Perchlorate and chlorate degradation by two organisms isolated from wastewater Microbial identification and kinetics
Filipa Costa Pinto Prata
Thesis submitted in fulfilment of the requirements for the degree of Master Science in CHEMISTRY
President: Profª Doutora Matilde Marques, IST-UTL Promotors: Profª Doutora Cristina Costa, FCT-UNL Profª Doutora Cristina Viegas, IST-UTL Doutor Paulo Costa Lemos, FCT-UNL
November 2007
ACKNOWLEDGMENTS
I wish to express my sincere gratitude to Prof ª Doutora Maria Cristina Costa, for the opportunity to perform my dissertation, support and guidance.
To Profª Doutora Cristina Viegas I would like to thank to accept to be my promotor.
I would also especially like to thank Doutor Paulo Lemos and Profª Doutora Maria
Ascensão Miranda Reis for their laboratorial guidance, suggestions and scientific advises which improved this work.
I would like to thank the BioEng staff for their friendship during my laboratorial work and also my gratitude to Marta for her hold.
Many thanks to my friends for their constant hold, encouragement and patience throughout the duration of this project. They always have a word of support and a smile to give me. Thank you Cristiana, Mariana and Bruna.
Never enough thanks to one who doesn’t want to be named but he knows who he is and so do I.
To my family I just want to thank for their care, support and ethical values that always motivated me to improve my knowledge and personality.
ABSTRACT
- - The biological removal of perchlorate (ClO 4 ) and chlorate (ClO 3 ) can be viewed as a very promising water treatment technology. The process is based on the ability of specific bacteria to use (per)chlorate as an electron acceptor in the absence of oxygen.
The present research work was focused on the isolation and kinetic characterization of perchlorate reducing bacteria. The enrichment process started with a sludge sample taken from an anaerobic digester of a domestic wastewater treatment plant (Beirolas,
Portugal). Two perchlorate-reducing bacteria (per1) and (per2) were isolated using different selection methods, platting and liquid transfer respectively. The purity of the isolates was confirmed by genetic characterization of 16S rDNA. The BLAST search showed that the microorganims shared a 99% sequence similarities to the 16S rDNA of
Dechlorospirillum sp. DB (per1) and Dechlorosoma sp. PCC (per2). Batch tests were performed under anaerobic conditions with acetate as the electron donor and perchlorate and/or chlorate as electron acceptor. During perchlorate reduction by Dechlorospirillum sp. DB it was observed transient accumulation of chlorate. The isolates showed different behaviour concerning perchlorate and chlorate reduction. Chlorate was preferentially reduced when both electron acceptors were present, being perchlorate reduced after completely depletion of chlorate. The former performance was observed in both bacteria.
Keywords : Bioremediation, Perchlorate and chlorate reduction, Isolation, Kinetic characterization, Dechlorospirillum sp. DB, Dechloromona sp. PCC.
Perchlorate and chlorate degradation by two organisms isolated from wastewater ii TABLE OF CONTENT
CHAPTER 1. LITERATURE STUDY………………………………………… 1 1.1. Introduction………………………………………………………………. 1 1.2. Perchlorate as a pollutant.....……………………………………………… 2
1.2.1. Properties...... …………………………………………………... 2 1.2.2. Perchlorate environmental occurence....…………………………… 4 1.2.3. Health effects..……………………………………………………... 7 1.2.4. Legislation...... 7 . 1.3. Perchlorate treatment technologies……………...…………………………. 8
1.3.1. Physical processes..……………………..…...…………………….. 9 1.3.2. Chemical processes………..…………………………………….... 13 1.3.3. Biological processes...... …..……………………………...... 14
1.4. The microbiology and biochemistry of perchlorate reduction………….. 16
1.4.1. Perchlorate reducing bacteria..……………………………………… 16 1.4.2. Electron donors used by PRB for growth…………...………...... 19 1.4.3. Nutritional requirements for PRB……..…………………………... 19 1.4.4. Biological perchlorate reduction…………………………………… 20 1.4.5. Enzymes responsible for (per)chlorate reduction………………….. 21 1.4.6. Factors that interfere with perchlorate enzyme induction………… 25
1.5. Outline of the thesis………………………………………………………... 26
27 CHAPTER 2. MATERIALS AND METHODS….……………………………… 2.1. Source of organisms……………………...………………………………. 27 2.2. Media...... 27 2.3. Bacterial isolation procedures and culturing conditions …………………. 28
