1-Hydroxyanthraquinone:
Activity in Paracoccus denitrificans and potential application for biomass reduction
in wastewater treatment facilities
A Thesis Submitted to the Faculty
of
Drexel University
By
Lynn Ann Arlauskas-Dekleva
In Partial Fulfillment of the Requirement
For the Degree of
Doctor of Philosophy
February 2007
ii
Dedication
This work is dedicated to the memory of my mother, Florence Arlauskas. She always felt that an education was the key to achieving your full potential and strongly supported my pursuits in both my undergraduate and graduate education. My mother and friend- this one is for you!
iii
Acknowledgments
This work is the result of a great many people who dedicated their time and effort to support my goal. Over the years, I have relied heavily on the advice and encouragement of my advisor, Dr. Michael Gealt. His patience, guidance and support enabled me to persevere and finally complete this body of work. I also wish to thank my other committee members; Dr. Charles Haas, Dr. Susan Kilham, Dr. Irene Legiec and Dr.
Raj Mutharasan for their time and valuable suggestions that helped direct and enrich this work. I appreciate the many hours you dedicated to reviewing and discussing the issues and wish to thank you for all of your suggestions.
This research is an extension of the pioneering work by Dr. Martin Odom who provided valuable guidance on the use of this compound in denitrifying systems. Martin provided priceless assistance and free access to both his laboratory and personnel. I wish to thank Martin‘s laboratory associates, Eva Nagel, JoAnn Payne, and Jolanta Wilczek who assisted in the setup and analysis of the initial laboratory investigations and welcomed me into their laboratory.
The DuPont company wastewater expert, Robert Reich, provided valuable information on industrial wastewater treatment facilities and insight into the potential and limitations associated with the implementation of the uncoupler technology in an industrial setting. His vast knowledge and hands-on experience were critical to the success of this program and I truly enjoyed the numerous and sometimes lengthy interactions. I wish to thank the head of the wastewater laboratory, Marge Ventura, and iv
her laboratory associates. They provided a wealth of information and support especially in the setup and maintenance of the wastewater reactors and gracefully looked the other way during the numerous midnight acquisitions.
I wish to thank Dr. Lisa Laffend for all her help and support. Lisa served as a member of my committee for my Candidacy exam and supported this work through its completion. Lisa‘s support as a colleague and friend was invaluable. She was always available for discussions on recent laboratory results, provided valuable guidance on the continuous reactor experiments and encouraged me to continue when things go tough.
Thanks to Lisa‘s laboratory associates, Keith Burlew and Keith Cantera, for their guidance and assistance with the setup and running of the continuous reactor.
I wish to acknowledge Dr. John Gannon, Dr. Ning Wang and Patrick Folsom for allowing free and unrestricted access to the respirometer, known affectionately as
―Ralph‖, and the laboratory facilities at the DuPont Glasgow site. The aerobic studies in this work could not have been possible without their support.
I wish to acknowledge the assistance of many individuals who were called on numerous times for their input, guidance and support. Steve Constable who provided information on biosolids production and disposal costs; Dr. Barabara Larsen and Beth
Fenner for analytical information, analysis and support; Dr. David Short and Sid Bledsoe for their help with the continuous reactor studies; Reggie Corbitt and Stan Miller for providing information on the operation of the Little Rock Wastewater Treatment facility;
Jim Hobsetter for access to information on municipal waste treatment facilities; my management, Dr. David Griffith, Dr. David Short, Dr. Julie Rakestraw, Dr. John Gannon v
and Dr. Michael Hennessey for their support of my graduate education; my first DuPont supervisor, Dr. Dale Peterson, who saw and encouraged my questioning nature; and Dr.
Henn Kilkson who identified the engineer deep inside my soul. Henn‘s encouragement of my graduate education was unwavering although it initially began in the Biological
Sciences.
Last but not least, I wish to thank and acknowledge the role of my family. My husband, Mark Dekleva and children Tyler and Garrison have always supported and encouraged my educational pursuits. They stood by during long sessions at the laboratory and laptop knowing how much this work meant to me. This degree is as much mine as theirs; I truly couldn‘t have done it without their love and support. Thanks boys!!! vi
Table of Contents
List of Tables ...... ix List of Figures ...... xi Abstract ...... xiii Chapter 1 Problem Description ...... 1 References ...... 3 Chapter 2 Effect of Chemical Uncouplers on Microbial Respiration and Biomass Production: Implications for their use in Wastewater Treatment Facilities ...... 8 Introduction ...... 8 Respiration ...... 10 Denitrification ...... 16 The Process of ATP Synthesis and Its Uncoupling ...... 20 Application of Chemical Uncouplers to Activated Sludge Processes ...... 29 References ...... 36 Chapter 3 The activity of 1-hydroxyanthraquinone on anaerobic respiration in Paracoccus denitrificans...... 49 Abstract ...... 49 Introduction ...... 49 Materials and Methods ...... 57 Organism and Growth Conditions ...... 57 Chemicals and Stock Solutions ...... 58 Experimental Procedures ...... 59 Results and Discussion ...... 61 Comparison of 1-Hydroxyanthraquinone with respiratory inhibitors and uncouplers of oxidative phosphorylation on denitrifying cultures of Paracoccus denitrificans .. 61 vii
Effect of 1-Hydroxyanthraquinone, 9,10-Anthraquinone and 2,4-Dinitrophenol on anaerobic cultures of Paracoccus denitrificans using nitrate or nitrite as the terminal electron acceptor ...... 67 References ...... 79 Chapter 4 Effect of 1-hydroxyanthraquinone on biomass accumulation and oxygen utilization in Paracoccus denitrificans and in mixed wastewater cultures ...... 91 Abstract ...... 91 Introduction ...... 91 Materials and Methods ...... 99 Organisms and Growth Conditions ...... 99 Paracoccus...... 99 Activated Sludge Cultures ...... 99 Chemicals and Stock Solutions ...... 100 Experimental Procedures ...... 101 Respirometry Studies ...... 101 Paracoccus Continuous Studies ...... 103 Biomass Determinations ...... 105 Results ...... 106 Effect of OHAQ on Paracoccus ...... 106 Paracoccus Substitution Studies ...... 109 Paracoccus Continuous Culture Studies ...... 114 Mixed Culture Batch Studies...... 116 Discussion ...... 118 References ...... 127 Chapter 5 The economic implications of metabolic uncoupling in wastewater treatment facilities ...... 137 References ...... 148 Chapter 6 Summary and Conclusions ...... 152 Chapter 7 Barriers to Implementation...... 155 viii
Lack of Pilot Scale Demonstrations ...... 155 Acclimation ...... 156 Effluent Discharges to Environment ...... 158 References ...... 160
ix
List of Tables
Chapter 2 Effect of Chemical Uncouplers on Microbial Respiration and Biomass Production: Implications for their use in Wastewater Treatment Facilities
Table 1: Classic Uncouplers of Respiration...... 28
Chapter 3 The activity of 1-hydroxyanthraquinone on anaerobic respiration in Paracoccus denitrificans
Table 1: Respiratory Inhibitors and Uncouplers of Oxidative Phosphorylation Studied in Pa. denitrificans under Denitrifying Conditions with Nitrate as the Electron Acceptor ...... 62
Table 2: Nitrogen production (mean mMoles N2/bottle) of Pa. denitrificans with 10 mg/L OHAQ, electron transport inhibitors (rotenone, NBD-Cl, and antimycin), ATPase inhibitors (DCC, Tinopal) and uncouplers of oxidative phosphorylation (DNP, FCCP and CCCP)...... 63
Table 3: The effect of 1-hydroxyanthraquinone (OHAQ) and 2,4-Dinitrophenol (DNP) on biomass production and nitrogen respiration rate in anaerobic cultures of Pa. denitrificans with nitrate as the Terminal Electron Acceptor...... 67
Table 4: The effect of 1-hydroxyanthraquinone (OHAQ), 9,10-Anthraquinone (AQ) and 2,4-Dinitrophenol (DNP) at a Test Concentration of 10 mg/L on biomass production and nitrogen respiration rate in anaerobic cultures of Pa. denitrificans with nitrate as the electron acceptor...... 69
Table 5: The effect of 1-hydroxyanthraquinone (OHAQ), 9,10-Anthraquinone (AQ) and 2,4-Dinitrophenol (DNP) at a Test Concentration of 10 mg/L on biomass production and nitrogen respiration rate in anaerobic cultures of Pa. denitrificans with nitrite as the electron acceptor...... 72
Chapter 4 Effect of 1-hydroxyanthraquinone on biomass accumulation and oxygen utilization in Paracoccus denitrificans and in mixed wastewater cultures
Table 1: The effect of 1, 5 and 10 mg/L OHAQ on biomass production and oxygen utilization rate in batch aerobic cultures of Pa. denitrificans...... 107
Table 2: Biomass Reductions, Log P, pKa and Water Solubilities of Test Compounds evaluated in Batch Aerobic Cultures of Pa. denitrificans ...... 112 x
Table 3: Summary of the Continuous Pa. denitrificans Reactor Results for the Control and OHAQ Studies. Reactor was Fed BTZ-3 Medium containing Acetone (for the control) or 10 mg/L OHAQ at a rate of 1.5 ml/min...... 115
Table 4: The effect of 1-hydroxyanthraquinone (OHAQ) at a test concentration of 10 mg/L on Oxygen Consumption rate and biomass production in aerobic mixed cultures derived from Municipal Wastewater Treatment facility...... 117
Chapter 5 The economic implications of metabolic uncoupling in wastewater treatment facilities
Table 1: Operating Conditions for Industrial and Municipal Wastewater Treatment Facilities used in Economic Analysis...... 139
Table 2: Economic analysis for Industrial Wastewater Treatment Facility ...... 143
Table 3: Economic analysis for a Municipal Wastewater Treatment Facility with a Hydraulic Flow Rate of 6,319 GPM...... 144
Table 4: Economic analysis for a Municipal Wastewater Treatment Facility with a Hydraulic Flow Rate of 817 GPM...... 145
xi
List of Figures
Chapter 2 Effect of Chemical Uncouplers on Microbial Respiration and Biomass Production: Implications for their use in Wastewater Treatment Facilities Figure 1: Simplified relationship between catabolism and anabolism ...... 9 Figure 2: Aerobic Electron Transport Pathway in Paracoccus denitrificans ...... 11 Figure 3: Electron Transport Pathway in Mitochondria ...... 11 Figure 4: The Q-Cycle in Mitochondria ...... 15 Figure 5: Anaerobic electron transport chain in Paracoccus denitrificans ...... 17
Figure 6: Schematic Model for the Arrangement of the F0F1 subunits of ATPase ...... 21 Figure 7: The molecular mechanism of H+ translocation mediated by a protonophoric uncoupler ...... 26
Chapter 3 The activity of 1-hydroxyanthraquinone on anaerobic respiration in Paracoccus denitrificans Figure 1: Simplified relationship between catabolism and anabolism ...... 51 Figure 2: Anaerobic electron transport chain in Paracoccus denitrificans ...... 55 Figure 3: The effect of OHAQ and Uncouplers on nitrogen production in anaerobic cultures of Pa. denitrificans with nitrate as the Terminal Electron Acceptor...... 66 Figure 4: The effect of 1-hydroxyanthraquinone (OHAQ), 9,10-Anthraquinone (AQ) and 2,4-Dinitrophenol (DNP) at a Test Concentration of 10 mg/L on nitrogen Production in anaerobic cultures of Pa. denitrificans with nitrate as the electron acceptor...... 70 Figure 5: The effect of 1-hydroxyanthraquinone (OHAQ), 9,10-Anthraquinone (AQ) and 2,4-Dinitrophenol (DNP) at a Test concentration of 10 mg/L on nitrogen respiration in anaerobic cultures of Pa. denitrificans with nitrite as the electron acceptor...... 73 Figure 6: Distribution of Electrons in Substituted Anthraquinone Compounds with Electron Donating Groups ...... 76
Chapter 4 Effect of 1-hydroxyanthraquinone on biomass accumulation and oxygen utilization in Paracoccus denitrificans and in mixed wastewater cultures Figure 1: Simplified relationship between catabolism and anabolism ...... 93 Figure 2: Aerobic Electron Transport Pathway in Paracoccus denitrificans ...... 97 xii
Figure 3: The effect of OHAQ on Oxygen Utilization in Aerobic Batch Cultures of Pa. denitrificans ...... 108 Figure4: Anthraquinone test compounds ...... 110 Figure 5: The Effect of OHAQ to Biomass Ratio on Biomass Reductions in Batch and Continuous Paracoccus and Batch Mixed Culture Studies...... 120 Figure 6: Distribution of Electrons in Substituted Anthraquinone Compounds with Electron Donating Groups ...... 122 Figure 7: The molecular mechanism of H+ translocation mediated by a protonophoric uncoupler ...... 123
Chapter 5 The economic implications of metabolic uncoupling in wastewater treatment facilities Figure 1: The Yearly Reagent Costs for Reagent Doses for Reagent Doses of 1 to 10 mg/L and Reagent Costs of $2.5/KG to $20.5/KG in a Moderate Flow Municipal Wastewater Treatment Facility...... 147
xiii
Abstract Activity in Paracoccus denitrificans and potential application for biomass reduction in wastewater treatment facilities Lynn Ann Arlauskas-Dekleva Michael A. Gealt, Ph.D.
The effect of 1-hydroxyanthraquinone (OHAQ) on respiration rate and biomass production was evaluated in Paracoccus denitrificans and in mixed cultures derived from a municipal wastewater treatment facility. OHAQ addition resulted in classic
―uncoupling‖ activity characterized by an increase in the respiration rate and decrease in the biomass production. In anaerobic batch cultures, OHAQ increased the nitrogen production rate 64.5 % and decreased biomass production 30.7% in Paracoccus. In aerobic batch cultures, OHAQ increased the oxygen utilization rate 38.5% and decreased the biomass production 39.3%. OHAQ addition to a continuous Paracoccus culture resulted in an increase in the respiratory activity compared to the steady state control values. There was a 10.4% in the carbon evolution rate (CER) and a 10.8 % increase in the specific oxygen utilization rate (SOUR). The growth of the organism was not adversely affected by the presence of OHAQ suggesting that the compound could increase the capacity of wastewater treatment facilities without modification to existing equipment. The addition OHAQ to mixed cultures derived from a municipal wastewater treatment facility resulted in an increase in the initial oxygen utilization rate and a xiv
reduction in the overall biomass production. The overall oxygen utilization was not adversely affected by OHAQ. In test vessels containing 300 mg/L MLSS, 10 mg/L
OHAQ increased the initial oxygen consumption rate 16.9% and decreased the biomass production 10.8 %.
The activity of OHAQ derivatives was related to the electronic properties and position of the substitution on the anthraquinone ring system. There was no correlation to the Log P, pKa or water solubility of the compound.
These studies demonstrate that the activity of OHAQ is the result of the
―uncoupling‖ of aerobic and anaerobic respiration. The results indicate that OHAQ has the potential to reduce both the capital and operating costs of biological wastewater treatment systems, especially in the industrial setting, by simultaneously improving the metabolic capacity and decreasing the biomass production rates. The uncoupling activity of anthraquinones combined with the low toxicity of compounds suggests that these compounds may be used for reducing biomass production in commercial bioprocesses in addition to traditional wastewater treatment systems.
1
Chapter 1 Problem Description
The primary purpose of biological waste treatment processes is the removal of organic material 51, thereby decreasing its environmental burden before its discharge.
The activated sludge process is the most common biological treatment process for domestic and industrial wastewater 55 and has proven to be both a reliable and efficient treatment technology capable of producing high-quality effluents 173. Wastewaters typically contain a complex mixture of components which are degraded by a mixed microbial population in biochemical reactions 99 that convert the organic material into new cell mass (biomass) and respiration products (carbon dioxide and water) 94, 98, 99.
The excess biomass produced in the activated sludge process can be significant and requires additional processing and disposal 173. The costs associated with the handling and disposal of biomass from biological treatment processes typically represent from
40 to 60% of a wastewater treatment plant operating costs 27, 99, 190 and may require significant capital expenditures.
The disposal of excess biomass may include landfill, incineration and use on agricultural land 104. In 2000, approximately 50% of the 7.5 millon dry metric tons of biosolids produced were land applied 188 151, 14% were disposed of in a landfill and
22% were incinerated 38. Projections of an increase in biosolids production 38 combined with increasing political, economic and regulatory pressure 54 emphasizes the need for technologies that minimize biomass production in wastewater treatment facilities. 2
There are many technologies that can reduce biomass production rates but one technology in particular, chemical uncoupling, has shown a lot of promise. Chemical uncouplers disrupt the link between the catabolic (energy yielding oxidation) step and the formation of ATP and the production of new cells in anabolism 134 therefore limiting the cells ability to capture energy from substrate oxidation 98, 203. Laboratory studies have suggested that the application of the uncoupler technology to activated sludge units can result in a significant reductions in the biomass production rate 93, 99,
101, 199, 201-203. The most active uncoupler compounds are usually the most toxic and a serious problem with the uncoupler technology in wastewater treatment is the potential presence of the compound in the effluent where it might be toxic to the environment.
A number of uncoupler compounds, (e.g. DNP, chlorinated phenols) are designated as toxic pollutants subject to effluent limitations 63, 64; impeding their utility at a full-scale wastewater treatment facility.
Anthraquinones, are found widely dispersed in the environment 204, occurring as the result of both natural and commercial processes 7, 18, 50, 204. They exhibit a wide variety of biological effects 7, 9, 10, 16, 18, 29, 34, 67, 70, 80-82, 131, 132, 148, 149, 189, but the focus of this investigation is their potential uncoupling activity in denitrifying and aerobic cultures. Uncoupling activity has been demonstrated with mitochondria 10, 16, 82 and sulfate respiring microorganisms 29 but not with aerobic or anaerobic microorganisms
29, 189.
The objective of this investigation is to determine if 1-hydroxyanthraquinone
(OHAQ) can reduce biomass production through the process of uncoupling without 3
adversely affecting the respiration rate of the organism. The specific research goals of
the program are to:
Develop anaerobic and aerobic systems to evaluate the activity of OHAQ.
Determine if 1-hydroxyanthraquinone possesses uncoupling activity under
aerobic and denitrifying conditions.
Evaluate if the activity can be expanded to mixed cultures derived from a
wastewater treatment facility.
References
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16. Hilgert, I., et al., Antitumor and immunosuppressive activity of hydroxyanthraquinones and their glucosides. Folia Biol., 1977. 23(2): p. 99-109.
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18. Karukstis, K.K., et al., Alternative Measures of Photosystem II Electron Transfer Inhibition in Anthraquinone-Treated Chloroplasts. Photochemistry and Photobiology, 1992. 55(1): p. 125-132.
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20. Kawai, K., et al., The inhibition of mitochondrial respiration by anthraquinone mycotoxins. The relationship between the position of hydroxyl group on the anthraquinone nucleus and the uncoupling effect on oxidative phosphorylation. Proc. Jpn. Assoc. Mycotoxicol., 1987. 26: p. 43-6.
