powdered extract was suitable to bioremediate certain industrial wastewaters
such as abattoir effluents and acid mine drainage, they failed to decolour and degrade dyes
from the textile industry.

It is the recommendation from this project that in order to completely decolour and degrade
the azo dyes from an industrial waste effluent, a dried powdered extract of SRB-BioSURE
Process® sludge from a biosulphidogenic reactor, including all of the necessary enzymes and
cofactors, in situ be used.

36
7 FUTURE RESEARCH

7.1. To identify, by HPLC, the individual aromatic amines produced from the degradation
of the dyes and then undergo a thorough analytical investigation of the specific extracts from
within the sulphidogenic bioreactor, thereby establishing the mechanism for total
degradation.

7.2. A UV-Vis scan of the effluent from the textile industry after passing through the
environmental treatment process (ETP) has only one major absorbance peak at 220 nm.
Though it is uncertain what this compound may be, the treatment of this effluent product by
SRB-BioSURE Process® sludge may remove it completely.

7.3. Produce a freeze-dried ‘powder’ of the SRB-BioSure sludge and test the sample with
both authentic dyes and with industrial effluents in order to establish the conditions required
for complete dye degradation.

37
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9 CAPACITY BUILDING

A resource base in the fields of enzymology and industrial wastewater treatment relating to
biological processes will be established providing support in cognate research areas in South
Africa. Student training and corporate technology collaboration will contribute to capacity in
wastewater management, the bioremediation of industrial effluents including pulp and paper,
textiles and dyes, abattoir, tannery, metallo industries and in the treatment of acid mine
drainage. One black Zimbabwe student (Mr Mutambanengwe) has obtained his MSc in April
2007. This project will help to conserve water through technology improvement in the
bioremediation of industrial effluents. Awareness to water research infrastructure, industrial
effluent disposal and water treatment will be made.

9.1 Students

9.1.1 MSc Students

S Oyekola Role of the enzymes in the cellulosome in the bioremediation of industrial

effluents.
CCZ Mutambanengwe Role of hydrogenase enzyme in the bioremediation of textile
effluents.

9.1.2 Post doctoral fellow

CA Togo. Total analysis of the degradation products of textile azo dyes by enzymes from a
biosulphidogenic reactor

9.2 Publications

9.2.1 Papers

Oyekola, OO, Ngesi, N, Whiteley, CG.

Isolation, purification and characterization of an endoglucanase and β-glucosidase from an
anaerobic sulphidogenic bioreactor.
Enz. Microbiol. Tech. 40 (8), 637-644, 2007.

Mutambanengwe, CCZ, Togo, CA, Whiteley, CG

Biosulphidogenic hydrogenases: Bioremediation of textile dye effluent.
Submitted, Enz. Microbiol. Tech. 2007.

Mutambanengwe, CCZ, Ngwenya, N, Rashamuse, KJ, Whiteley, CG.

Biosulphidogenic hydrogenases: Purification and properties
Submitted, Enz. Microbiol. Tech. 2007.

Togo, CA, Whiteley CG.

Total analysis of the degradation products of textile azo dyes by enzymes from a
biosulphidogenic reactor (In preparation)

41
9.2.2 Conference presentations

Mutambanengwe, CCZ, Whiteley, CG.

Novel hydrogenases from Sulphate Reducing Bacteria: Decolourisation of industrial
wastewater effluents
XVIII Enzyme Engineering, Gyeong-Ju, SOUTH KOREA, 2005

Mutambanengwe, CCZ, Whiteley, CG

Use of sludge biomass in the production of enzymes under sulphidogenic conditions for the
treatment of textile effluent.
SASBMB, 19, 281, Stellenbosch, SOUTH AFRICA, 2005

Oyekola, OO, Pletschke, BI, Whiteley, CG

Isolation, purification and characterization of an endoglucanase from an anaerobic
sulphidogenic bioreactor.
WISA, Cape Town, SOUTH AFRICA, 2004

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