Perchlorate and chlorate degradation by two organisms isolated from wastewater iii 2.4. Morphology………………………………………………………………… 29 2.5. 16S ribosomal DNA extraction and sequencing…………………………… 29
2.5.1. Extraction and confirmation 16S ribosomal DNA…………………… 29 2.5.2. PCR amplification and purification………………………………… 30
2.6. Phylogenetic analysis……..………………………………………………... 31 2.7. Batch growth kinetics……………………………………………………… 31 2.8. Analytical techniques……………………………………………………… 32 2.9. Calculations……………………………………………………………….. 33
2.9.1. Specific growth rate………………………………….…...... 33 2.9.2. Specific uptake rate…………..………………………………………. 33 2.9.3. Substrate uptake yield………..…………………………………….. 33 2.9.4. Chloride formation yield…………………………………………… 33 2.9.5. Biomass yield………………………………………………………. 33
CHAPTER 3. RESULTS AND DISCUSSION …………………………..……… 34 3.1. Results………………………………………..……………………….……. 34
3.1.1. Morphological and genetic characterization of the isolates…...... 34 3.1.2. Growth kinetics………………..…………………………………… 35
3.2. Discussion…………………………….………………………………….. 43
CHAPTER 4. CONCLUSIONS AND FURTHER RESEARCH....………….… 49
CHAPTER 5. BIBLIOGRAPHY……………..………..………………………... 51
Perchlorate and chlorate degradation by two organisms isolated from wastewater iv LIST OF TABLES
Table 1.1: Physical and chemical properties of selected perchlorate compounds...... 4 Table 1.2: Perchlorate respiring bacterial isolates...... 17 Table 2.1: Media and reagents used for enrichment and isolation .…… ……….…... 27 Table 3.1:Specific growth rates of described perchlorate and chlorate reducing bacteria…………………………………………………………………… 44 Table 3.2: Biomass yields in the presence of different electron acceptors determined in this study and reported by others...... 46 Table 3.3: Resume of all kinetics parameters (n, number of data points considered for parameter calculations)…… …...... 48
LIST OF FIGURES
Figure 1.1: Energy profile for the rate-limiting step in perchlorate reduction, abstraction of the first oxygen atom. ………………….…………….…. 3 Figure 1.2: Perchlorate manufacturers and users in US, April 2003 ………………. 5 Figure 1.3: Concentrations levels of perchlorate found in wine samples from various Continents…………………………………………………….. 6 Figure 1.4: Mechanism of anion exchange – chloride for perchlorate.…………….. 10 Figure 1.5: Reverse osmosis (RO). The influent water is forced through a membrane that is impermeable to dissolved salts.……………………... 11 Figure 1.6: Electrodialysis. Water flows through alternate semipermeable membranes while under the influence of an electric field .…………….. 12 Figure 1.7: Simple electrolytic cell of the reduction of perchlorate.……………….. 13 Figure 1.8: Schematic diagram of ion transport and bioreduction in the ion exchange membrane bioreactor …………………………………..… 15 Figure 1.9: Phylogenetic distribution of (per)chlorate and chlorate reducing microorganisms based on total 16S rDNA ………………………….. 18
Perchlorate and chlorate degradation by two organisms isolated from wastewater v Figure 1.10: Schematic of perchlorate-reducing pathway, based on accepted roles of (per)chlorate reductase and chlorite dismutase enzymes………….. 20 Figure 1.11: Model of the pathway involved in the respiratory reduction of (per)chlorate by (per)chlorate reducing bacteria ……………………… 21 Figure 1.12: Model of the pathway involved in the reduction of chlorite by perchlorate reducing bacteria………………………………………… 23 Figure 2.1: Schematic representation of the reactor used for batch tests………….. 28 Figure 2.2: Schematic representation of the reactor used for batch tests………….... 31 Figure 3.1: Optical microscopy observation of the enriched cultures; A: (per1) and B: (per2) (100x)……………...…………………… ……………..…….. 34 Figure 3.2: Acetate and perchlorate uptake and transient accumulation of chlorate - as function of time during the reduction of 10mM of ClO 4 by Dechlorospirillum sp. DB. Note the different concentration scale for - ClO 3 . Dry Weight (DW) as function of time is also represented…...... 36 Figure 3.3: Acetate and chlorate uptake as function of time during the reduction of - 10mM of ClO 3 by Dechlorospirillum sp. DB. Dry weight (DW) and chloride formation as a function of time are also represented…………. 37 Figure 3.4: Acetate, perchlorate and chlorate uptake as function of time during the - - reduction of 5mM of ClO 4 + 5mM of ClO 3 by Dechlorospirillum sp. DB. Dry weight (DW) and chloride formation as a function of time are also represented……………………………………………………… 38 Figure 3.5: Perchlorate and chlorate uptake as function of time during the - - reduction of 5mM of ClO 4 + 5mM of ClO 3 by Dechlorospirillum sp. DB.………...…………………………………………………………… 39 Figure 3.6: Acetate and perchlorate uptake as function of time during the reduction