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35. Weimer, P.J., et al., Anthraquinones as inhibitors of sulfide production by sulfate- reducing bacteria in sewage, oil wells, process tanks, or biomass fermentation, in 22 pp. Cont.-in-part of U.S. Ser. No. 652,406, abandoned. 1993, du Pont de Nemours, E. I., and Co., USA: U.S. 7
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8
Chapter 2 Effect of Chemical Uncouplers on Microbial Respiration and Biomass Production: Implications for their use in Wastewater Treatment Facilities
Lynn Ann Arlauskas-Dekleva and Michael A. Gealt
Introduction
The primary purpose of biological treatment processes is the removal of organic
material 51, thereby decreasing its environmental burden before its discharge. The
activated sludge process is the most common biological treatment process for domestic
and industrial wastewater 97, 154 and has proven to be a reliable and efficient treatment
technology capable of producing high-quality effluents 173. Wastewaters typically
contain a complex mixture of components which are degraded by a mixed microbial
population in biochemical reactions that convert the organic material into new cell
mass (biomass) and respiration products (carbon dioxide and water) 94, 98, 99. The
excess biomass produced in the activated sludge process can be significant and requires
additional processing and disposal 173. The costs associated with the handling and
disposal of biomass from biological treatment processes can represent from 40 to 60%
of a wastewater treatment plant operating costs 27, 99, 190 and may require significant
capital expenditures.
The metabolism of organic wastes, in catabolism, provides the energy required
for cell synthesis (anabolism) in the form of an energy rich molecule, ATP 24, 97, 112. 9
The relationship between ATP formation by catabolism and its use in biomass synthesis 24, shown in Figure 1, illustrates the relationship between the two processes.
FIGURE 1: SIMPLIFIED RELATIONSHIP BETWEEN CATABOLISM 24 AND ANABOLISM (ADAPTED FROM REFERENCE )
Biomass
Substrate ATP
Catabolism Anabolism
Substrate
CO + H O 2 2 ADP + Pi
Energy
ATP, synthesized in a process known as respiration 127, 167, provides the energy required for cell synthesis 112 and serves as a link between substrate oxidation and biomass synthesis 24. Chemical compounds known as uncouplers limit the cells ability to capture energy from substrate oxidation 98, 202 and disrupt the energetic link between catabolism (oxidation of organic matter) and anabolism (ATP generation) 105.
10
Respiration
In most organisms, the majority of the ATP is synthesized in a process known
as respiration 127, 167 that utilizes a sequence of oxidation-reduction reactions to transfer
electrons from the substrate to oxygen 60 or, in the absence of oxygen, to inorganic
acceptors, e.g. nitrate 19. The electrons travel through a series of membrane-associated
electron carrier proteins and lipids 191, known as the electron transport chain or
respiratory network 181. The components of the electron transport chain are organized
into dehydrogenase and oxidase complexes connected by quinones 191 and catalyze the
downhill transfer electrons from NADH and couple to the O2 through a redox potential
span of 1.1V 127.
The electron transport chain is localized in a membrane essentially
impermeable to most ions, including OH- and H+ 61. In prokaryotes, the membrane is
associated with the outer cellular membrane. In eukaryotic systems, the membrane is
the inner membrane of the mitochondrion. The flow of electrons is coupled to the
translocation of protons across the membrane that generates an electrical or membrane
potential ( ) and proton gradient ( pH) on the opposite sides of the membrane 40, 57,
61, 68, 114, 125.
In bacteria there is considerable variation in the components of the electron
transport chain, depending on the species and the culture growth conditions, 119. The
composition of the aerobic electron transport chain of Paracoccus denitrificans, a gram
negative 171, 181 non-fermenting facultative anaerobe 15, 57, 77, 184, shown in Figure 2 57, 75, 11
119, 181, 191, closely resembles the four enzymatic complexes (I-IV) of a mitochondrial electron transport chain, Figure 3 21, 57, 74, 75, 83, 119, 127, 161, 181, 198.
FIGURE 2: AEROBIC ELECTRON TRANSPORT PATHWAY IN 57, 75, 119, 181, PARACOCCUS DENITRIFICANS (MODIFIED FROM REFERENCES 191)
cyt o O2
dehydrogenase UQ bc1 c cyt aa3 O2
dehydrogenase
FIGURE 3: ELECTRON TRANSPORT PATHWAY IN MITOCHONDRIA 119, 191
Complex III
NADH dehydrogenase UQ Cytochrome bc1 Cytochrome c Cytochrome c oxidase
Complex I Complex IV Succinate dehydrogenase Complex II
12
In the mitochondrial and Paracoccus electron transport chains, the electrons from NADH flow to oxygen via the NADH dehydrogenase enzyme complex, quinones, cytochrome bc1 complex, cytochrome c and a terminal cytochrome c oxidase
181. Most bacteria, unlike the mitochondrial respiratory chain, contain a number of alternative pathways in which electrons are transferred from specific dehydrogenases to a variety of terminal oxidases. This respiratory network enables bacteria to respond to a variety of environmental stimuli 8, 181.
The Paracoccus aerobic pathway, Figure 2, branches at ubiquinone (UQ) leading either through the bc1 complex to cytochrome aa3 (cyt aa3), or bypassing the
191 bc1 complex to cytochrome o (cyt o) . The extent to which the o-type cytochrome is active under aerobic conditions is thought to vary with different Paracoccus strains and growth conditions 160, 179, 184. For example, the cytochrome o pathway may function as a ―proton-motive release valve‖ under conditions of diminished ATP consumption.
Electron flow through the two coupling sites (bc1 and cytochrome aa3) generates a proton-motive force, P, which drives ATP production. As the ATP consumption decreases, the proton-motive builds to a point of retarding electron flow through the bc1 complex. The diversion of electron through the cytochrome o pathway, a non- coupling site, maintains the oxidation rates of NADH and ubiquinol. In a similar manner, the pathway may provide a survival mechanism in the presence of bc1 complex inhibitors or toxins 179, 191. 13
Complex I (or NADH-Coenzyme Q Reductase) of the mitochondrial electron transport chain contains at least 46 different subunits 198 and is composed of NADH dehydrogenase, a non-covalently bound FMN, Coenzyme Q (CoQ) and eight FeS
(iron-sulfur) clusters 2, 74, 127, 161, 198. The enzyme complex is an L-shaped structure that spans the inner mitochondrial membrane and protrudes into the mitochondrial matrix 2,
127, 161, 198. The NADH dehydrogenase complex (NDH-1) from Paracoccus, composed of 10-14 dissimilar subunits 197, 198, non-covalently bound FMN and FeS groups 196, 197, is similar to the mitochondrial Complex I in terms of electron carriers, inhibitor specificity and amino acid sequence of subunits but is much simpler in structure 85, 198.
The NADH-Quinone Oxidoreductase and NDH-1 complexes generate a transmembrane electrochemical potential through redox-driven proton translocation 181 by means of coupling the transfer of electrons from NADH to the translocation of four protons across the membrane 181, 191, 196, 198.
Mitochondrial Complex II (or Succinate-Ubiquinone Oxidoreductase) is composed of succinate dehydrogenase, flavin adenine dinucleotide (FAD) and three
FeS clusters 2, 127. The four-subunit enzyme in mammalian systems is composed of two integral membrane protein subunits and two hydrophilic proteins that lie in the matrix space 74, 161. The direction of electron flow through this reversible enzyme complex is dictated by the relative concentrations of reactants and products and the enzyme participates in both the respiratory chain and the tricarboxylic acid (TCA) cycles; 161. Complex II is not a coupling site in the respiratory pathway 127 and 14
transfers electrons from succinate to ubiquinone, which is located within the inner membrane 74.
Complex III, Cytochrome C Reductase (or Coenzyme Q -Cytochrome C
191 Reductase, also called the bc1 complex) , is a multi-subunit enzyme present in the respiratory chain of mitochondria and prokaryotes, possessing Class I terminal oxidases 8. The enzyme is composed of cytochrome c1, b-type cytochromes and an
8, 127, 169 iron-sulfur (FeS) protein . The cytochrome bc1 complex spans the inner membrane, extending into both the cytoplasm and matrix of the mitochondria 161. The
FeS protein, or Rieske protein, first described and isolated by Rieske et al. in 1964, contains a Fe2S2 cluster in which one iron atom (Fe1) is coordinated to the protein by
43, 91 two cysteine residues and the other iron (Fe2) by two histidine residues . The two inorganic sulfide ions bridge the two iron ions forming a flat, rhombic cluster and hydrogen bond with the main-chain nitrogens. The iron ligated to the cysteines remains in the ferric state, regardless of the reduction state of the cluster, while the histidine-ligated iron goes from a ferric state to a ferrous state when reduced 43, 185.
The oxidation of ubiquinone (QH2) and reduction of quinone (Q) occur during the reaction cycle 161 and is associated with translocation of four protons 74, 127. The reaction cycle, Figure 4, begins with the oxidation of ubiquinol to the semi-quinone anion, UQ¯, and the release of two protons on the cytoplasmic side of the membrane at the cytoplasmic (P-phase) of the enzyme 127, 161. The electron is passed to the high-
161 potential FeS complex, which subsequently transfers the electron to cytochrome c1 .
The semi-quinone anion is oxidized to UQ and then is transferred to heme bl , then 15
heme bh , where it is used to reduce a molecule of ubiquinone at the matrix to its semi- quinone form. A second QH2 is oxidized at the cytoplasmic site and the semi-quinone is reduced to QH2, picking up to protons from the matrix side of the membrane. The full reaction cycle consists of the oxidation of two molecules of ubiquinol with the associated release of four protons to the cytoplasmic side of the membrane, the reduction of one molecule of ubiquinone coupled to the uptake of two protons from the matrix space, and the reduction of two molecules of cytochrome c 161.
161 FIGURE 4: THE Q-CYCLE IN MITOCHONDRIA
Fe2S2 c 1 c Cytoplasm (P-phase)
e- - QH2 Q Q e-
b L
b H
- QH2 Q Q
Matrix (N-phase)
Complex IV, also known as Cytochrome C Oxidase, catalyzes the transfer of four electrons from ferrocytochrome c to dioxygen, in the terminal step of respiration,
8, 127, 161. Cytochrome c oxidase is composed of 13-subunits in mitochondria; bacterial 16
cytochrome c oxidase contains three or four subunits161. The enzyme is an integral
membrane protein 122 that contains two spectrally distinct, though chemically identical
127 8, heme a species, heme a and heme aa3 , and two copper complexes CuA and CuB
127 . Electrons from cytochrome c are transferred to a copper center, CuA, located on
the electrically positive (P) cytoplasmic side of the membrane and transferred via heme
122 a to a heme-copper binuclear center (heme a3 and CuB) . Molecular oxygen binds to
the reduced heme a3 and is reduced by four electrons and four protons taken from the
negative (N) matrix side of the membrane, to form water 122, 161. Complex IV, through
a series of conformational changes, couples this reduction reaction with the
unidirectional transport of up to four additional matrix protons (vectorial protons) 161.
Denitrification
A reduction in the molecular oxygen concentration induces the anaerobic
respiratory network in Paracoccus denitrificans. This organism can utilize nitrate as a
terminal electron acceptor, and to do so synthesizes four enzyme complexes that
catalyze the reduction of nitrate, nitrite, nitric oxide, and nitrous oxide in a process
known as denitrification 4, 19, 36, 84, 140, 181, 191, 192. The conversion to denitrifying
conditions influences the composition of the electron transport chain of Pa.
11, 21, 157, 160 denitrificans; there is a sharp decrease in synthesis of cytochromes a + a3
and an increase in the c-type cytochromes 11, 157, 160, including cytochrome o 57, 191. The 17
anaerobic electron transport pathway includes several branches to the individual reductases 139, 191 and the denitrifying enzymes are ‗plugged‘ into the respiratory chain, shown in Figure 5, at the level of either ubiquinone or cytochrome c 42, 171.
FIGURE 5: ANAEROBIC ELECTRON TRANSPORT CHAIN IN 191 PARACOCCUS DENITRIFICANS MODIFIED FROM
- NO3 nitrate reductase
- NO2
dehydrogenase UQ bc1 c nitrite reductase NO nitric oxide reductase
1/2 N O nitrous oxide 2 reductase
1/2 N2
Ubiquinol transfers electrons directly to nitrate reductase, bypassing the bc1 complex and the c cytochromes 32, 76, 171, 191. Electron flow from ubiquinol to nitrite, nitrous, and nitric reductases proceeds via the cytochrome bc1 complex to cytochrome c 11, 19, 42, 77, 139, 171, 180, 181. There are two proton-translocating sites in the anaerobic electron transport chain of Pa. denitrificans. The first site is the NADH dehydrogenase
181, 191 and the second site is the bc1 complex . 18
The first step in denitrification is catalyzed by a membrane bound nitrate reductase, which converts nitrate to nitrite by the reaction 19, 76, 87, 171:
- + - - NO3 + 2H + 2e NO2 + H2O
The reduction of nitrate is achieved at the active site of the -subunit located on the cytoplasmic (N) side of the membrane 181 and the conversion of nitrate is coupled to the translocation of protons 69 derived from the cytoplasm 78. The enzyme complex consists of three different subunits; one transmembrane subunit, , and two soluble subunits, - and associated with the cytoplasmic membrane 181. Electrons from ubiquinol, on the periplasmic side of the membrane, are passed through the membrane via the b-type hemes in the -subunit and iron-sulfur centers in the -subunit to the molybdenum cofactor in the -subunit 181 and two electrons are released to the periplasm 42.
The subsequent reaction, mediated by nitrite reductase, a soluble periplasmic enzyme 19, 30, 42, 69, 84, 90, 171, converts nitrite into gaseous nitric oxide by the reaction 181:
- + - NO2 + 2H + e NO + H2O
The enzyme contains both c- and d1-hemes and can receive electrons from
42 cytochrome bc1 via cytochrome c550 on the periplasmic face of the membrane . The active site of the enzyme is located at the periplasmic side of the cytoplasmic 19
3, 14 membrane where the transfer of two electrons through cytochrome bc1, as in the aerobic and mitochondrial electron transport chain, is coupled to the translocation of four protons to the periplasmic side of the membrane 12, 14, 138, 171.
Nitric oxide reductase, an integral membrane protein 127, 191, catalyzes the reduction of nitric oxide to nitrous oxide by the reaction 181:
+ - 2NO + 2H + 2e N2O + H2O
The enzyme consists of two subunits and contains both b- and c-type hemes and a non-heme iron 42, 181. The nitric oxide is reduced at the periplasmic surface of the membrane by electrons from the cytochrome bc1 complex and net outward charge transfer 42
The final step in denitrification, the reduction of nitrous oxide to dinitrogen is
181 carried out by the periplasmic N2O reductase :
+ - N2O + 2H +2e N2 + H2O
The enzyme is a soluble periplasmic enzyme that contains two copper centers,
181 center A and center Z . The complex is linked to cytochrome bc1 via cytochrome
127 c550 and the protons required for reduction of nitrous oxide are taken from the 20
periplasmic side of the plasma membrane 11, 31. The reduction of nitrous oxide is
accompanied by proton translocation 11, 106.
The Process of ATP Synthesis and Its Uncoupling
The net movement of charges across the membrane in the electron transport
chain generates an electrical or membrane potential ( ) and proton gradient ( pH) on
the opposite sides of the membrane 57, 61, 68, 114. The energy contained in the pH and
known collectively as the electrochemical gradient or proton-motive force ( P) 57,
61, 68, 114-116, is recovered by the cell as the protons return across the membrane 103. The
cell exploits this movement of protons at the cell membrane to synthesize ATP or to
drive nutrient transport and other endergonic reactions 57, 61, 114-116.
ATP is synthesized from ADP and inorganic phosphate (Pi) in a reversible
reaction mediated and facilitated by the transmembrane enzyme complex, ATPase
(ATP synthase) 35, 45, 53, 187, 200. The synthesis of ATP from ADP and inorganic
phosphate (Pi) is dependent on the driving force of the charge and proton gradient
components of P, and on the physical import of protons across the membrane 35, 142.
The ATPase complex from the membranes of mitochondria, chloroplasts and bacteria
are similar in structure and mechanism 37, 47, 48, 53, 206 and consist of a membrane
37, 44, 48, 53, 117, 118, 187, 200, 206 extrinsic F1 and transmembrane F0 components , shown
schematically in Figure 6 56, 187. 21
FIGURE 6: SCHEMATIC MODEL FOR THE ARRANGEMENT OF THE 56, 187 F0F1 SUBUNITS OF ATPASE
The F1 component, is located at the interior surface of the cell membrane and possesses catalytic and proton translocating properties 117, 136. In mitochondria and
22, bacteria, F1 is composed of five different subunits with the stoichiometry 3 3 1 1 1 22
136 which form structure consisting of a catalytic hexagon ring of alternating and subunits surrounding a central cavity containing the -subunit 53, 156, 187, 205.
The lipophillic F0 component, an integral membrane component, forms the
118, 124 channel through which protons are imported to F1 . The F0 component contains
22, 136 three essential subunits with a stochiometry a1b2c9-12 . Subunits a and c are hydrophobic proteins that span the membrane 56 and play an essential role in proton
45, 56, 185 translocation through F0 . Subunit b has a hydrophilic domain exposed to the cytoplasm and is anchored to the membrane by a hydrophobic N-terminal segment 45,
56. Subunit b does not directly participate in proton translocation 56 but is required for
45 F1 binding . The F0 subunits are proposed to be arranged in a rotary fashion, with a ring of c subunits surrounding the transmembrane helices of subunits a and b with the polar domain of subunit b extending from the center 45.
All eight subunits are required for E. coli ATP synthase 187. Chloroplast and cyanobacterial ATP synthase contain nine different subunits and fifteen subunits have been identified in the animal mitochondrial enzyme complex 141, 187. ATP synthase has been shown to exist in a dimeric state in yeast and mammalian mitochondria 158, 159 while monomeric ATP synthase has been found in Pa. denitrificans, Acetobacter woodii and spinach chloroplasts 159.
The F0 and F1 components of the ATPase enzyme complex are connected through two stalks: a central stalk formed by the connection between F1 subunits and
and the c9-12 ring of F0 and a peripheral one, that links the / hexagon of F1 to the F0 23
subunit via the hydrophilic b subunits 136, 205. The and subunits are proposed to reside in the stalk structure, subunit is required for F1 binding and cross-links to the
22, 45 , and subunits of F1 and the c subunit ring of F0 .
The F1 subunit has six alternating nucleotide-binding sites, three catalytic and three non-catalytic, on the hexagon formed by three / dimers 53, 136. The catalytic sites, designated DP, TP and E 1, have different catalytic site conformations that switch their affinities at a fixed step in the catalytic sequence 45, 48, 187. The three subunits exhibit cooperativity, positive in catalysis and negative in nucleotide binding, and demonstrate differing interactions with the central -subunit helices 48, 53.
Experimental results suggest that all three catalytic sites are occupied under physiological conditions (ATP> 1mM) 48, 187 and that the E catalytic site is where Pi must initially bind for ATP synthesis 1. Only one catalytic site is active at a time 53, 187 but it is necessary for the other catalytic sites to have bound substrates for product release from the high affinity site 187. The major energy requiring step is not the synthesis of ATP at catalytic sites, but rather the simultaneous and highly cooperative binding of substrates to, and the release of products from, these sites 37, 48, 187.
ATP synthesis or hydrolysis by F0F1 is a combination of catalysis, mechanical work ( -subunit rotation and torque generation) and proton transport 48, 135. ATP synthesis or hydrolysis is coupled to proton transport by the mechanical rotation of the
53, 56 33, 117, 125 - -c assembly extending through F1F0 and takes place at the F1 module .
187 Proton binding to a critical carboxyl group of subunit c in F0 that drives the rotation 24
of the - -c assembly rotation triggering conformational and affinity changes in the catalytic sites on the subunits that results in the release of the ATP product 22, 37, 48, 135,
142.
In coupled conditions, the ATPase enzyme complex can work reversibly as a synthase or hydrolase of ATP, consuming or pumping protons respectively 44, 121, 136.
Overall, the complete membrane-bound enzyme complex catalyzes the reaction 33, 114:
+ + ATP + H20 + 2H in ADP + Pi + 2H out
The stoichiometry of proton translocation through the H+-ATPase was initially determined to be equal to 2-3 but is now considered to be equal to 4 in plant chloroplasts 45, 59. The shift in stoichiometry may be due to the diversity of test material (chloroplasts, mitochondria, bacteria), techniques (steady-state vs. pH or salt jumps) and experimental approaches. The H+-ATP stoichiometry was reported to be 3 in rat-liver mitochondria 59, 120, 165, 1 in the plasma membrane of the fungus Neurospora
143 143 crassa , and 2 in the F0F1 ATPase of E. coli .
ATP provides the energy required for cell synthesis 112 and serves as a link between substrate oxidation and biomass synthesis 24. Anabolic pathways involve the use of free energy to build molecules required by the cell 105 for growth or other energy linked reactions 24. Energy transfer between the two pathways is in the form of ATP
105. Uncouplers of oxidative phosphorylation are thought to act as proton conductors, 25
dissipating the proton gradient across the membrane and decreasing the magnitude of
P 144 that drives the production of ATP in the cell membranes 66, 203 without appreciably affecting the electron transfer mediators 175. Oxidation of the substrate still occurs but the phosphorylation of ADP to ATP is reduced and less energy is available for the formation of biomass 202.
The effectiveness of various uncouplers is correlated with their capacity to act as proton ionophores 68 126 170 and catalyze electrogenic transfer of protons across the membrane 114 203. As a result of the proton and membrane potential components of the
P, the interior of a coupling membrane has a higher pH and a more negative electric potential than the exterior 113. The electrogenic transfer of protons introduces positive electrical charges into the interior of the cell forming a potential (locally positive) opposite that of the membrane potential 98, 191. This results in the destruction of both the pH gradient and membrane potentials which are203 required for complete metabolic uncoupling 33.
The mechanism of H+ translocation mediated by a weakly acidic uncoupler is illustrated in Figure 7 107, 175, 176.
26
FIGURE 7: THE MOLECULAR MECHANISM OF H+ TRANSLOCATION MEDIATED BY A PROTONOPHORIC UNCOUPLER 107, 175, 176 (ADAPTED FROM )
A- A-
H+ H+
HA HA + — Membrane
The original Mitchell model, proposed in 1966, assumes that the cell membrane is impermeable to most ions and the weak acid uncouplers are soluble in the membrane in both the neutral and anionic forms 107, 175, 176. The anions (A-) move from right to left through the interior of the membrane driven by the higher negative interior potential. This increase in the concentration of anions at the left-hand interface drives the heterogenous reaction:
- + A if + H HAif
- An anion at the interface (A if) combines with a proton from the aqueous phase
+ (H ) to form the undissociated acid (HAif ). The neutral form of the uncoupler diffuses through the membrane to the interior surface, driven by the concentration gradient of the compound across the membrane. As the acid form encounters the more alkaline 27
environment at the interior interface, the acid deprotonates and releases a proton into the cytoplasm. The anion then returns electrophoretically to the positively charged outside from the negatively charged inside of the energized membrane. The net result of the uncoupler cycle is the transport of H+ into the cell and dissipation of both the H+ gradient and membrane potential resulting in uncoupling 107, 175, 176.
Operationally, this results in a dramatic reduction in the P/O ratio; i.e. the moles of inorganic phosphate taken up per atom of oxygen consumed, while respiration, and therefore oxygen consumption, continues in the presence of substrate 66, 144, 176.
During uncoupling, a larger fraction of the substrate consumed in respiration is in support of proton translocation to maintain the proton gradient rather than the production of ATP. It is important to note that the synthesis of biomass is reduced or completely inhibited and energy-linked reactions such as active transport of ions are suppressed 175.
A wide variety of compounds are known to be uncouplers of oxidative phosphorylation, a listing of the most common uncouplers is provided in Table 1.
Uncouplers are, in general, moderately weak acids with an acid dissociation constant
176 (pKa) in the range or 5-7 and at physiological pH the compounds will exist in both the neutral (HA) and the anionic (A-) forms 61. Uncouplers generally contain an acidic hydrogen group, an electron-withdrawing moiety (-NO2, -CF3, -CN) and bulky hydrophobic group(s) 66, 176 which impart lipid solubility to the compounds, thought to be of primary importance for induction of uncoupling 175, 176.
28
TABLE 1: CLASSIC UNCOUPLERS OF RESPIRATION (ADAPTED 144 FROM )
2,4- Dinitrophenol CCCP (carbonyl cyanide m-chlorophenyl hydrazone) FCCP (carbonyl cyanide-p-trifluromethoxyphenyl hydrazone) p-Nitrophenol Dicoumarol (3,3'-methylene bis-4-hydroxycoumarin) Salicylanilides TCS (Tetrachlorosalicylanilide) S 13 (2',5-dichloro-3-tert-butyl 4'-nitrosalicylanilide) Benzimidazoles TTFB (4,5,6,7-tetrachloro-2'-trifluoromethyl-benzimidazole)
The most frequently studied uncoupler has been 2,4-dinitrophenol (DNP) 134, which was reported to have an effect on oxidative phosphorylation long before its mode of action was understood 68. DNP was shown to inhibit ATP synthesis in rabbit kidney preparations by Loomis and Lipmann, in 1948 96, stimulate the removal of the organic fraction of synthetic sewage by Rich and Yates in 1955 152, temporarily inhibit metabolic activity but not uncouple growth of Psuedomonas putida in batch cultures by
Low and Chase in 1996 97 and increase the rate of glucose consumption and decrease biomass production by 70% in continuous cultures of Saccharomyces cerevisia in 1975 by Shah et al 164. Mitchell, in 1961, after observing that the addition of dinitrophenol equilibrated the pH gradient across bacterial and mitochondrial membranes 68, 114 proposed that dinitrophenol and other ―uncoupler‖ compounds shuttle protons across the cell membranes 114 short-circuiting the energy producing mechanisms. 29
Uncoupler studies in denitrification systems have focused primarily on the
inhibition rather than the stimulation of respiration. Several weak acid-type uncouplers
and their analogues have been reported to uncouple aerobic but inhibit nitrogen oxide
respirations in microbial cultures. Carbonyl cyanide m-chlorophenylhydrazone
(CCCP) resulted in the uncoupling of aerobic respiration in Pseudomonas denitrificans
and Pseudomonas aeruginosa while CCCP, carbonyl cyanide p-
trifluoromethoxyphenylhydrazone (FCCP) and DNP inhibited the reductions of nitrate,
nitrite and nitrous oxide in Ps. denitrificans, Ps. aeruginosa 186 and Pa. denitrificans 87.
The rank order of the uncouplers as inhibitors of dentrification was found to be similar
to that for oxidative phosphorylation inhibition, release of respiratory control
(uncoupling), or increase in proton permeability of membranes, i.e. CCCP FCCP >
DNP 186. The addition of CCCP to denitrifying cultures of Pa.denitirificans was found
- to cause a lag phase in NO3 reduction but not in membrane vesicles with nitrate
reductases facing the outer bulk medium 13 and it was suggested that uncouplers
- eliminated the P-dependent transport of NO3 across the membrane.
Application of Chemical Uncouplers to Activated Sludge Processes
Biological treatment by the activated sludge process has become the major
technology for treating both municipal and industrial wastewaters worldwide 203.
Previously, the volume of wastewater treated, maximum substrate removal, and
effluent quality were the main criteria for the activated sludge process. However, 30
recently the rising cost for the treatment and disposal of excess sludge produced and restrictive legislation have emphasized the need to minimize biomass generated from activated sludge operations 203. The concept of uncouplers for biomass reduction in activated sludge systems is not new, and while there is a large amount of uncoupler literature, reports on their application in activated sludge systems are limited.
In 1955, Rich and Yates 152, in their pioneering work with uncouplers in activated sludge, found the addition of 5 and 15mg/L DNP increased the substrate uptake rate and inhibited growth in activated sludge units while the addition of higher concentrations of DNP resulted in a reversible decrease in the rate of substrate uptake.
Shah, et al 164 employing yeast to metabolize sugary wastes in a continuous waste treatment system found an 85% increase in degradation rate and 70% decrease in cell growth when the aqueous concentration of DNP was 5 x 10-6M (0.92 mg/L). A material balance on carbon utilization confirmed that most of the glucose consumed was degraded aerobically to carbon dioxide and water with negligible amounts going for cell growth or production of acids and alcohols.
Recently, the bulk of the literature has focused on the use of chemical uncouplers such as DNP, para-nitrophenol (p-NP), pentachlorophenol (PCP), chlorophenols (CP) and 3,3‘,4‘,5-tetrachlorosalicylanilide (TCS) in activated sludge cultures. Mayhew and Stephenson 105 investigated the effect of eight chemical inhibitors on the oxygen uptake rate of activated sludge cultures obtained from a municipal wastewater treatment facility. There was no difference between the control and experimental oxygen uptake rate between 0 and 25 hrs, after which the oxygen 31
uptake rate increased in the DNP and rotenone samples. Supplementation of lab-scale activated sludge simulations with 35 mg/L DNP resulted in decreased biomass production rates without an adverse effect on process efficiency. The average yield coefficient was 0.42 and 0.30 for the control and DNP simulation, respectively, while the biochemical oxygen demand (BOD) removal was unaffected by the addition of
DNP.
Okey and Stensel 133, in 1993, reported that the addition of halogenated phenols, such as 2,4-dichlorphenol (DCP), to unacclimmated cultures derived from an activated sludge and soil inoculum, resulted in a 25 to 45% increase in oxygen consumption when tested at ratios of 0.02 to 0.08 mg DCP/mg MLSS. In Warburg studies studies, where the MLVSS concentration ranged from 500 to 3000mg/L, the ratio of uncoupler to MLVSS concentration was found to correlate better with the extent of uncoupler activity than the concentration of uncoupler alone 133.
In batch studies with Psueudomonas putida, Low and Chase 97 found that the addition of p-nitrophenol (p-NP) inhibited biomass production without adversely affecting the removal of organic material. The observed biomass yields were found to decrease as the influent pH decreased in continuous studies while the specific substrate uptake rate increased in the presence of p-NP. The biomass reduction increased from
62% to 77% as the pH was decreased from 7.0 to 6.2 in feeds supplemented with 100 mg/L p-NP. In a bench scale activated sludge unit, the biomass production rate was decreased 49% in the presence of 100mg/L p-NP, but the total substrate removal rate was reduced by 25%. This decline in overall performance was attributed to the design 32
of the activated sludge unit resulting in solids removal in the final effluent and a decrease in the reactor biomass concentration 98.
Yang 199 reported that substituted phenols (p-chlorophenol, m-chlorophenol, m- nitrophenol, and o-nitrophenol) were effective in reducing biomass production in batch activated sludge studies. The acidity constant (pKa) of the metabolic uncoupler highly influenced its effectiveness in reducing biomass production and that lower pKas resulted in higher decreases in the biomass production. m-Chlorophenol was the most effective in reducing sludge production and had the least effect on process performance than the other compounds in the study. The addition of 20 mg/L of m-chlorophenol resulted in an 86.9% sludge reduction while the COD removal efficiency was only decreased by 13.2% as compared to the control test.
Strand and Stensel 172 compared eleven chemical uncouplers for their effectiveness in batch studies using activated sludge inoculums. The addition of
20mg/l CCCP and FCCP resulted in a reduction in biomass production of 41% and
33%, respectively while DNP did not have an effect on biomass production. 2,4,5-
Trichlorophenol (TCP) and 4-chloro-2-nitrophenol (CNP) were the most effective compounds tested and resulted in a 50% reduction in biomass when tested at concentrations of 3 and 4 mg/L, respectively. In completely mixed activated sludge
(CMAS) units, TCP functioned as an uncoupler when added to the influent at 10 mg/L.
When tested at lower concentrations (2.5 mg/L), TCP was degraded to levels below that required for uncoupling and long-term (80 days) exposure led to the development of a microbial population that were resistant to the uncoupling effect of TCP. 33
Uncoupling by TCP was more effective in a plug-flow activated sludge unit when tested at 2mg/L. Yields decreased from 0.4 g volatile suspended solids (VSS)/g chemical oxygen demand (COD) to 0.3 g VSS/g COD while the specific oxygen uptake rate (SOUR) increased from 8 to 16 mg O2/gVSS/hr.
In batch activated sludge cultures, Harem 60 found that DNP reduced the relative yields at concentrations of 40 and 80 mg/l but had little or no effect at the lower test concentrations of 10 and 20 mg/l 173. The most effective uncoupler found during these screening studies were CNP and TCP, which reduced the biomass production 68% and 73% relative to the controls when tested at a concentration of
20mg/L. In subsequent studies, TCP was tested in CMAS systems treating simulated municipal wastewater. Initially, TCP reduced the average yield by 50% and SOUR increased from 8 O2/gVSS/hr for the control reactor to approximately 20 O2/gVSS/hr.
After long-term exposure to the uncoupler, the biomass yield in the TCP reactor increased and effluent TCP levels decreased indicating the biomass became resistant to the uncoupler compound.
Chen et. al. 23 studied the feasibility of using TCS as a metabolic uncoupler to reduce biomass production in batch and continuous activated sludge cultures. TCS was found to be an effective chemical agent limiting biomass production when tested at concentrations > 0.4 mg/L. TCS reduced biomass production by 40% when added to continuous cultures daily to achieve a test concentration of 0.8 mg/L. Substrate removal efficiency and effluent nitrogen concentration were not adversely affected by the presence of TCS. 34
Ye et al. 201 studied the effect of o-chlorophenol (oCP), DCP, TCS, pNP and
DNP on biomass production in activated sludge cultures. The uncouplers were tested at a concentration of 20 mg/L in batch screening studies and resulted in reductions in biomass productions of 42.8 to 69.1% relative to the control culture. The most effective uncouplers were DNP, p-NP and TCS which resulted in biomass reductions of 69.1%, 63.7% and 56.1%. TCS reduced biomass production in a laboratory-scale
CMAS by 30% when tested at a concentration of 40mg/day 202. The substrate removal capability was not adversely affected by the presence of TCS, but effluent nitrogen concentration increased during the 60-day continuous operation. The SOUR of the culture was increased in the presence of TCS but the sludge settability from the test reactor was comparable to that of the control. Subsequent studies by Ye and Li 203 investigated the effects of three uncoupled metabolic systems: conventional activated sludge with the addition of TCS, an oxic-settling-anaerobic (OSA) process and the
TCS/OSA combined process on the biomass production rate. TCS was an effective chemical uncoupler reducing the biomass production by 34% compared to the control but had an adverse effect on the effluent nitrogen concentration and sludge settability when tested at 50mg/L for the first 3 months and 100 mg/L in the last 3 months. The
OSA process decreased biomass production by 26% and did not have an adverse effect on substrate removal or sludge settability. The most effective system was the combined process, which resulted in a 46.9% decrease in biomass production.
While there is an abundance of literature on the topic of the activity of uncouplers, there is just a handful of investigations into the use or activity of 35
uncouplers in biological waste treatment systems. Although there have been reports on the ―evidence‖ or the ―likelihood‖ of uncoupling in full-scale treatment systems, most of the information is speculative and lacks a solid scientific investigation. Okey and
Stensel 133, 134 observed behavior indicative of uncoupling at two full-scale activated sludge systems but could not detect any organic uncouplers at a high enough concentration to produce these effects. The lack of information on the use of uncouplers at the pilot or full scale is at best discouraging. Chemical uncouplers may result in a reduction in both the capital and operating costs of biological waste treatment systems by significantly improving the capacity of biological waste treatment systems and simultaneously decreasing the biomass production rates. The need for efficient, cost-effective and environmentally sound compound for waste treatment in both the municipal and industrial markets warrants the further investigation of the use of chemical uncouplers for biomass reduction.
36
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49
Chapter 3 The activity of 1-hydroxyanthraquinone on anaerobic respiration in Paracoccus denitrificans
Lynn Ann Arlauskas-Dekleva, J. Martin Odom, Jolanta Wilchek, JoAnn Payne, Eva Nagel, and Michael A. Gealt
Abstract
The activity of 1-hydroxyanthraquinone (OHAQ) on nitrogen respiration and biomass production was evaluated in anaerobic batch cultures of Paracoccus denitrificans using nitrate or nitrite as the terminal electron acceptor. The addition of 10 mg/L of OHAQ resulted in a significant increase in the nitrogen production rate and decrease in biomass production in batch denitrification studies. The activity of OHAQ was different than the other electron transport and ATPase inhibitors and uncouplers of oxidative phosphorylation evaluated in this test system. The hydroxyl group at position 1 of the anthraquinone ring system was required for uncoupling activity in the denitrifying system used in these studies. The uncoupling activity combined with the low toxicity of OHAQ suggests that this compound may be used to reduce both the capital and operating costs of commercial bioprocesses in addition to traditional biological wastewater treatment systems.
Introduction
The primary purpose of biological treatment processes is the removal of organic
material 51, thereby decreasing its environmental burden before its discharge. The 50
activated sludge process is the most common biological treatment process for domestic and industrial wastewater 97, 154 and has proven to be a reliable and efficient treatment technology capable of producing high-quality effluents 173. Wastewaters typically contain a complex mixture of components which are degraded by a mixed microbial population in biochemical reactions 99 that convert the organic material into new cell mass (biomass) and respiration products (carbon dioxide and water) 94, 98, 99. The excess biomass produced in the activated sludge process can be significant and requires additional processing and disposal 173. The costs associated with the handling and disposal of biomass from biological treatment processes can represent from 40 to 60% of a wastewater treatment plant operating costs 27, 99, 190 and may require significant capital expenditures.
The metabolism of organic wastes, in catabolism, provides the energy required for cell synthesis (anabolism) in the form of an energy rich molecule, ATP 24, 97, 112.
The relationship between ATP formation by catabolism and its use in biomass synthesis 24, shown in Figure 1, illustrates the relationship between the two processes.
51
FIGURE 1: SIMPLIFIED RELATIONSHIP BETWEEN CATABOLISM 24 AND ANABOLISM (ADAPTED FROM REFERENCE )
Biomass
Substrate ATP
Catabolism Anabolism
Substrate
CO + H O 2 2 ADP + Pi
Energy
In most organisms, the majority of the ATP is synthesized in a process known as respiration 127, 167 that utilizes a sequence of oxidation-reduction reactions to transfer electrons from the substrate to oxygen 60 or in the absence of oxygen to inorganic acceptors, e.g. nitrate 19. The electrons travel through a series of membrane-associated electron carrier proteins and lipids 191, known as the electron transport chain or respiratory network 181. The electron transport chain is localized in a membrane essentially impermeable to most ions, including OH(-) and H(+) 61. In prokaryotes, the membrane is associated with the outer cellular membrane. In eukaryotic systems the membrane is the inner membrane of the mitochondrion. The flow of electrons is coupled to the translocation of protons across the membrane that generates an electrical 52
or membrane potential ( ) and proton gradient ( pH) on the opposite sides of the membrane 40, 57, 61, 68, 114, 125.
The energy contained in the pH and known collectively as the electrochemical gradient or proton-motive force ( P) 57, 61, 68, 114-116, is recovered by the cell as the protons return across the membrane 103. The cell exploits this movement of
57, 61, 114-116 protons at the cell membrane to synthesize ATP from ADP and Pi in a reversible reaction 35, 45, 53, 187, 200. ATP provides the energy required for cell synthesis
112 and serves as a link between substrate oxidation and biomass synthesis 24.
One approach for decreasing biomass production involves the use of chemical compounds, known as uncouplers, which disrupt the direct energetic link between catabolism (oxidation of organic matter) and anabolism (ATP generation) 105, therefore limiting the cells ability to capture energy from substrate oxidation 98, 203. Uncouplers transport protons across biological membranes, dissipating the proton gradient across the membrane and decreasing the magnitude of P 49, 144. The rate of ATP synthesis and biomass production are reduced 66, 175, 203 while respiration, and therefore oxygen consumption, continues in the presence of substrate 66, 98, 144, 176.
Chemical uncouplers are a structurally diverse group of molecules 95 144. In general, they are moderately weak lipophilic acids with an acid dissociation constant
176 (pKa) in the range of 5-7 . Uncouplers generally contain an acidic hydrogen, an
66, 176 electron-withdrawing moiety (-NO2, -CF3, -CN) and bulky hydrophobic group(s) 53
and at physiological pH the compounds will exist in both the neutral (HA) and the anionic (A-) forms 61.
Laboratory studies have shown that the application of the uncoupler technology to activated sludge units can result in a significant reduction in the biomass production rate 93, 99, 101, 199, 201-203. The most active uncoupler compounds are usually the most toxic and a serious problem with the uncoupler technology in wastewater treatment is the potential presence of the compound in the effluent where it might be toxic to the environment. A number of uncoupler compounds, (e.g. DNP, chlorinated phenols) are designated as toxic pollutants subject to effluent limitations 63, 64; impeding their utility at a full-scale wastewater treatment facility.
Anthraquinones, are found widely dispersed in the environment 204 occurring as the result of both natural and commercial processes 7, 18, 50, 204. Anthraquinones, are an important class of compounds found as raw materials 25 in the textile 58, plastics, cosmetic, 204 and pharmaceutical 67, 70, 128, 174 industries. They exhibit a wide variety of biological effects 7, 9, 10, 16, 18, 29, 34, 67, 70, 80-82, 131, 132, 148, 149, 189 including inhibition of electron transport in chloroplasts 80, 132, uncoupling of mitochondrial 10, 16, 82 and sulfate respiration29 and inhibition of bacterial growth 7, 9, 34, 81 and respiration 29, 131, 189.
The uncoupling activity of anthraquinones in sulfate-reducing bacteria 29 and mitochondria 10 combined with their low systemic toxicity 62 suggest that these compounds may have some utility for biomass reduction in wastewater treatment systems. Laboratory studies have focused primarily on the application of anthraquinones for growth inhibition of aerobic and anaerobic microorganism 29, 50, 131, 54
189 rather than its potential use as an uncoupler. Several weak acid-type uncouplers and their analogues have been reported to uncouple aerobically but inhibit nitrogen oxide respiration in microbial cultures. The purpose of this study is to evaluate the effect of
1-hydroxyanthraquinone (OHAQ) on anaerobic batch cultures of Paracoccus denitrificans using nitrate as the terminal electron acceptor. The activity of OHAQ on nitrogen and biomass production is compared to respiratory inhibitors and uncouplers of oxidative phosphorylation.
Paracoccus was chosen as the model system because of its ability to grow aerobically or under anaerobic conditions with nitrate, nitrite, or nitrous oxide as the terminal electron acceptor 21, 77, 171, 191]. Formerly known as Micrococcus denitrificans
184, Pa. denitrificans is a gram negative 171, 181, non-fermenting facultative anaerobic bacteriuum 15, 57, 77, 184, commonly found in soil and sludge 181. The organism, in response to a reduction in the molecular oxygen concentration, synthesizes four enzyme complexes that catalyze the sequential reduction of nitrate, nitrite, nitric oxide, and nitrous oxide to nitrogen gas in a process known as denitrification 4, 19, 36, 84, 140, 181,
191, 192.
The overall conversion of nitrate to nitrogen gas proceeds through a series of four reactions represented by 4, 19, 36, 84, 140, 181, 191, 192:
¯ ¯ NO3 NO2 NO N2O N2
55
The anaerobic electron transport pathway includes several branches to the individual reductases 139, 191 and the denitrifying enzymes are ‗plugged‘ into the respiratory chain, shown in Figure 2, at the level of either ubiquinone or cytochrome c
42, 171.
FIGURE 2: ANAEROBIC ELECTRON TRANSPORT CHAIN IN 191 PARACOCCUS DENITRIFICANS MODIFIED FROM
- NO3 nitrate reductase
- NO2
dehydrogenase UQ bc1 c nitrite reductase NO nitric oxide reductase
1/2 N O nitrous oxide 2 reductase
1/2 N2
Ubiquinol transfers electrons directly to nitrate reductase, bypassing the bc1 complex and the c cytochromes 32, 76, 171, 191. Electron flow from ubiquinol to nitrite, nitrous, and nitric reductases proceeds via the cytochrome bc1 complex to cytochrome c 11, 19, 42, 77, 139, 171, 180, 181. There are two coupling sites in the anaerobic electron 56
transport pathway. Site one is the NADH dehydrogenase and site two is the bc1 complex.
The uncoupling activity of anthraquinones in sulfate-reducing bacteria 29 and mitochondria 10 combined with their low systemic toxicity 62 suggest that these compounds may have some utility for biomass reduction in wastewater treatment systems. The purpose of this study is to elucidate the effect of
1-hydroxyanthraquinone (OHAQ) on a model system consisting of Paracoccus denitrificans. The activity of the compound was monitored by nitrogen generation and biomass generation in batch anaerobic cultures. The site of activity was evaluated by using nitrate or nitrite as the terminal electron acceptor. 57
Materials and Methods
Organism and Growth Conditions
Paracoccus denitrificans (ATCC 19367) was obtained from the American Type
Culture Collection, Rockville, MD and maintained on Nutrient Agar or Tryptic Soy
Agar plates. The microbial culture was grown in BTZ-3 130 minimal medium at 35-
37°C, 200 RPM, in an environmental chamber (Barnstead Thermolyne Corporation,
Dubuque, IA) until late exponential or early stationary phase (approximately 18-24
hours).
In these denitrification studies, the medium was supplemented with 76 mM of
NaNO3 or 15 mM of NaNO2 and was purged with oxygen-free helium (Linde Gas,
Cleveland, OH) for a minimum of 15 minutes prior to inoculation. Paracoccus
cultures was adapted to anaerobic conditions by the transferring the culture to 120 ml
borosilicate glass serum bottles (Wheaton, Millville, NJ) with chlorobutyl rubber
stoppers (Wheaton) containing helium purged BTZ-3 medium. The adapted cultures
were transferred to fresh medium a minimum of two times prior to their use in
denitrification studies. In these studies, the nitrogen concentration of the headspace
was used to evaluate the effect of the test compounds on the overall denitrification
process and not on a particular step in the pathway; the concentration of the
intermediates such as nitrite was not determined.
58
Chemicals and Stock Solutions
1-hydroxyanthraquinone (OHAQ) (Aldrich 1,926-4, purity >97%) was a gift
courtesy of J. Martin Odom (DuPont, Wilmington, DE). The other test compounds
were obtained from the following commercial sources: 2,4-dinitrophenol (DNP)
(Fluka 42160, purity >99%), carbonyl cyanide m-chlorophenyl hydrazone (CCCP)
(Fluka 21855, purity ≥98%), carbonyl cyanide-p-trifluromethoxyphenyl hydrazone
(FCCP) (Fluka 21857, purity ≥99%), Tinopal (Sigma F6259, purity 90%), 7-chloro-
4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) (Sigma C5261, purity >99%), N,N‘-
dicyclohexylcarbodiimide (DCC) (Sigma D3128, purity >95%), rotenone (Sigma R-
8875, purity 95-98%), antimycin (mixture of compounds A1, A2, A3 and A4, Fluka AG
CH-94 50626-785, purity 90%).
Stock solutions of test compounds were prepared in acetone (J.T. Baker JT
9010-1, purity 99.5%) due to their low aqueous solubility, with the exception of
Tinopal which was soluble in aqueous solutions. The stock solutions were prepared at
an approximate concentration of 1000 mg/L to ensure the acetone concentration did not
exceed 1% (V/V) in any of the experiments. The test compounds, evaluated at a test
concentration of 10 mg/L, were added to the test vessels using a 25G tuberculin syringe
(Becton Dickinson & Company, Franklin Lakes, NJ) to minimize introduction of
oxygen. 59
Experimental Procedures
Paracoccus cultures were adapted to growth under anaerobic conditions by the
transferring aerobically grown cells to serum bottles containing helium purged BTZ-3
medium supplemented with 76 mM of NaNO3 or 15 mM of NaNO2. The bottles were
incubated at 35-37 C, 200 RPM, in an environmental chamber. Late exponential or
early stationary phase (Abs590 3.40-3.80) cultures (approximately 72 hours) were
transferred to fresh medium containing either the test compound (at a concentration of
10 mg/L) or acetone for the control bottles. The nitrogen concentration of the
headspace was determined using a Hewlett Packer 5890 gas chromatograph (GC)
(Aligent, Fort Collins, CO) equipped with a thermal conductivity detector (TCD).
Oxygen and nitrogen were separated using a molecular sieve column (Supleco 5A,
30m x 0.53mm Catalog 2-5463) with ultra pure helium as the carrier gas. The carrier,
auxiliary and reference gas flow rates were maintained at 3, 7 and 15 ml/minute,
respectively. The temperature of the oven was maintained at 35°C and the injector and
detector temperatures were 230 C. Gas samples (200 l) manually injected into the
GC using gas tight syringes (Hamilton Company, Reno, NV) and the nitrogen
concentration was manually integrated from calibration curve.
Calibration standards were prepared by addition of ultrapure N2 (Linde Gas,
Cleveland, OH) to helium purged serum bottles containing BTZ-3 medium. The
calibration consisted of duplicate injections using a minimum of eight data points 60
ranging from 37.2 to 2232 nMoles N2/ injection. Correlation coefficients for the calibration curves were generally > 0.99.
The growth of the Paracoccus cultures was monitored at 590 nm using a Hach
DR/3000 (Loveland, CO) or Pharmacia LKB UV-Vis (Thermo Fisher Scientific
Waltham, MA) spectrophotometer. The relationship between Abs590 and dry weight was determined and found to be linear up to an OD of approximately 2.4. Biomass concentrations were determined by filtration on 0.2 or 0.45 M pre-weighed filters.
The filters were dried to constant weight in a 100 C oven after washing twice with deionized water.
61
Results and Discussion
Comparison of 1-Hydroxyanthraquinone with respiratory inhibitors and uncouplers of oxidative phosphorylation on denitrifying cultures of Paracoccus denitrificans
The effect of respiratory inhibitors and uncouplers of oxidative phosphorylation
on anaerobic respiration was evaluated in batch culture studies using Pa.denitrificans.
The compounds used in the studies (Table 1) included several inhibitors of electron
transport and ATPase as well as the highly active uncouplers of oxidative
phosphorylation, CCCP (carbonyl cyanide m-chlorophenyl hydrazone) and FCCP
(carbonyl cyanide-p-trifluromethoxyphenyl hydrazone). Rotenone 46, 71, 110, 160, 184 and
Tinopal AN, (1,1-bis- (3,N-5-dimethylbenzoxazol-2-yl)-methine p-toluene sulphonate)
144, 145 directly inhibit NADH denhydrogenase while antimycin A inhibits the
79, 144, 162, 166 respiratory chain electron transport between cytochromes b and c1 .
Dicyclohexylcarbodiimide (DCC) 44, 103 and 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole
(NBD-Cl) 41, 76, 92 specifically react with ATPase and inhibit both the hydrolysis and
synthesis of ATP.
62
TABLE 1: RESPIRATORY INHIBITORS AND UNCOUPLERS OF OXIDATIVE PHOSPHORYLATION STUDIED IN PA. DENITRIFICANS UNDER DENITRIFYING CONDITIONS WITH NITRATE AS THE ELECTRON ACCEPTOR
Inhibitor Active Site Electron Transport Inhibitors Rotenone Iron-suphur centre N-2 of NADH dehydrogenase
Tinopal AN After cluster N-2 of NADH dehydrogenase
Antimycin A Cytochrome bc 1
ATPase Inhibitors NBD-Cl (7-chloro-4-nitrobenzo-2-oxa-1,3-diazole) Modification of phenolic oxygen groups of an essential
tyrosine residue in F1
DCC (N,N'-dicyclohexylcarbodiimide)
F0 component in membrane,
Uncouplers CCCP (Carbonyl cyanide m-chlorophenyl hydrazone) FCCP (Carbonyl cyanide-p-trifluromethoxyphenyl hydrazone) DNP (2,4-Dinitrophenol)
The addition of all test compounds with the exclusion of OHAQ and DNP resulted in the partial or complete inhibition of respiratory activity in denitrifying cultures of Pa. denitrificans (Table 2). The lag phase in the rotenone cultures under denitrifying conditions was significantly longer than the 1 to 2 hour lag reported in Pa. denitrificans growing aerobically on succinate after which growth was restored with a 63
maximum specific growth rate ( max) which was 40% of that before rotenone addition
109, 146. The electron transport inhibitors, rotenone, Tinopal or antimycin A resulted in the immediate inhibition of nitrogen production (Table 2). Nitrogen production was partially restored in the rotenone cultures after a 68 hour lag phase, but the culture did not fully recover by the termination of the experiment (130 hours). The average nitrogen production rate of the rotenone cultures was 0.86 Moles N2/hr, 20% the rate measured in the control bottles.
TABLE 2: NITROGEN PRODUCTION (MEAN MMOLES N2/BOTTLE) OF PA. DENITRIFICANS WITH 10 MG/L OHAQ, ELECTRON TRANSPORT INHIBITORS (ROTENONE, NBD-CL, AND ANTIMYCIN), ATPASE INHIBITORS (DCC, TINOPAL) AND UNCOUPLERS OF OXIDATIVE PHOSPHORYLATION (DNP, FCCP AND CCCP).
Mean Moles N2/bottle Time Acetone Control OHAQ DNP FCCP CCCP 4 11.5 14.1 14.1 14.5 74.9 22 26.8 15.0 15.9 17.0 24.0 45 39.8 187 169 29.9 25.6 68 139 403 408 22.2 61.6 130 401 414 417 81.7 110.8
Time H2O Control Tinopal Rotenone Antimycin DCC NBD-Cl 4 11.8 15.7 16.2 18.2 19.8 42.4 22 24.5 27.1 16.4 21.5 19.6 22.1 45 59.8 69.0 22.9 23.7 26.0 25.7 68 125 52.4 42.2 25.2 44.1 56.1 130 397 57.2 95.7 42.1 102 105
64
The nitrogen production in the bottles receiving Tinopal or antimycin was completely inhibited at a test concentration of 10 mg/L and the cultures did not recover by the termination of the experiment at 130 hours. Similar results have been reported for aerobically grown Pa. denitrificans 147 where the addition of 1, 5 and 10 M reduced and 20 M Tinopal immediately inhibited growth of the organism. The lack of nitrogen production in cultures containing 10 mg/L antimycin may be the result of the complete inhibition of electron transfer between cytochrome bc1 and cytochrome c or the inhibition of denitrification by intermediates, such as nitrite, in the pathway.
Concentrations significantly above 10 mM nitrite are inhibitory to denitrifying microorganisms 72 and in particular toxic to Paracoccus 108, therefore, the conversion of nitrate to nitrite in the first step of the pathway might result in inhibition in the growth and respiration of the test organism. In these studies, the nitrogen concentration of the headspace was used to evaluate the effect of test compounds on the overall denitrification process and not on a particular step in the pathway; the concentration of the intermediates such as nitrite was not determined.
The ATPase inhibitors, DCC or NBD-Cl, produced effects similar to the electron transport inhibitors and significantly decreased the rate of nitrogen production in the batch anaerobic cultures of Pa. denitrificans (Table 2). The mean nitrogen production rate in the DCCD and NBD-Cl bottles at a test concentration of 10 mg/L was 0.79 Moles N2/hr and 1.33 Moles N2/hr, respectively, 20 to 30 % of the 4.4
Moles N2/hr production rate for the control bottles. The nitrogen production rate remained relatively constant in the DCCD and NBD-Cl bottles over the duration of the 65
experiment and at 130 hours (the termination of the experiment) the bottles had produced approximately 25% of the total nitrogen production of the control bottles.
The activity of OHAQ was compared to uncouplers of oxidative phosphorylation; CCCP (Carbonyl cyanide m-chlorophenyl hydrazone), FCCP
(Carbonyl cyanide-p-trifluromethoxyphenyl hydrazone) and DNP (2,4-Dinitrophenol).
The addition of OHAQ or DNP resulted in the stimulation and CCCP or FCCP the inhibition in the nitrogen production in denitrifying Pa. dentificans (Tables 2 and 3,
Figure 3). Thus, the addition OHAQ resulted in classical uncoupling behavior characterized by a decrease in biomass production and increase in the respiration rate.
At a test concentration of 10 mg/L, OHAQ addition increased the nitrogen production rate 45.7 % (p= 2.29 x 10-2) and decreased the biomass production 24.5 % (p=1.33 x
10-3) compared to the control bottles. The mean nitrogen production rate was increased from 4.4 Mole N2/hr for the control bottles to 8.2 Mole N2/hr for the OHAQ bottles without affecting the overall nitrogen production. The biomass production at the termination of the experiment was decreased 24.5 % from 1,765 mg/L for the control bottles to 1,333 mg/L for OHAQ.
66
FIGURE 3: THE EFFECT OF OHAQ AND UNCOUPLERS ON NITROGEN PRODUCTION IN ANAEROBIC CULTURES OF PA. DENITRIFICANS WITH NITRATE AS THE TERMINAL ELECTRON ACCEPTOR
500
/bottle 400 2 300
200 Moles N 100
0 Mean Mean 4 22 45 68 130 Time, hours
Control OHAQ DNP FCCP CCCP
Mean nitrogen production from batch anerobic studies using Pa. denitrificans with nitrate as the terminal electron acceptor and acetone (!) (in the control vessels), OHAQ (!), DNP (!), FCCP (!) or CCCP (!) at an initial test concentration of 10 mg/L. Results are the mean of three replicate test vessels.
67
TABLE 3: THE EFFECT OF 1-HYDROXYANTHRAQUINONE (OHAQ) AND 2,4-DINITROPHENOL (DNP) ON BIOMASS PRODUCTION AND NITROGEN RESPIRATION RATE IN ANAEROBIC CULTURES OF PA. DENITRIFICANS WITH NITRATE AS THE TERMINAL ELECTRON ACCEPTOR.
Mean Moles % Nitrogen t-Test Mean Biomass Biomass t-Test p
N2/hr Increase p value mg/L Reduction % value Control 4.4 1765 OHAQ 8.2 46.7 2.29E-02 1333 24.5 1.33E-03 DNP 10.5 58.4 1.97E-02 1833 -3.9 3.66E-01
In this study, DNP, at a concentration of 13 mg/L, significantly increased the
nitrogen production rate (p=1.97 x 10-2) but, did not decrease the biomass production
rate (Table 3). The mean nitrogen production rate in the DNP bottles was increased
58.4% from the average control production rate of 4.4 Mole N2/hr to 10.5 Mole
N2/hr. As observed in the OHAQ test bottles, the overall nitrogen production in the
control and DNP bottles was comparable indicating that the test compounds did not
inhibit the metabolism of the organism.
Effect of 1-Hydroxyanthraquinone, 9,10-Anthraquinone and 2,4-Dinitrophenol on anaerobic cultures of Paracoccus denitrificans using nitrate or nitrite as the terminal electron acceptor
Nitrate or nitrite was used as electron acceptors to determine if the
anthraquinones interacting specifically with the portion of the electron transport chain 68
involving nitrate reductase. The electrons for nitrate reductase are transferred directly
171, 191 from ubiquinol while electrons for the other reductases are supplied via the bc1 complex and cytochrome c 181. If the anthraquinones specifically interact with nitrate reductase or the transfer of electrons from ubiquinol to nitrate reductase, the cultures would not display the same behavior when nitrite only was supplied. Concentrations significantly above 10mM nitrite are inhibitory to denitrifying microorganisms 72 and in particular toxic to Paracoccus 182, therefore, the level of nitrite in the medium was reduced from the nitrate test concentration of 76 mM to approximately 15 mM to decrease the potential inhibitory effects.
The addition OHAQ to a denitrifying culture of Pa. denitrificans with nitrate as the terminal electron acceptor resulted in classic uncoupling behavior characterized by a decrease in biomass production and increase in the respiration rate. At a test concentration of 10 mg/L, OHAQ addition increased the nitrogen production rate
-3 -5 64.5% (p= 6.66 x 10 ) and decreased the biomass production 30.7% (p=2.06 x 10 ) compared to the control bottles (Table 4, Figure 4). The mean nitrogen production rate was increased from 4.2 Mole N2/hr for the control bottles to 11.8 Mole N2/hr for the
OHAQ bottles without affecting the overall nitrogen production. The biomass production at the termination of the experiment was decreased 30.7 % from 1,900mg/L for the control bottles to 1,317 mg/L for OHAQ. The increase in respiration rate was not observed in cultures treated with the parent compound, 9,10-anthraquinone (AQ), which lacks the hydroxyl group in position 1, but at a test concentration of 10 mg/L the biomass production was reduced by 9.2% (p=7.68 x 10-3). In this study, 2,4- 69
dinitrophenol (DNP), at a concentration of 13 mg/L significantly increased the nitrogen production rate (p=2.18 x 10-5) but as observed in previous studies did not affect the biomass production rate. The mean nitrogen production rate in the DNP bottles was increased from the mean control production rate of 4.2 Mole N2/hr to 14.6 Mole
N2/hr. As in the OHAQ test bottles, the overall nitrogen production in the control and
DNP bottles was similar.
TABLE 4: THE EFFECT OF 1-HYDROXYANTHRAQUINONE (OHAQ), 9,10-ANTHRAQUINONE (AQ) AND 2,4-DINITROPHENOL (DNP) AT A TEST CONCENTRATION OF 10 MG/L ON BIOMASS PRODUCTION AND NITROGEN RESPIRATION RATE IN ANAEROBIC CULTURES OF PA. DENITRIFICANS WITH NITRATE AS THE ELECTRON ACCEPTOR.
70
FIGURE 4: THE EFFECT OF 1-HYDROXYANTHRAQUINONE (OHAQ), 9,10-ANTHRAQUINONE (AQ) AND 2,4-DINITROPHENOL (DNP) AT A TEST CONCENTRATION OF 10 MG/L ON NITROGEN PRODUCTION IN ANAEROBIC CULTURES OF PA. DENITRIFICANS WITH NITRATE AS THE ELECTRON ACCEPTOR.
500 /bottle
2 400 300
200 Moles N 100 0
Mean Mean 0 23 48 72 Time, hours
Con OHAQ AQ DNP
Mean nitrogen production from batch anerobic studies using Pa. denitrificans with nitrate as the terminal electron acceptor and acetone (") (for the control vessels), OHAQ (!), AQ (#) or DNP (%) at an initial test concentration of 10 mg/L. Results are the mean of three replicate test vessels.
The behavior of OHAQ was similar in the both the nitrite and nitrate studies.
The addition of OHAQ to a denitrifying culture of Pa. denitrificans using nitrite as the electron acceptor resulted in a significant increase in the rate of nitrogen production and a 29% decrease in the biomass production compared to the control bottles (Figure
5, Table 5). During the 24 to 48 hour time period, the OHAQ bottles, at a test concentration of 10 mg/L, produced an average of 2.9 Moles N2/hr and the nitrogen production was complete at the 72 hour time point. The biomass production was 71
decreased from 767 mg/L in the control bottles to 544 mg/L (p= 5.84 x 10-4). In contrast, the control bottles exhibited a significant lag in nitrogen production and at 24 hours the cultures had a mean nitrogen production rate of 0.05 Moles N2/hr. After the
96 hour lag phase, the nitrogen production rate in the control bottles increased to 1.8
Moles N2/hr, but the rate never achieved the production rate seen in the OHAQ bottles. The unsubstituted anthraquinone, AQ, had similar nitrogen production rates as the control bottles and did not decrease the biomass production. The mean nitrogen production rate in the AQ bottles was 0.1 Moles N2/hr between 24 and 48 hours and increased to 2.0 Moles N2/hr after the 72 hour sampling interval. The lag in nitrogen production was similar to that seen in the control bottles. 2,4-Dinitrophenol produced similar results to those obtained in the nitrate study. DNP significantly increased the nitrogen production rate but did not result in a significant reduction in the biomass production. The nitrogen production rate during the 24 to 48 hour time period was an average of 2.6 Moles N2/hr and was complete by the 72 hour sampling point (Figure
4). DNP did result in 14% reduction of the biomass, from a mean control value of 767 mg/L to 658 mg/L, but this reduction was not significant (p=1.91 x 10-1). At the termination of the nitrite experiment, the total nitrogen production was similar in the control and experimental bottles.
72
TABLE 5: THE EFFECT OF 1-HYDROXYANTHRAQUINONE (OHAQ), 9,10-ANTHRAQUINONE (AQ) AND 2,4-DINITROPHENOL (DNP) AT A TEST CONCENTRATION OF 10 MG/L ON BIOMASS PRODUCTION AND NITROGEN RESPIRATION RATE IN ANAEROBIC CULTURES OF PA. DENITRIFICANS WITH NITRITE AS THE ELECTRON ACCEPTOR.
Mean Mean Moles % Nitrogen Biomass Biomass t-Test p
N2/hr Increase mg/L Reduction % value Control 0.05 767 OHAQ 2.9 98.2 544 29.0 5.84E-04 AQ 0.1 62.2 825 -7.6 3.52E-01 DNP 2.6 97.9 658 14.1 1.91E-01
73
FIGURE 5: THE EFFECT OF 1-HYDROXYANTHRAQUINONE (OHAQ), 9,10-ANTHRAQUINONE (AQ) AND 2,4-DINITROPHENOL (DNP) AT A TEST CONCENTRATION OF 10 MG/L ON NITROGEN RESPIRATION IN ANAEROBIC CULTURES OF PA. DENITRIFICANS WITH NITRITE AS THE ELECTRON ACCEPTOR.
160 /bottle 2 120
80
Moles N 40
0
Mean Mean 20 70 120 170 Time, hours
Con OHAQ AQ DNP
Mean nitrogen production from batch anerobic studies using Pa. denitrificans with nitrite as the terminal electron acceptor and acetone (") (for the control vessels), OHAQ (!), AQ (#) or DNP (%) at an initial test concentration of approximately 10 mg/L. Results are the mean of three replicate test vessels.
In anaerobic cultures using either nitrate or nitrite as the terminal electron acceptor, the addition of OHAQ resulted in the uncoupling of metabolism as evidence by the increased respiration rate and decreased biomass production. The lack of uncoupling activity with the unsubstituted parent compound, AQ, may be the result of the lack of a hydroxyl group on the ring system. The addition of a hydroxyl functional group to AQ has been shown to increase its activity in mitochondria 183 and 74
chloroplasts 132 and that the position of the functional group or number of functional groups affects the activity. The effect of ring substitutions on the uncoupling efficiency of phenolic compounds has been reported in a variety of systems. Clowes &
Krahl 26, 86, in 1936, noted the location of the substitution in nitro- and halogenated phenols directly affected uncoupling activity in Arbacia eggs and that the presence of a phenolic hydroxyl group was essential for biological activity. Betina and Kuzela 10 found that anthraquinone compounds bearing a free hydroxyl group at the -position exhibited uncoupling behavior in rat liver mitochondria and compounds, lacking a - hydroxyl group, were ineffective. This requirement for a -hydroxyl group in uncoupling was confirmed by Kawai et al 82 who found that 1,2-DiOH, which contains a -hydroxyl group, was the only compound that exhibited significant uncoupling behavior.
In aerobic studies with Pa. denitrificans, this laboratory has demonstrated the activity of the compound was related to both the positional and electronic properties of the substitution on the anthraquinone ring system (Chapter 4). Electron donating groups (EDG) in position 1 (ortho) of the aromatic ring system resulted in a larger biomass reduction than substitutions in either position 2 (meta) or multiple (ortho, meta and para) substitutions. Electron donating groups in position 1 were not inhibitory to
Paracoccus and did not adversely affect the aerobic respiration of the cultures. In the group of compounds tested, a hydroxyl group in position 1 had a larger effect on biomass reduction than an amino group substitution, although both groups resulted in 75
significant biomass reductions. Position 2 EDGs did not result in significant biomass reductions and were, in general, inhibitory to the culture.
The substitution results in the aerobic studies suggest that the difference in the activity of the compounds with substitutions at positions 1 or 2 may be the result of different electron density of the compounds. EDGs have at least one pair of non- bonded electrons on the hetroatom next to the aromatic ring structure 102 which donate electrons onto the ring structure 28. Substitutions in position 1 donate electrons to the ring structure according to the proposed mechanism 1, Figure 6, and result in an electrically neutral compound with partial negative and partial positive charges in close proximity 65. While substitutions in position 2 donate electrons according to the proposed mechanism 2, Figure 6, resulting in the generation of a zwitterion 65, a compound which is electrically neutral but carries formal positive and negative charges
193. 76
FIGURE 6: DISTRIBUTION OF ELECTRONS IN SUBSTITUTED ANTHRAQUINONE COMPOUNDS WITH ELECTRON DONATING GROUPS
Mechanism 1
- + O OH O OH
O O
Mechanism 2
O O
OH OH+
- O O
A difference in the electron distribution may interfere with diffusion of the
compound across or within the membrane and/or the release of protons. The activity of
the substituted anthraquinone compounds utilized in this study indicates that the charge
distribution of the compounds plays a key role in their ability to reduce biomass
production in microbial systems. 77
The activity OHAQ was unlike the respiratory inhibitors, CCCP or FCCP; that partially or completely inhibited respiration in the test organism. Uncouplers have been reported to inhibit nitrate, nitrite and nitrous oxide reductions in intact cells 87, 89,
186, inhibit ATP synthesis in membrane vesicles 137, and eliminate the P-dependent
- 13, 155, 195 transport of NO3 across the membrane . In these studies, DNP stimulated the nitrogen production rate but did not significantly decrease the biomass production compared to the controls. The DNP concentration used in the current studies was significantly lower than the concentration reported to inhibit denitrification in
Psuedomonas denitrificans 186.
While uncoupler studies in denitrification systems have focused primarily on the inhibition rather than the stimulation of respiration by uncouplers, these studies demonstrate that OHAQ uncouples denitrification in anaerobic cultures of
Pa.denitrificans. The uncoupling of denitrification was not sensitive to the terminal electron acceptor used in the study; the compound was active in cultures using either nitrate or nitrite as the terminal electron acceptor. The compound has activity distinctly different from the respiratory inhibitors or uncouplers of oxidative phosphorylation used in the study and anthraquinones have an advantage over other uncouplers of oxidative phosphorylation because of their low systemic toxicity 62, 153. Miscellaneous inhibitory effects on particular enzyme systems have been reported, but the overall lack of toxicity of anthraquinones is supported by their natural occurrence in plants, their widespread use as dyes and their use until recently as laxatives 131. The uncoupling activity combined with the low toxicity of compounds suggests that these compounds 78
may have the potential for biomass reductions in commercial bioprocesses in addition to traditional wastewater treatment systems. 79
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91
Chapter 4 Effect of 1-hydroxyanthraquinone on biomass accumulation and oxygen utilization in Paracoccus denitrificans and in mixed wastewater cultures
Lynn Ann Arlauskas-Dekleva and Michael A. Gealt
Abstract
The activity of 1-hydroxyanthraquinone (OHAQ) on biomass production and oxygen utilization in Paracoccus denitrificans and mixed cultures derived from a municipal wastewater treatment system was evaluated. The addition of 10 mg/L of OHAQ resulted in a significant increase in the oxygen utilization rate and decrease in biomass production in batch respirometry studies. In continuous studies, OHAQ resulted in an increase in the respiratory activity that was sustained over the duration of the experiment. The activity of the compound was related to both the positional and electronic properties of the substitution on the anthraquinone ring system. The uncoupling activity combined with the low toxicity of OHAQ suggests that this compound may be used to reduce both the capital and operating costs of biological wastewater treatment systems by simultaneously improving the metabolic capacity and decreasing the biomass productions rates.
Introduction
The primary purpose of biological treatment processes is the removal of organic
material 51, thereby decreasing its environmental burden before its discharge. The
activated sludge process is the most common biological treatment process for domestic
and industrial wastewater 97, 154 and has proven to be a reliable and efficient treatment 92
technology capable of producing high-quality effluents 173. Wastewaters typically contain a complex mixture of components which are degraded by a mixed microbial population in biochemical reactions 99 that convert the organic material into new cell mass (biomass) and respiration products (carbon dioxide and water) 94, 98, 99. The excess biomass produced in the activated sludge process can be significant and requires additional processing and disposal 173. The costs associated with the handling and disposal of biomass from biological treatment processes can represent from 40 to 60% of a wastewater treatment plant operating costs 27, 99, 190 and may require significant capital expenditures.
The metabolism of organic wastes, in catabolism, provides the energy required for cell synthesis (anabolism) in the form of an energy rich molecule, ATP 24, 97, 112.
The relationship between ATP formation by catabolism and its use in biomass synthesis 24, is shown in Figure 1.
93
FIGURE 1: SIMPLIFIED RELATIONSHIP BETWEEN CATABOLISM 24 AND ANABOLISM (ADAPTED FROM REFERENCE )
Biomass
Substrate ATP
Catabolism Anabolism
Substrate
CO + H O 2 2 ADP + Pi
Energy
In most organisms that have been studied, the majority of the ATP is synthesized in a process known as respiration 127, 167 that utilizes a sequence of oxidation-reduction reactions to transfer electrons from the substrate to oxygen 60 or in the absence of oxygen to inorganic acceptors, e.g. nitrate 19. The electrons travel through a series of membrane-associated electron carrier proteins and lipids 191, known as the electron transport chain or respiratory network 181. The electron transport chain is localized in a membrane essentially impermeable to most ions, including OH(-) and
H(+) 61. In prokaryotes, the membrane is associated with the outer cellular membrane.
In eukaryotic systems the membrane is the inner membrane of the mitochondrion. The flow of electrons is coupled to the translocation of protons across the membrane that 94
generates an electrical or membrane potential ( ) and proton gradient ( pH) on the opposite sides of the membrane 40, 57, 61, 68, 114, 125.
The energy contained in the pH and known collectively as the electrochemical gradient or proton-motive force ( P) 57, 61, 68, 114-116, is recovered by the cell as the protons return across the membrane 103. The cell exploits this movement of
57, 61, 114-116 protons at the cell membrane to synthesize ATP from ADP and Pi in a reversible reaction 35, 45, 53, 187, 200. ATP provides the energy required for cell synthesis
112 and serves as a link between substrate oxidation and biomass synthesis 24.
One approach for decreasing biomass production involves the use of chemical compounds, known as uncouplers, which disrupt the direct energetic link between catabolism (oxidation of organic matter) and anabolism (ATP generation) 105, therefore limiting the cells ability to capture energy from substrate oxidation 98, 203. Uncouplers transport protons across biological membranes, dissipating the proton gradient across the membrane and decreasing the magnitude of P 49, 144. The rate of ATP synthesis and biomass production are reduced 66, 175, 203 while respiration, and therefore oxygen consumption, continues in the presence of substrate 66, 98, 144, 176.
Chemical uncouplers are a structurally diverse group of molecules 95, 144. In general, they are moderately weak lipophilic acids with an acid dissociation constant
176 (pKa) in the range or 5-7 . Uncouplers generally contain an acidic hydrogen, an
66, 176 electron-withdrawing moiety (-NO2, -CF3, -CN) and bulky hydrophobic group(s) 95
and at physiological pH the compounds will exist in both the neutral (HA) and the anionic (A-) forms 61.
Laboratory studies have shown that the application of the uncoupler technology to activated sludge units can result in a significant reduction in the biomass production rate 93, 99, 101, 199, 201-203. The most active uncoupler compounds are usually the most toxic and a serious problem with the uncoupler technology in wastewater treatment is the potential presence of the compound in the effluent where it might be toxic to the environment. A number of uncoupler compounds, (e.g. DNP, chlorinated phenols) are designated as toxic pollutants subject to effluent limitations 63, 64; impeding their utility at a full-scale wastewater treatment facility.
Anthraquinones, are found widely dispersed in the environment 204, occurring as the result of both natural and commercial processes 7, 18, 50, 204. They are found as raw materials 25 in the textile 58, plastics, cosmetic, 204 and medical 5, 67, 70, 128, 174 industries. They exhibit a wide variety of biological effects 7, 9, 10, 16, 18, 29, 34, 67, 70, 80-82,
131, 132, 148, 149, 189 including inhibition of electron transport in chloroplasts 80, 132, uncoupling of mitochondrial 10, 16, 82 and sulfate respiration29, and inhibition of bacterial growth 7, 9, 34, 81 and respiration 29, 131, 189.
The uncoupling activity of anthraquinones in sulfate-reducing bacteria 29 and mitochondria 10 combined with their low systemic toxicity 62 suggest that these compounds may have some utility for biomass reduction in wastewater treatment systems. Previous laboratory studies have focused primarily on the application of 96
anthraquinones for growth inhibition of aerobic and anaerobic microorganisms 29, 50, 131,
189 rather than its potential use as an uncoupler.
The purpose of this study is to elucidate the effect of 1-hydroxyanthraquinone
(OHAQ) on a complex biological system by analyzing not only batch and continuous aerobic cultures of Paracoccus denitrificans but also cultures derived biomass obtained at a municipal wastewater treatment facility. Paracoccus was chosen as the model system because of its ability to grow aerobically or under anaerobic conditions with nitrate, nitrite, or nitrous oxide as the terminal electron acceptor 21, 77, 171, 191. Formerly known as Micrococcus denitrificans 184, Pa. denitrificans is a gram negative 171, 181, non-fermenting facultative anaerobe, 15, 57, 77, 184 commonly found in soil and sludge 181.
The composition of the aerobic electron transport chain of Paracoccus denitrificans, 57, 75, 119, 181, 191 (Figure 2), closely resembles the four enzymatic complexes (I-IV) of a mitochondrial electron transport chain21, 57, 74, 75, 83 Yagi, 2003 #1157,
119, 127, 161, 181.
97
FIGURE 2: AEROBIC ELECTRON TRANSPORT PATHWAY IN 57, 75, 119, 181, PARACOCCUS DENITRIFICANS (MODIFIED FROM REFERENCES 191).
cyt o O2
dehydrogenase UQ bc1 c cyt aa3 O2
dehydrogenase
methanol
In Paracoccus, the electrons from NADH flow to oxygen via the NADH dehydrogenase enzyme complex, quinones, cytochrome bc1 complex, cytochrome c and a terminal cytochrome c oxidase 181. Most bacteria, unlike the mitochondrial respiratory chain, contain a number of alternative pathways in which electrons are transferred from specific dehydrogenases to a variety of terminal oxidases. The
Paracoccus aerobic pathway, branches at ubiquinone (UQ) leading either through the two coupling sites, the bc1 complex and cytochrome aa3 (cyt aa3) generating a proton- motive force, or bypassing the bc1 complex to cytochrome o (cyt o), a non-coupling site 191. The extent to which the o-type cytochrome is active under aerobic conditions is thought to vary with the different Paracoccus strains and growth conditions 160, 179,
184 and enables the bacterium to respond to a variety of environmental stimuli 8, 181. 98
The uncoupling activity of anthraquinones in sulfate-reducing bacteria 29 and mitochondria 10 combined with their low systemic toxicity 62 suggest that these compounds may have some utility for biomass reduction in wastewater treatment systems. The purpose of this study is to elucidate the effect of
1-hydroxyanthraquinone (OHAQ) on batch aerobic cultures of Paracoccus denitrificans. The activity was also evaluated in activated sludge cultures derived from a municipal wastewater treatment and continouous aerobic cultures of Paracoccus to assess the effectiveness of OHAQ in mixed culture and high cell density systems. 99
Materials and Methods
Organisms and Growth Conditions
Paracoccus
Paracoccus denitrificans (ATCC 19367) was obtained from the American Type
Culture Collection, Rockville, MD and maintained on Nutrient Agar or Tryptic Soy
Agar plates (Becton Dickinson & Company, Franklin Lakes NJ). Plates were stored at
room temperature and transferred on a weekly basis. The microbial culture was grown
in BTZ-3 130 minimal medium at 35-37 C, 200 RPM, in an environmental chamber
(Barnstead Thermolyne, Corporation Dubuque, IA) until late exponential or early
stationary phase.
Activated Sludge Cultures
Mixed liquor suspended solids (MLSS) were obtained from the secondary
aeration unit at Meadowview Municipal Wastewater treatment facility in Elkton, MD.
The culture was maintained in an aerated 1-liter fill and draw reactor. The laboratory
reactor was constructed from a 1-liter glass graduated cylinder. There were two
sampling ports, one located near the bottom of the reactor (approximately at the 250 ml
level) and at the midpoint of the unit. The unit was aerated using house air and a air 100
diffusion tube at a rate sufficient to maintain the oxygen concentration in the reactor at
5 mg/L. Mixing in the reactor was accomplished by the aeration system. Neither,
significant solids settling during the aeration period nor wall growth were not observed
during the study. On a daily basis, the supernatant was replaced with plant influent
after settling for 1 hour. The biomass concentration in the reactor was maintained at
approximately 4 g/L by wasting excess sludge as needed.
Chemicals and Stock Solutions
1-hydroxyanthraquinone (OHAQ, Aldrich 1,926-4, >97% pure),
1,8-dihydroxyanthraquinone (1,8-DiOH Aldrich D108103, 96% pure),
1,2-dihydroxyanthraquionone (1,2-DiOH Aldrich 122777), 2-aminoanthraquinone (2-
Amino Aldrich 16,554-9, technical grade), 2-ethylanthraquinone (2-Ethyl Aldrich
E-1,226-6, 97% pure), 2-methylanthraquinone (2-Methyl Aldrich 10,964-9, 95% pure)
, 2-hydroxymethylanthraquinone (2-OHMethyl Aldrich 22,652-1, 97% pure) were the
gift of J. Martin Odom (DuPont, Wilmington, DE). The following test compounds
were obtained from commercial sources: 1-Aminoanthraquinone (1-Amino Sigma
Aldrich 3909, 97% pure), purpurin (1,2,4-trihydroxyanthraquinone 1,2,4-TriOH
Aldrich 229148), 2-hydroxyanthraquinone (2-OHAQ ABCR Chemicals TCH1012,
95% pure) . 101
Stock solutions of test compounds were prepared at an approximate
concentration of 1000 mg/L in acetone (J.T. Baker JT 9010-1, purity 99.5%) due to
their low aqueous solubility. The test compounds, evaluated at a test concentration of
10 mg/L, were added to the test vessels using a syringe to minimize the introduction of
oxygen. The acetone concentration in the control and experimental test systems did
not exceed 1% (V/V).
Experimental Procedures
Respirometry Studies
Aerobic respirometer studies were performed in a closed test system using a
Columbus Instruments Micro-Oxymax respirometer (Columbus Instruments, Inc.
Columbus, OH). The test vessels consisted of a 1000 ml tissue culture bottle (Corning
Pyrex ® Corning, NY) modified with a crimp top addition tube (Bellco Glass,
Vineland NJ) and butyl rubber septum (Wheaton Millville, NJ) to permit sampling and
the addition of reagents while maintaining a closed system. The test bottles containing
the culture medium were aseptically connected to the respirometer and verified for
leaks prior to initiation of the experiment. The test compound or acetone (control) was
added using a 25G tuberculin syringe (Becton Dickinson & Company, Franklin Lakes,
NJ) and the test vessels were mixed for approximately 5 minutes at 200 RPM using an
orbital shaker (Barnstead Thermolyne, Corporation Dubuque, IA). The test vessels 102
were inoculated with an overnight culture of Paracoccus or biomass obtained from the laboratory activated sludge reactor to obtain the desired initial biomass concentration.
In most studies, the initial biomass concentration was approximately 60 mg/L for the
Paracoccus batch studies and 300 mg/L in the mixed culture studies.
The volume of the headspace in the test vessels was measured by the respirometer at the initiation of the experiment. The carbon dioxide and oxygen concentration of the headspace was determined initially and at two hour intervals using a single-beam infrared carbon dioxide sensor and an electrochemical oxygen sensor.
The carbon dioxide and oxygen sensors had operating ranges of 0-1.0% and 10-21%, respectively. The gas from the test vessels was passed through a magnesium perchlorate drying column prior to the sensors and returned to the test vessels. The headspace was refreshed after two test intervals. The sensors were purged with laboratory air after each sample interval and were calibrated using house nitrogen and a gas mixture consisting of 0.65 % CO2 and 20.6 % O2 (Linde Gas, Cleveland, OH).
Calibration of the gas sensors was performed at the initiation of the studies and checked on a monthly interval or if there was a perturbation of the system. The magnesium perchlorate in the gas drying columns was changed when the column was
50% utilized.
The respirometer studies were performed at ambient laboratory temperatures
(20-25 C) and the test vessels were agitated using a Lab Line orbital shaker
(BarnsteadThermolyne Corporation, Dubuque IA). The studies were run in triplicate and the results were averaged. Biomass determinations were performed at the 103
termination of the experiment. The duration of the experiments varied but was
generally 48 hours.
Paracoccus Continuous Studies
Continuous Paracoccus studies were performed in a 2-liter Braun Biotech
Biostat B fermentor (Sartorius, Bethlehem, PA) using a 1-liter working volume. The
BTZ-3 medium was fed to and effluent drawn from the reactor using a MasterFlex L/S
Digital Economy Drive (Cole Parmer Instrument Company, Vernon Hills, IL) and
PharMed tubing (Cole Parmer Instrument Company). The volume of the reactor was
maintained at a constant level by adjusting the height of the effluent tube prior to the
initiation of the studies. The temperature of the reactor was maintained at 25°C, the
temperature used in the batch respirometry studies. The aeration of the reactor was
controlled by a Brooks 5850 series mass flow controller (Emerson Process
Management, Pittsburgh, PA) and maintained at one standard liter per minute (SLPM)
over the duration of the experiment. Dissolved oxygen was measured by a
polarographic oxygen electrode (Mettler Toledo, Columbus, OH) and maintained at a
minimum of 20% by the speed of the impeller. The concentration of nitrogen, oxygen
and carbon dioxide in the off-gas was monitored and recorded at 3 minute intervals by
a VG Gas (Thermo Fisher Scientific, Waltham, MA) Prima 600S mass spectrometer. 104
Camile TG (developed by Guzdial, Georgia Institute of Technology, Atlanta, Georgia) and DuPont proprietary software was used for process control and data acquisition.
The continuous studies were performed consecutively using the same experimental apparatus. The reactor was operated was operated at a hydraulic residence time (HRT) of 11 hours by feeding the BTZ-3 medium containing 1% acetone for the control studies or BTZ-3 containing 10 mg/L OHAQ for the experimental studies at a rate of 1.5 mL/min The control reactor was maintained for a minimum of five residence times before changing to the experimental conditions
(control study) or OHAQ (OHAQ study), prior to initiating the medium feed to the reactor.
Paracoccus was established in the reactor containing 1-liter of sterilized BTZ-3 medium by growing under batch conditions for approximately 19 hours. The reactor was bulk dosed with acetone (10 ml) and fed BTZ-3 medium containing 1% acetone for a minimum of 55 hours (5 residence times) prior to sampling for biomass determinations. The reactor was converted from the control to test reactor by bulk dosing with a stock solution of OHAQ in acetone (final OHAQ concentration of 10 mg/L) and changing the feed to BTZ-3 containing 10 mg/L OHAQ. The reactor was sampled and experiment terminated after a minimum of five residence times.
The oxygen uptake rate (OUR), specific oxygen uptake rate (SOUR) and carbon dioxide evolution rate (CER) were calculated from the weight of the reactor and the oxygen and carbon dioxide concentrations of the inlet and outlet gas streams. The respiratory quotient (RQ) was calculated from the ratio of CER to OUR. 105
Biomass Determinations
The growth of the Paracoccus cultures was monitored at 590 nm using a Hach
DR/3000 (Loveland, CO) or Pharmacia LKB UV-Vis (Thermo Fisher Scientific
Waltham, MA) spectrophotometer. The relationship between Abs590 and dry weight
was determined and found to be linear up to an Abs590 of approximately 2.4.
Biomass concentrations (dry weight per liter of culture) of both the Paracoccus
and activated sludge cultures were determined by filtration on 0.2 or 0.45 M pre-
weighed filters. The filters were dried to constant weight in a 100 C oven after two
deionized water washes. 106
Results
Effect of OHAQ on Paracoccus
The effect of OHAQ was evaluated in aerobic batch respirometry studies using
Paracoccus as the model system. The oxygen uptake rates were used as a measure of
biological activity and were compared in the control and experimental bottles.
Biomass production was evaluated at the termination of the experiment. The addition
of OHAQ to aerobic batch cultures of Pa. denitrificans resulted in an increase in the
initial oxygen uptake rate indicating the stimulation of biological activity in the culture
(Table 2, Figure 3). At a test concentration of 10 mg/L, OHAQ increased the initial
-4 oxygen utilization rate 38.5% (p= 3.05 x 10 ) from 0.09 mg O2/hr for the control
bottles to 0.14 mg O2/hr for the OHAQ bottles. This initial increase in biological
activity did not affect the cumulative oxygen utilization but was associated with a
significant reduction in the biomass production. At 10 mg/L OHAQ, the biomass
production was reduced 39.3% (p = 3.13 x 10-2), from 3,983 mg/L for the control
bottles to 2,417 mg/L for 10 mg/L OHAQ. Similar results in respiratory activity and
biomass production were observed with OHAQ at test concentrations of 1 mg/L and 5
mg/L (Table 1, Figure 3) but not at test concentration of 0.1 mg/L OHAQ (data not
shown), this indicates there is a threshold concentration associated with OHAQ
activity.
107
TABLE 1: THE EFFECT OF 1, 5 AND 10 MG/L OHAQ ON BIOMASS PRODUCTION AND OXYGEN UTILIZATION RATE IN BATCH AEROBIC CULTURES OF PA. DENITRIFICANS.
Mean O2 Utilization Rate
O2 Utilization
Mean Biomass Biomass t-Test Mean O2 / unit Biomass/ O2 Dose Biomass SD Reduction p value 7-9 hrs 27-40 hrs Utilization biomass Utilization
mg O2 *L/ mg biomass/
mg/L mg/L % mg O2/hr mg O2/hr mg mg biomass L*mg O2 Control 3983 629 0.085 1.84 37.1 0.009 107 10 2417 548 39.3 3.13E-02 0.139 1.82 39.5 0.016 61.1 5 2433 369 38.9 2.12E-02 0.166 1.87 40.7 0.017 59.8 1 2075 530 47.9 3.96E-02 0.147 1.77 39.2 0.019 52.9
108
FIGURE 3: THE EFFECT OF OHAQ ON OXYGEN UTILIZATION IN AEROBIC BATCH CULTURES OF PA. DENITRIFICANS
10
8
6
4
2 Mean Oxygen Utilization,Mean Oxygen mg 0 2 4 7 9 11 14 16 18 20 Time, hours
Control 10 mg/L 5 mg/L 1 mg/L
Cumulative oxygen utilization from batch respirometry studies using Pa. denitrificans at an initial biomass concentration of 60 mg/L and acetone (") (for the control vessels) or 1-hydroxyanthraquinone at an initial test concentration of 10 mg/L (!), 5 mg/L (%) or 1 mg/L(#). Results are the mean of three replicate test vessels.
A comparison of oxygen utilization and biomass production at the termination of the experiment demonstrates that the oxygen utilization per unit biomass was significantly higher in the cultures containing OHAQ compared to the controls. This increase in biological activity combined with the significant decrease in biomass production establishes that OHAQ is an effective chemical uncoupler in Paracoccus batch cultures. 109
Paracoccus Substitution Studies
The relationship between the hydroxyl group and uncoupling activity of OHAQ
was investigated using ten anthraquinone compounds with amino-, methyl-, ethyl-,
hydroxymethyl- or multiple hydroxyl substitutions (Figure 4) in aerobic batch cultures
of Pa. dentrificans.
110
FIGURE 4: ANTHRAQUINONE TEST COMPOUNDS
R8 O R1
R7 R2
R6 R3
R5 O R4
Subsitution Test Compound Compound ID R1 R2 R3 R4 R5 R6 R7 R8
1-Hydroxyanthraquinone OHAQ -OH -H -H -H -H -H -H -H 1,8-Dihydroxyanthraquinone 1,8-DiOH -OH -H -H -H -H -H -H -OH
1-Aminoanthraquinone 1-Amino -NH2 -H -H -H -H -H -H -H 1,2,4-Trihydroxyanthraquinone 1,2,4-TriOH -OH -OH -H -OH -H -H -H -H
2-Hydroxymethylanthraquinone 2-OH Methyl -H -CH2OH -H -H -H -H -H -H
2-Ethylanthraquinone 2-Ethyl -H -CH2CH3 -H -H -H -H -H -H 1,2-Dihydroxyanthraquinone 1,2-DiOH -OH -OH -H -H -H -H -H -H
2-Aminoanthraquinone 2-Amino -H -NH2 -H -H -H -H -H -H 2-Hydroxyanthraquinone 2-OH -H -OH -H -H -H -H -H -H 2-Methylanthraquinone 2-Methyl -H -CH -H -H -H -H -H -H 3
These ten compounds were evaluated for their ability to uncouple microbial
respiration as measured by cumulative oxygen utilization and biomass reduction. The
compounds were chosen on the basis of substitution moity, substitution location on
ring system, commercial availability and potential application in wastewater treatment 111
systems. Halogen and nitro substituted compounds were not included in these evaluations because of the strict effluent discharge limits associated with these compounds. The evaluations were performed in a series of experiments that included not only controls but OHAQ to ensure that different series of experiments were comparable. The octanol/water partition coefficient (Log P), acid dissociation constant
(pKa) and water solubility of the chemicals were compiled from the literature and/or estimated using the EPA‘s EPIsuite software 39.
The results from these experiments, presented in Table 2, showed that biomass reductions did not correlate to either Log P or water solubility of the compounds, as correlation coefficients for the relationship were R2 = 0.1002 and R2 = 0.3263, respectively. The pKa of test compounds has been identified as an important parameter in their activity as uncouplers 199. However, the results obtained with the substituted anthraquinone compounds in this study did not show any direct relationship between the pKa and the observed biomass reductions. The test compounds used in this series had pKa‘s ranging from -0.051 for 1-Amino to 14.04 for 2-DiOH. The greatest biomass reductions were obtained with 1-OHAQ (pKa 7.15), 1,8-DiOH ( pKa 6.26) and
2-OH Methyl (pKa 14.04). Additionally, 1,2,4-TriOH with a reported pKa of 7.05, had no effect on the microbial respiration or biomass production.
112
TABLE 2: BIOMASS REDUCTIONS, LOG P, PKA AND WATER SOLUBILITIES OF TEST COMPOUNDS EVALUATED IN BATCH AEROBIC CULTURES OF PA. DENITRIFICANS .
Water Water Compound Biomass Solubilityd Solubilityd CAS d d Test Compound ID Reduction t Test Log P Log P pKa1 pKa2 (exp) (est) % (exp) (est) mg/L mg/L 1-Hydroxyanthraquinone OHAQ 40 3.52 3.64 7.0a 10.6a 8.5 9.5 129-43-1 1,8-Dihydroxyanthraquinone 1,8-DiOH 52.2 3.94 8.1a 10.7a 3.5 117-10-2 1-Aminoanthraquinone 1-Amino 22.2 3.74 3.53 -0.51b 0.3 1.6 72-48-0 1,2,4-Trihydroxyanthraquinone 1,2,4-TriOH 3.9 NS 3.46 7.05b 6.4 7.2 82-45-1 2-Hydroxymethylanthraquinone 2-OH Methyl 54 2.43 14.04b 58.4 117-79-3 2-Ethylanthraquinone 2-Ethyl 18.7 NS 4.37 4.38 0.4 17241-59-7 1,2-Dihydroxyanthraquinone 1,2-DiOH Variable 3.16 7.28a 10.5a 15.8 84-51-5 2-Aminoanthraquinone 2-Amino Inhibition 2.43 0.97b 0.2 21.7 84-54-8 2-Hydroxyanthraquinone 2-OH Inhibition 2.86 7.32a 11.1a 1.1 34.5 81-54-9 Slight Inhibition 605-32-3 2-Methylanthraquinone 2-Methyl 33.3 3.89 1.2
= significant, NS=Not significant , a Gorelikm M. et.al (1989) Zhurnal Organichesko Khimii 25(10):2046-2055 b Calculated using Advanced Chemistry Development (ACD/Labs) Software V8.14 for Solaris ( © 1994-2006 ACD/Labs) reported in Registry Database (Copyright © 2006 ACS) c Kirk-Othermer Encyclopedia of Chemical Technology 4th Edition Vol 2 John Wiley & Sons, New York 1992 d Data provided by EPA's EPIsuite estimation software http://www.epa.gov/oppt/exposure/pubs/episuitedl.htm
The results indicate that biomass reduction was related to both the positional
and electronic properties of the substitution(s). Electron donating groups (EDG), such
as hydroxyl or amino substitutions, in position 1 (ortho) resulted in a significant
reduction in biomass production. Conversely, substitution of either a hydroxyl or
amino group at position 2 (para) resulted in almost the complete inhibition of growth
and respiration.
The activity of compounds with multiple hydroxyl substitutions was found to
be dependent on the number and location of the substitutions. Hydroxyl substitution at
both positions 1 and 8 resulted in a 52.2% reduction (p=2.33 x 10-4) in biomass
production while substitution at both positions 1 and 2 lead to partial inhibition of the 113
culture and highly variable results. Additional substitutions by EDGs in position 4
(meta), as in the test compound 1,2,4-TriOH, resulted in the complete loss of uncoupling activity.
To investigate if the inhibition observed with position 2 substituted compounds was the result of the electron donating properties of the substitution, compounds with methyl, ethyl, or hydroxymethyl substitutions at position 2 were evaluated. Methyl and ethyl groups are not EDGs and the methylene spacer between the oxygen and aromatic ring in 2-hydroxymethyl interrupts electron flow between the oxygen and ring system, preventing the electron density from distributing across the ring system. As anticipated, the test compounds did not inhibit the respiration but rather resulted in a reduction in the biomass production. The biomass production was reduced by 33.3%
(p=1.40 x10-2) and 54.0% (p=2.37 x10-3) for the methyl and hydroxymethyl substituted compounds, respectively. The ethyl substituted compound reduced the biomass production by 18.7% (p=1.75 x10-1), which was not significant. The results obtained with these compounds demonstrate that the inhibition is the result of the electron donating properties of the substitution at position 2 of the ring system.
The results also indicate that biomass reduction was related to both the positional and electronic properties of the substitution(s) of the anthraquinone ring system. EDGs in position 1 resulted in uncoupling while EDGs in position 2 resulted in the inhibition of the microorganism. The hydroxyl substituted compounds had the greatest effect on biomass reductions when the compounds were substituted at position 114
1 or both positions 1 and 8. The addition of a hydroxyl group at position 2 to OHAQ
resulted in highly variable experimental results.
Paracoccus Continuous Culture Studies
The use of chemical uncouplers for biomass reduction in wastewater treatment
facilities requires that the compound is effective at a high population density under
continuous growth conditions. Therefore, the activity of OHAQ in continuous cultures
with Paracoccus, the model organism, was evaluated. The reactor was operated at a
residence time of 0.09hr-1, which was slightly below the observed growth rate for the
organisms in the batch respirometry studies. The carbon dioxide evolution rate (CER),
oxygen utilization rate (OUR) and specific oxygen utilization rate (OUR) was
continuously monitored and final biomass concentration determined after a minimum
of five residence times.
The addition of OHAQ to a continuous Paracoccus culture resulted in an
increase in the CER, OUR and specific oxygen uptake rate (SOUR) but did not
decrease the biomass production rate (Table 3). The CER was increased 10.4 % from
27.6 mM/L·hr for the control reactor to 30.8 mM/L·hr for the OHAQ reactor.
Similarly, there was a 10.8 % increase in the SOUR from a control value of 3.98
mM/mg·hr to 4.46 mM/mg·hr for the OHAQ reactor. The stir speed of the reactor
increased from the steady state control rate of 610 RPM to 700 RPM in the presence of 115
OHAQ to compensate for the increased OUR. The growth of the organism was not adversely affected by the presence of OHAQ and the difference in the biomass concentrations of the control and OHAQ reactors was not significant. The final biomass concentration in the control and OHAQ studies was 9,050 ± 328 mg/L and
9,550 ± 471 mg/L, respectively.
The studies demonstrate that OHAQ increases the respiratory activity of the biomass, as evidence by the increases in CER, OUR and SOUR, under conditions growth conditions. The growth rate of the organism was not adversely affected by the presence of the test compound and was controlled by the reactor operating conditions.
The activity of OHAQ on the microbial biomass was immediate and was sustainable.
TABLE 3: SUMMARY OF THE CONTINUOUS PA. DENITRIFICANS REACTOR RESULTS FOR THE CONTROL AND OHAQ STUDIES. REACTOR WAS FED BTZ-3 MEDIUM CONTAINING ACETONE (FOR THE CONTROL) OR 10 MG/L OHAQ AT A RATE OF 1.5 ML/MIN.
Control OHAQ Final Biomass mg/L 9050 9550 CER mM/L*hr 27.6 30.8 CO2 out % 0.57 0.63 OUR mM/L*hr 35.9 42.6 SOUR mM/mg*hr 3.98 4.46 RQ 0.77 0.72 pH 7.2 7.1 Stir Speed RPM 610 700 CER= carbon dioxide evolution rate, OUR= Oxygen Uptake Rate, SOUR= Specific Oxygen Uptake Rate, RQ=Respiratory Quotient=CER/OUR
116
Mixed Culture Batch Studies
The application of the chemical uncouplers for biomass reduction in wastewater
treatment facilities requires that the compound is active on a mixed population without
adversely affecting the degradation capabilities of that population. Therefore, the
activity of OHAQ was evaluated in batch studies using mixed cultures derived from a
municipal wastewater treatment facility. The oxygen utilization rates were used as a
measure of biological activity and were compared in the control and experimental
bottles. Biomass production was evaluated at the termination of the experiment. The
addition OHAQ of mixed cultures resulted in an increase in the initial oxygen
utilization rate and a reduction in the overall biomass production (Table 4). The overall
oxygen utilization was not adversely affected by OHAQ. In test vessels containing 300
mg/L MLSS, 10 mg/L OHAQ increased the initial oxygen consumption rate 16.9%
(p=3.32 x 10-3) and decreased the biomass production 10.8 % (p = 7.76 x 10-3)
compared to the control bottles (Table 4). The mean oxygen utilization rate was
increased initially from 0.024 mg O2/hr for the control bottles to 0.028 mg O2/hr for the
OHAQ bottles between 7 and 9 hours followed by a decrease to 0.021 mg O2/hr over
the 29 to 34 hour test interval. The oxygen utilization rate in the control bottles was
constant throughout the experiment. The cumulative oxygen utilization in the control
and OHAQ bottles was comparable at the termination of the experiment while the 117
biomass production was decreased from 1083 mg/L for the control bottles to 967 mg/L
for the OHAQ bottles.
TABLE 4: THE EFFECT OF 1-HYDROXYANTHRAQUINONE (OHAQ) AT A TEST CONCENTRATION OF 10 MG/L ON OXYGEN CONSUMPTION RATE AND BIOMASS PRODUCTION IN AEROBIC MIXED CULTURES DERIVED FROM MUNICIPAL WASTEWATER TREATMENT FACILITY.
Mean O2 Utilization Rate
Mean Biomass t-Test Mean O2 O2 Utilization / Biomass / O2 Biomass SD Reduction p value 7 to 9 hrs 29 to 34 hrs Utilization biomass Utilization mg O2 *L/ mg mg biomass/
Sample ID mg/L % mg O2/hr mg O2/hr mg biomass L*mg O2 Control 1083 28.9 0.024 0.023 1.19 1.10E-03 910 OHAQ 967 28.9 10.8 7.76E-03 0.028 0.021 1.21 1.25E-03 799
A comparison of oxygen utilization and biomass production at the termination
of the experiment demonstrates that the oxygen utilization per unit biomass was
significantly higher in the cultures containing OHAQ compared to the controls (p=2.27
x 10-3). This increase in biological activity combined with the significant decrease in
biomass production establishes that OHAQ is an effective chemical uncoupler in mixed
cultures derived from municipal wastewater treatment facilities.
118
Discussion
The addition of 10 mg/L OHAQ to Pa. denitrificans and activated sludge
cultures resulted in a significant increase in the oxygen utilization rate compared to the
controls. In batch studies, this increase was observed in the initial oxygen utilization
rate and was associated with a significant decrease in the final biomass concentration.
The biomass reductions in the Paracoccus batch studies were consistently in excess of
40% throughout the entire experimental program. OHAQ was equally effective over a
test concentration range of 1 to 10 mg/L but had no significant effect at a test
concentration of 0.1 mg/L.
In the continuous Paracoccus studies, OHAQ addition resulted in an increase in
the respiratory activity compared to the steady state control values and was sustained
over the duration of the experiment. The addition of OHAQ did not significantly
reduce the biomass production in the continuous study but increased the SOUR
approximately 10%. These results suggest that the compound could increase the
capacity of culture treatment facilities without modification in or addition to existing
equipment.
The biomass reduction in the mixed culture batch studies was less than that
obtained in the Paracoccus batch studies. The average biomass reduction with 10
mg/L OHAQ was 10.8% in mixed cultures with an initial biomass concentration of 300
mg/L. This value although lower than that obtained in the pure culture studies, is
significant in regards to the relative biomass production produced in wastewater 119
treatment facilities. An industrial facility with a hydraulic flow rate of 1100 GPM with an organic loading of 20,000mg/L biological oxygen demand (BOD5) produces approximately 79,200 lbs of biomass per day 150 and a moderate flow municipal wastewater treatment facility with a hydraulic flow rate of 6319 GPM and organic
111 loading of 187mg/L BOD5 generates 13,300 lbs of biomass per day .
The relationship between the uncoupling activity of the compounds and the uncoupler to biomass ratio (Cu/Xo) has been reported for both pure and mixed cultures
93, 133, 172, 201. The results obtained in the batch and continuous studies verified that the activity of OHAQ on biomass production rates was related to the initial OHAQ concentration to initial biomass ratio, Cu/Xo, rather than the initial test concentration.
There was a strong correlation (R2= 0.9979) between the OHAQ to biomass ratio and the biomass reduction obtained with the Paracoccus and mixed culture studies (Figure
5). The average biomass reductions obtained over the experimental program were in excess of 40% when evaluated at a Cu/Xo of 0.17. A decrease in the Cu/Xo to 0.001, did not result in a reduction in the biomass production in either the batch or continuous
Paracoccus studies.
120
FIGURE 5: THE EFFECT OF OHAQ TO BIOMASS RATIO ON BIOMASS REDUCTIONS IN BATCH AND CONTINUOUS PARACOCCUS AND BATCH MIXED CULTURE STUDIES.
50 y = 408.57x - 1.1742 2 40 R = 0.9979
30
20 Biomass Reduction, % Reduction, Biomass 10
0 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.10 OHAQ/Biomass Ratio
In the mixed culture batch studies, a Cu/Xo OHAQ of 0.03, resulted in a lower but significant reduction in the overall biomass production. The lower activity of
OHAQ in the mixed culture studies was attributed to the diverse microbial population present in wastewater treatment systems. Chemical uncouplers may affect the growth rates of the individual species in the population differently and result in a change in the population dynamics of the system 100.
The effect of ring substitutions on the uncoupling efficiency of phenolic compounds has been reported in a variety of systems. Clowes & Krahl 26, 86, in 1936, noted the location of the substitution in nitro- and halogenated phenols directly affected 121
uncoupling activity in sea urchin (Arbacia) eggs and that the presence of a phenolic hydroxyl group was essential for biological activity. Betina and Kuzela 10 found that anthraquinone compounds bearing a free hydroxyl group at the -position exhibited uncoupling behavior in rat liver mitochondria and compounds, lacking a -hydroxyl group, were ineffective. This requirement for a -hydroxyl group in uncoupling was confirmed by Kawai et al 82 who found that 1,2-DiOH, which contains a -hydroxyl group, was the only compound that exhibited significant uncoupling behavior.
In this study, there was a strong correlation between biomass reductions and the electronic substitution and its position. Electron donating groups (EDG) in position 1
(ortho) of the aromatic ring system resulted in a larger biomass reduction than substitutions in either position 2 (meta) or multiple (ortho, meta and para) substitutions.
Electron donating groups in position 1 were not inhibitory to Paracoccus and did not adversely affect the respiration of the cultures. In the group of compounds tested, a hydroxyl group in position 1 had a larger effect on biomass reduction than an amino group substitution, although both groups resulted in significant biomass reductions.
Position 2 EDGs did not result in significant biomass reductions and were, in general, inhibitory to the culture.
The substitution results suggest that the difference in the activity of the compounds with substitutions at positions 1 or 2 may be the result of different electron density of the compounds. EDGs have at least one pair of non-bonded electrons on the hetroatom next to the aromatic ring structure 102 which donate electrons onto the ring 122
structure 28. Substitutions in position 1 donate electrons to the ring structure according
to the proposed mechanism 1, Figure 6, and result in an electrically neutral compound
with partial negative and partial positive charges in close proximity 65. While
substitutions in position 2 donate electrons according to the proposed mechanism 2,
Figure 6, resulting in the generation of a zwitterion 65, a compound which is electrically
neutral but carries formal positive and negative charges 193.
FIGURE 6: DISTRIBUTION OF ELECTRONS IN SUBSTITUTED ANTHRAQUINONE COMPOUNDS WITH ELECTRON DONATING GROUPS
Mechanism 1
- + O OH O OH
O O
Mechanism 2
O O
OH OH+
- O O
123
The difference in the charge distribution of the ortho and meta substituted
anthraquinone compounds may be the basis for the difference in biomass reductions in
these studies. The proposed mechanism of proton translocation mediated by an
uncoupler assumes that the cell membrane is impermeable to most ions and the
chemical compounds are soluble in the membrane in both the neutral (HA) and anionic
(A-) forms 107, 175, 176. The anions (A-) move from right to left through the interior of
the membrane driven by the higher negative interior potential (Figure 7).
FIGURE 7: THE MOLECULAR MECHANISM OF H+ TRANSLOCATION MEDIATED BY A PROTONOPHORIC UNCOUPLER 107 175 176 (ADAPTED FROM )
A- A-
H+ H+
HA HA + — Membrane
This increase in the concentration of anions at the left-hand interface drives the heterogenous reaction: 124
(-) (+) A if + H HAif
- where an anion at the interface (A if) combines with a proton from the aqueous
+ phase (H ) to form the undissociated acid (HAif ). The neutral form of the uncoupler diffuses through the membrane to the interior surface, driven by the concentration gradient of the compound across the membrane. The compound encounters the more alkaline environment at the interior interface, deprotonates, and releases a proton into the cytoplasm. The anion then returns electrophoretically to the positively charged outside from the negatively charged inside of the energized membrane. Chemical uncouplers do not directly interact with the components of the electron transport chain
175 but rather decrease the rate of ATP synthesis and biomass production 66, 98, 144, 176, 203 by disrupting the link between substrate oxidation and biomass synthesis 24. The net result of the uncoupler cycle is the transport of H(+) into the cell and dissipation of both the proton gradient and P 107, 175, 176. A larger fraction of the substrate consumed in respiration is in support of proton translocation to maintain the proton gradient rather than the production of ATP resulting in a decrease in biomass production 175.
A difference in the electron distribution may interfere with diffusion of the compound across or within the membrane and/or the release of protons. The activity of the substituted anthraquinone compounds utilized in this study indicates that the charge distribution of the compounds plays a key role in their ability to reduce biomass 125
production in microbial systems. The mechanism of uncoupling outlined above suggests that the polar nature of some of the substituted compounds may interfere with cellular metabolism as a result of their inability to diffuse across the cell membrane.
The studies in this report demonstrate that the activity of OHAQ are the result of the ―uncoupling‖ of aerobic respiration. This laboratory has demonstrated the compound also possesses uncoupling activity under denitrifying conditions (Chapter
3).
Biological treatment by the activated sludge process has become the major technology for treating both municipal and industrial wastewaters worldwide 203.
Previously, the volume of wastewater treated, maximum substrate removal, and effluent quality were the main criteria for the activated sludge process. However, recently the rising cost for the treatment and disposal of excess sludge produced and restrictive legislation have emphasized the need to minimize biomass generated from activated sludge operations 202. The concept of uncouplers for biomass reduction in activated sludge systems is not new, and while there is a large amount of literature on uncouplers, reports on their application in activated sludge systems is limited. The resulted reported here indicate that OHAQ has the potential to reduce both the capital and operating costs of biological wastewater treatment systems by simultaneously improving the metabolic capacity and decreasing the biomass production rates.
Anthraquinones have an advantage over other uncouplers of oxidative phosphorylation because of their low systemic toxicity 62, 153. Miscellaneous inhibitory effects on particular enzyme systems have been reported, but the overall lack of 126
toxicity of anthraquinones is supported by their natural occurrence in plants, their widespread use as dyes and their use until recently as laxatives 131. The uncoupling activity combined with the low toxicity of compounds suggests that these compounds may be used for reducing biomass production in commercial bioprocesses in addition to traditional wastewater treatment systems. The need for efficient, cost-effective and environmentally sound waste treatment in both the municipal and industrial markets warrants the further investigation of the use of this chemical uncoupler for enhancement of metabolic capacity and biomass reductions. 127
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137
Chapter 5 The economic implications of metabolic uncoupling in wastewater treatment facilities
Lynn Ann Arlauskas-Dekleva and Michael A. Gealt
The primary purpose of biological treatment processes is the removal of organic
material 51, thereby decreasing its environmental burden before its discharge. The
activated sludge process is the most common biological treatment process for domestic
and industrial wastewater 97, 154 and has proven to be both a reliable and efficient
treatment technology capable of producing high-quality effluents 173. Wastewaters
typically contain a complex mixture of components which are degraded by a mixed
microbial population in biochemical reactions 99 that convert the organic material into
new cell mass (biomass) and respiration products (carbon dioxide and water) 94, 98, 99.
The excess biomass produced in the activated sludge process can be significant and
requires additional processing and disposal 173. The costs associated with the handling
and disposal of biomass from biological treatment processes typically represent from
40 to 60% of a wastewater treatment plant operating costs 27, 99, 190 and may require
significant capital expenditures.
In the United States, approximately 55% of the 7.2 millon dry metric tons of
biosolids produced in 2004 were applied to soils for agronomic, silvicultural, and/or
land restoration purposes 123. The remaining 45% were disposed of in municipal solid 138
waste (MSW) landfills (63%), surface disposal units (4%), and/or incineration facilities
(33%) 123. Although composted biosolids were applied to the White House lawn, the
Washington Monument and the National Mall Constitution Gardens 178, the overall picture for beneficial use of biosolids is hampered by the political, economic and regulatory pressures 54. Concerns about pollutants, odors 38, and allegations of illness and death related to land application 52 163 combined with projections of an increase in biosolid production rates 38 emphasizes the need for technologies that minimize biomass production in wastewater treatment facilities.
There are many technologies that can reduce biomass production rates but one technology in particular, chemical uncoupling, has shown considerable promise.
Chemical uncouplers disrupt the link between the catabolic (energy yielding oxidation) step and the formation of ATP and the production of new cells in anabolism 134 therefore limiting the cells ability to capture energy from substrate oxidation 98, 203.
Laboratory studies have shown that the application of the uncoupler technology to activated sludge units can result in a significant reduction in the biomass production rate 93, 99, 101, 199, 201-203. The most active uncoupler compounds are usually the most toxic and a serious problem with the uncoupler technology in wastewater treatment has been the potential presence of the compound in the effluent where it might be toxic to the environment 95. A number of uncoupler compounds, (e.g. DNP, chlorinated phenols) are designated as toxic pollutants subject to effluent limitations 63, 64; impeding their utility at a full-scale wastewater treatment facility. The uncoupling activity of anthraquinones in sulfate-reducing bacteria 29 and mitochondria 10 combined 139
with their low systemic toxicity 62 suggest that these compounds may have some utility for biomass reduction in wastewater treatment systems.
Reports of 40%, 50% even 60% reductions in biomass production in the presence of uncouplers are not uncommon in the literature. The viability of the technology at a full-scale treatment facility however, depends on the ability of the compounds to reduce operating costs above the chemical costs associated with the technology. Although critical, only a few reports have mentioned the economic impact associated with application of the uncoupler technology.
An initial economic assessment was performed that evaluated the impact of uncouplers on an industrial and two municipal wastewater treatment systems. In this analysis, operating values were developed for the industrial and municipal facilities 111,
150 (Table 1) that are similar to these facilities standard operating conditions and, using these values, the economic impact of the uncoupler was determined.
TABLE 1: OPERATING CONDITIONS FOR INDUSTRIAL AND MUNICIPAL WASTEWATER TREATMENT FACILITIES USED IN ECONOMIC 17, 111, 150 ANALYSIS. INFORMATION FROM
Facility Industrial Municipal 1 Municipal 2 Average flow rate (GPM) 1,100 6,319 817
BOD/COD loading (BOD5 lbs/day) 264,000 33,360 1572 Biomass generation/BOD or COD treated (lbs dry * biomass/lb BOD5 removed) 0.3 0.4 0.4 Biomass Disposal Landfill Land Application Incineration Disposal Costs ($/dry ton) $100 $50 $200 Excess plant capacity No Yes No Biomas Production lbs dry/day 79,200 13,344 572 140
The industrial facility is a completely mixed activated sludge unit composed of three 1.2 million gallon secondary aeration tanks. The hydraulic flow rate is 1,100 gallons per minute (GPM) (1.6 million GPD) with an organic loading of 20,000 mg/L
BOD5. The plant generates 79,200 lbs of dry biomass per day based on a yield of 0.3 pounds dry biomass per pound BOD5. The solids are separated by using a membrane filter press and are sent to a landfill. The plant does not have any excess capacity and is considering a capital expansion project 150.
Two municipal cases were considered in this analysis. The first municipal facility has a hydraulic flow rate of 6,319 GPM with an organic loading of 187 mg/L
BOD5. The plant generates 13,334 lbs biomass per day based on a yield of 0.4. The solids are sent to a digester that generates methane used to supplement on-site power requirements and then land applied 111. The second municipal facility has a hydraulic flow rate of 817 GPM with an average organic loading of 160 mg/L BOD5. The plant generates 572 lbs biomass per day based on a yield of 0.4. The solids generated from this facility are sent for incineration. The plant is operating at maximum capacity and is currently sending a portion of its wastewater to a local industrial facility for treatment 17.
The economic analysis initially considered a 10% reduction in biomass production rate and an estimated solids disposal cost of $100 per ton for the industrial
150, $50 per ton for the large municipal 129 and $200 per ton of dry solids for the small 141
municipal case 20. The dry solids contain approximately 55% moisture; therefore one ton of dry biomass was equivalent to two tons of dry solids or 4.44 tons of wet cake 150.
The chemical uncoupler, 1-hydroxyanthraquinone (OHAQ), supplied from the manufacturer (Epochem Company, Shanghai, China) in 50KG containers at a cost of
$20/KG OHAQ (purity 97%), was used at a dose of 10 mg/L wastewater, a dose shown in our studies to be effective in both anaerobic and aerobic microcosms.
Although the oxygen utilization rate in the presence of chemical uncouplers is increased, the oxygen consumed per unit BOD5 is constant. Therefore, the aeration requirement per BOD5 loading remained constant in this evaluation. The capital investment for the equipment (dry powder storage facility, slurry tank, pumps) and facilities (outbuilding) was estimated at $250,000 and $50,000, respectively. The project costs associated with front end loading (FEL) or engineering design of $75,000 and yearly maintenance ($5,000) were included. The front end loading of a project is the fee charged at the time of the initial purchase for an investment and is used as an estimate for initial engineering design of the project 177.
Net Present Value (NPV), a standard method for financial evaluations, was used to determine the impact of the uncoupler technology. NPV is used to analyze the profitability of an investment or project and is the difference of cash inflows and cash outflows 73 194. The cash flows of the project are discounted into present value (PV) amounts using a discount rate that represents the projects cost of capital and its risk 73.
The investment‘s future positive cash flows are reduced into one present value number 142
which is subtracted from the initial cash outlay required for the investment 73 194 according to:
t NPV = ∑ Ct/(1+r) – C0
where C is the cash flow, t is the time of cash flow, r is the is discount rate, C0 is initial cash investment. The discount rate is the rate at which the capital needed for the project would earn if invested in an alternative venture 194. This analysis used 1% for the selling price, 3% for variable cost and 3% fixed cost escalation factors and a 35%
Federal and 3% State/Local Tax rate 73.
The Net Present Value for the industrial treatment facility was calculated for biomass reductions ranging from 10% to 50% (Table 2). Although, the annual savings in solids disposal for a 10% reduction in biomass was $492,000 the NPV for the project was negative indicating the project would result in a negative cash flow. The
NPV is positive for a biomass reduction of 11% but the time required for the cash inflows to equal the original outlay is greater than 10 years. The NPV and discounted payback become more favorable as the biomass reductions exceed 15%. For biomass reductions of 20% or the NPV is $1,042,000 and the project moves to a positive cash flow basis after only 2 years.
143
TABLE 2: ECONOMIC ANALYSIS FOR INDUSTRIAL WASTEWATER TREATMENT FACILITY
Biomass Reduction Baseline 10% 11% 12% 15% 20% 30% 40% 50%
Yield 0.300 0.270 0.267 0.264 0.255 0.240 0.210 0.180 0.150
Biomass Production lbs/day 79,200 71,280 70,488 69,696 67,320 63,360 55,440 47,520 39,600
tons/day 29.6 26.6 26.3 26.0 25.1 23.7 20.7 17.7 14.8 Reagent Costs $1000/yr 448 448 448 448 448 448 448 448 448 Disposal costs $/day 13,126 11,813 11,682 11,551 11,157 10,501 9,188 7,876 6,563 $1000/yr 4,791 4,312 4,264 4,216 4,072 3,833 3,354 2,875 2,396 Annual Savings
$1000/yr 479 527 575 719 958 1,437 1,916 2,396 NPV ($1000) (-192) 55 302 1,042 2,271 4,735 7,199 9,667 Discounted Payback (yr) > 10 9 5 2 1 0 0 0
The results are very different for the municipal wastewater treatment facility
cases. In the larger municipal facility, the reagent costs are approximately $2.5 million
while the estimated cost savings from the solids disposal range from $40,000 to
$202,000 for biomass reductions of 10% and 50%, respectively (Table 3). In addition,
the municipal facility analyzed uses biomass from the facility to generate a portion of
the electrical requirements of the facility. It follows that a reduction in biomass to the
digesters would reduce the gas generation rate resulting in an increase in the amount of
electricity that must be purchased.
144
TABLE 3: ECONOMIC ANALYSIS FOR A MUNICIPAL WASTEWATER TREATMENT FACILITY WITH A HYDRAULIC FLOW RATE OF 6,319 GPM.
Biomass Reduction Baseline 10% 20% 30% 40% 50% Yield 0.400 0.360 0.320 0.280 0.240 0.200
Biomass Production lbs/day 13,344 12,010 10,675 9,341 8,006 6,672 tons/day 5.0 4.5 4.0 3.5 3.0 2.5 Reagent Costs $1000/yr 2,574 2,574 2,574 2,574 2,574 2,574 Disposal costs $/day 1,106 995 885 774 663 553 $1000/yr 404 363 323 283 242 202 Annual Savings $1000/yr 40 81 121 161 202
The lower flow rate municipal facility (817 GPM) resulted in encouraging results. The reagent costs for the 817 GPM facility are approximately $333,000/yr while the estimated cost savings from the solids disposal range from $341,000 to
$369,000 for biomass reductions of 10% and 50%, respectively (Table 4). The overall savings to the plant range from $8,000/yr to $36,000/yr not considering the cost of equipment and labor associated with the project.
145
TABLE 4: ECONOMIC ANALYSIS FOR A MUNICIPAL WASTEWATER TREATMENT FACILITY WITH A HYDRAULIC FLOW RATE OF 817 GPM.
Biomass Reduction Baseline 10% 20% 30% 40% 50% Yield 0.400 0.360 0.320 0.280 0.240 0.200
Biomass Production lbs/day 572 515 458 401 343 286 tons/day 0.2 0.2 0.2 0.1 0.1 0.1 Reagent Costs $1000/yr 333 333 333 333 333 333 Disposal costs $/day 190 171 152 133 114 95 $1000/yr 69 62 55 48 42 35 Annual Savings $1000/yr 341 348 355 362 369
Total Cost Savings $1000/yr 8 15 22 29 36
The results of the economic analysis indicate that the application of the uncoupler technology is dependent on the hydraulic flow and BOD5 loading as well as the reagent costs. The cost savings for moderate flow, high BOD5 wastewaters was sensitive to the reagent costs. In the industrial case used in this study, the uncoupler technology required a minimum biomass reduction of approximately11% for the cost savings to exceed the cost to implement the technology. In contrast, moderate flow and low BOD5 wastewaters required large amounts of reagent and resulted in minimal cost savings associated with the reduction in biomass production rates. In the moderate flow municipal case analyzed in this study, the application of the uncoupler technology would result in a deficit in excess of $2 million annually. The low flow and low BOD5 wastewaters in the second municipal case showed favorable economics at the 10% 146
biomass reduction level. In this case the biosolids were sent for incineration at a relatively high cost to the facility.
The results suggest that further improvements are required in uncoupler technology to achieve positive economics in all municipal wastewater treatment settings. If the base reagent cost or the required treatment dose can be decreased, a positive cash flow basis can be obtained for moderate flow municipal facilities (Figure
1). The implementation of the uncoupler technology generates a positive cash flow basis at a treatment dose of 1 mg/L at all reagent costs except $20.5/KG. Positive cash flows are also seen with reagent doses of 2.5 mg/L and 5 mg/L for reagent costs of
$2.5/KG and 2.5 mg/L for reagent costs of $5.1/KG.
This economic analysis used a very conservative approach and did not consider the potential of an increase in revenue for the facility. The enhanced degradation per unit biomass during uncoupled growth can increase the capacity of the facility without capital expenditures. These results suggest that the use of uncouplers for biomass reductions in wastewater treatment facilities has the potential to significantly decrease the operating expenses and eliminate capital expansion of capacity limited facilities. 147
FIGURE 1: THE YEARLY REAGENT COSTS FOR REAGENT DOSES FOR REAGENT DOSES OF 1 TO 10 MG/L AND REAGENT COSTS OF $2.5/KG TO $20.5/KG IN A MODERATE FLOW MUNICIPAL WASTEWATER TREATMENT FACILITY.
500 400 300 200 $1000/yr 100 0 Yearly Reagent Cost Cost Reagent Yearly 1 2.5 5 10 Reagent Dose, mg/L
$2.5/KG $5.1/KG $10.3/KG $20.5/KG
Yearly reagents costs in $1000/yr for a moderate flow municipal facility using uncoupler treatment doses of 1, 2.5, 5.0 and 10 mg/L and reagent costs of $2.5/KG (!) , $5.1/KG(!), $10.3/KG (!), and $20.5/KG (!). Cost savings for solids disposal for 50% biomass reduction identified with .
148
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152
Chapter 6 Summary and Conclusions
The addition of 1-hydroxyanthraquinone (OHAQ) to aerobic and anaerobic batch cultures of Pa. denitrificans resulted in classic ―uncoupling‖ activity characterized by an increase in the respiration rate and decrease in biomass production.
The compound was active in aerobic and anaerobic cultures of Paracoccus, batch continuous Paracoccus cultures and in mixed cultures derived from a municipal wastewater treatment facility.
At a test concentration of 10 mg/L, OHAQ addition increased the nitrogen production rate 45.7 % (p= 2.29 x 10-2) and decreased the biomass production 24.5 %
(p=1.33 x 10-3) in anaerobic cultures using nitrate as the terminal electron acceptor.
The activity was comparable using either nitrate or nitrite as the terminal electron acceptor. In cultures using nitrite as the terminal electron acceptor, OHAQ addition
-3 increased the nitrogen production rate 64.5% (p= 6.66 x 10 ) and decreased the biomass production 30.7% (p=2.06 x 10-5) compared to the control. The unsubstituted parent compound, which lacks a hydroxyl group at position 1 of the ring system, did not result in a decrease in biomass production or increase in nitrogen production rates.
In aerobic batch cultures, OHAQ at a test concentration of 10 mg/L, increased the initial oxygen utilization rate 38.5% (p= 3.05 x 10-4) and decreased the biomass production 39.3% (p = 3.13 x 10-2). In studies using anthraquinone compounds with different ring substitutions, the biomass reduction was found to be related to both the 153
positional and electronic properties of the substitution(s). Electron donating groups
(EDG), such as hydroxyl or amino substitutions, in position 1 (ortho) resulted in a significant reduction in biomass production. Conversely, substitution of either a hydroxyl or amino group at position 2 (para) resulted in almost the complete inhibition of growth and respiration.
OHAQ addition to a continuous Paracoccus culture resulted in an increase in the respiratory activity compared to the steady state control values and was sustained over the duration of the experiment. There was a 10.4% in the carbon evolution rate
(CER) and a 10.8 % increase in the specific oxygen utilization rate (SOUR). The growth of the organism was not adversely affected by the presence of OHAQ and the difference in the biomass concentrations of the control and OHAQ reactors was not significant. These results suggest that the compound could increase the capacity of wastewater treatment facilities without modification in or addition to existing equipment.
The addition OHAQ to a mixed cultures derived from a municipal wastewater treatment facility resulted in an increase in the initial oxygen utilization rate and a reduction in the overall biomass production. The overall oxygen utilization was not adversely affected by OHAQ. In test vessels containing 300 mg/L MLSS, 10 mg/L
OHAQ increased the initial oxygen consumption rate 16.9% (p=3.32 x 10-3) and decreased the biomass production 10.8 % (p = 7.76 x 10-3) compared to the control bottles. The biomass reduction in the mixed culture batch studies was less than that obtained in the Paracoccus batch studies. This value, although lower than that 154
obtained in the pure culture studies, is significant in regards to the relative biomass production produced in wastewater treatment facilities.
These studies demonstrate that the activity of OHAQ is the result of the
―uncoupling‖ of aerobic and anaerobic respiration. The resulted reported here indicate that OHAQ has the potential to reduce both the capital and operating costs of biological wastewater treatment systems, especially in the industrial setting, by simultaneously improving the metabolic capacity and decreasing the biomass production rates. The uncoupling activity of anthraquinones combined with the low toxicity of compounds suggests that these compounds may be used for reducing biomass production in commercial bioprocesses in addition to traditional wastewater treatment systems. The need for efficient, cost-effective and environmentally sound waste treatment in both the municipal and industrial markets warrants the further investigation of the use of this chemical uncoupler for enhancement of metabolic capacity and biomass reductions.
155
Chapter 7 Barriers to Implementation
The concept of uncouplers for biomass reduction in activated sludge systems is
not new, and while there is a large amount of literature on uncouplers, reports on their
application in activated sludge systems is limited. The results from laboratory
investigations indicate that OHAQ has the potential to reduce both the capital and
operating costs of biological wastewater treatment systems by simultaneously
improving the metabolic capacity and decreasing the biomass production rates.
Although uncouplers have the potential to significantly reduce biomass production, the
barriers to their implementation at the plant scale include: lack of pilot scale
demonstrations and long term operating data, bacterial acclimation and potential
discharge of the chemical into the environment.
Lack of Pilot Scale Demonstrations
There is an abundance of literature on the topic of uncouplers but just a handful
of investigations into the use of uncouplers in WWT systems. The lack of information
on the use of uncouplers at the pilot or full scale is at best discouraging. Although
there have been reports on the ―evidence‖ or the ―likelihood‖ of uncoupling in full-
scale treatment systems, most of the information is speculative and lacks a solid 156
scientific investigation. For example, Okey and Stensel 134 observed behavior
indicative of uncoupling at a full-scale activated sludge system but could not detect any
organic uncouplers at a high enough concentration to produce these effects. In their
analysis of 23rd Avenue Treatment Plant in Phoenix, they found a decrease in the
biomass production from 0.25-0.35 lb MLSS/lb COD to zero and a dramatic increase
in the oxygen requirement while the COD removal remained the same or was slightly
increased. Reports of reductions in substrate uptake rates 95, 101, 133, 203, population
shifts, and reduced settling characteristics 23, 101, 203 that adversely affect laboratory
reactor performance merit further investigations on the use of these compounds for
biomass reduction. In addition, the lack of long term pilot scale demonstrations
impedes the implementation of this technology at either an industrial or municipal
facility.
Acclimation
The primary objective of the uncoupler technology is a stable long-term
biomass reduction in a biological treatment system. However, there have been reports
in the literature describing the adaptation and/or acclimation of pure and mixed cultures
to uncouplers. Gage and Neidhardt 49, in experiments with E. coli, found DNP
exposure initially leads to a decrease in the growth rate but that the cultures eventually
recovered to near normal rates. Krulwich et al 88 found mutants of the Bacillus species, 157
originally isolated in the presence of CCCP, were able to synthesize substantial amounts of ATP at uncoupler-reduced P levels that would not support ATP synthesis by the wild type. In continuous flow experiments using activated sludge, Strand and
Stensel 172 observed reductions in yields declined significantly when microbial populations developed which were resistant to the uncoupler, TCP. Harem 60 found the effluent soluble TCP concentration dropped from 2mg/l to an undetectable level between day 75 and 82 in the laboratory reactor study, indicating the development of uncoupler-degrading capability in the bacterial consortium. In their studies of nitrite inhibition of denitrification in Pseudomonas fluorescens, Almeida et al 6 observed uncoupling by nitrous acid in batch systems but not in continuous systems and concluded there was a change in the physiology of the cells with time. The potential for acclimation to chemical uncouplers may be circumvented by alternative dosing regiments or alternating chemical compounds. While it is possible that long term exposure to chemical compounds would encourage the development of a resistant or uncoupler-insensitive population a protocol of bulk or intermittent dosing or alternating of chemical uncoupler compounds may discourage the development of an acclimated population. 158
Effluent Discharges to Environment
The most active uncoupler compounds are generally the most toxic and a
serious problem with the uncoupler technology is the potential presence of the
compound in the effluent. A number of uncoupler compounds, (e.g. DNP, chlorinated
phenols) are designated as toxic pollutants and are subject to effluent limitations under
section 307 of the Clean Water Act 63, 64.
Harem 60 in his studies with 2,4,5-trichlorophenol (245TCP) observed that the
soluble uncoupler concentration was an important operational parameter. The required
effluent concentration for uncoupling with 245TCP was 2 mg/L, which is
approximately the recommended water quality criterion of 2.6 ml/L for the protection
of human health 64. Shah et al 164 found a DNP concentration of 0.92 mg/L was
required for uncoupling in a continuous treatment system. The treatment standard
suggested by Federal guidelines is 70 g/L 63, an order of magnitude below Shah‘s
concentration. 2,4-Dinitrophenol is highly toxic to humans with a lethal oral dose of
14 to 43 mg/kg and has a high acute toxicity, LD50 (rats) of 30 mg/kg, based on short-
term animal tests 153, 168. The toxic nature of DNP and its stringent effluent standard
hamper its utility as an uncoupler at a full-scale wastewater treatment facility.
The anthraquinones have uncoupling activity that resembles that of the classic
10 62 uncouplers but have a low systemic toxicity . The LD50 for anthraquinone is 159
reported to be greater than 5000 mg/kg and in a 90 day feeding trial in rats receiving up to 15mg anthraquinone/kg resulted in no ill-effects153. The low toxicity of the anthraquinone compounds combined with uncoupling activity in bacterial systems may provide an advantage over the classic uncouplers in large scale systems. The laboratory investigations in aerobic and anaerobic test systems suggest that these compounds may be used for reducing biomass production in commercial bioprocesses in addition to traditional wastewater treatment systems. The need for efficient, cost- effective and environmentally sound waste treatment in both the municipal and industrial markets warrants the further investigation of the use of this chemical uncoupler for enhancement of metabolic capacity and biomass reductions. 160
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