of 10mM of ClO 4- by Dechlorosoma sp. PCC. Dry weight (DW) as function of time is also represented..…………………………… …….. 40 Figure 3.7: Acetate and chlorate uptake as function of time during the reduction of - 10mM of ClO 3 by Dechlorosoma sp. PCC. Dry weight (DW) and chloride formation as a function of time are also represented...... 41 Figure 3.8: Acetate, perchlorate and chlorate uptake as function of time during the - - reduction of 5mM of ClO 4 + 5mM of ClO 3 by Dechlorosoma sp. PCC. Dry weight (DW) as a function of time is also represented.…….. 42
Perchlorate and chlorate degradation by two organisms isolated from wastewater vi Chapter 1 – Literature study
Chapter 1. LITERATURE STUDY
1.1. INTRODUCTION
- - Perchlorate (ClO 4 ) and chlorate (ClO 3 ) have been produced on large scale by the chemical industry for use in a wide range of applications. The improper storage and/or disposal of these oxyanions have led to harmful concentrations in surface and groundwater supplies, as they are extremely soluble and not significantly broken down in the environment. These characteristics make them persistent and problematic
- environmental pollutants of drinking waters. Moreover, ClO 4 and chlorate are also a health concern, as they can cause serious diseases such cancer. In the medium-term the
- removal of ClO 4 from drinking water will become necessary in order to protect the environment and human health. The long-term solutions must involve a reduction in the
- release of ClO 4 into the environment and wastewater treatment should be done more efficiently.
The biological removal of these anions can be viewed as a very promising water treatment technology. The process is based on the ability of specific bacteria to utilize
(per)chlorate as a physiological electron acceptor in the absence of oxygen and reduce it completely to innocuous chloride. The main advantages of this process are the selectivity, its fastness and the low operating costs. Although a number of investigators are currently working on bioreduction processes, studies are needed to identify and
- characterize more of the microorganisms that reduce ClO 4 so as to optimize conditions
Perchlorate and chlorate degradation by two organisms isolated from wastewater 1
Chapter 1 – Literature study for maximal destruction while minimizing by-product formation, wasteful side- reactions and nutrient consumption. Also more effort must be expended in elucidating
- the mechanism by which microorganisms reduce ClO 4 , including isolation, purification and characterization of the active enzyme(s). It may be possible to exploit the mechanism whereby the bacteria are capable of perchlorate reduction, but only if we have a better understanding of that mechanism.
1.2. PERCHLORATE AS A POLLUTANT
1.2.1. Properties
- As an anion, ClO 4 consists of a central chlorine atom surrounded by a tetrahedral array of four oxygen atoms. As the oxidation state of the chlorine is +7, the species is a strong oxidizing agent (1).
– + - – ClO 4 + 8H + 8e ↔ Cl + 4H 2O, E° = 1.287 V (1)
- 2- Nevertheless, ClO 4 is slightly weaker than dichromate (Cr 2O7 ) or
- permanganate (MnO 4 ) and its redox reaction is extremely non-labile, i.e. reacts slowly
- with most reducing agents. The reduction of ClO 4 can only be observed in concentrated
- strong acid. In 0.1 to 4.0 M acid solution, ClO 4 is not reduced by common reagents
- such as thiosulfate, sulfite, or iron(II). In fact, the redox behaviour of ClO 4 is so rarely observed in chemical systems that sodium perchlorate is used to adjust the ionic strength of solutions prior to electrochemical or other laboratory studies. This behaviour
Perchlorate and chlorate degradation by two organisms isolated from wastewater 2
Chapter 1 – Literature study results from the high strength of the chlorine-oxygen bonds and the requirement that reduction must proceed initially by oxygen atom abstraction rather than a direct involvement of the central chlorine atom. This kinetic behaviour is illustrated in Figure
1.1. The conversion of perchlorate to chlorate is generally regarded as the first step in perchlorate reduction pathway. The reaction is thermodynamically favoured as shown by ∆E<0, i.e., the products have lower internal energy than the reactants. However, the reaction rate is controlled by the kinetic barrier of the high activation energy E a of the transition state.
Figure 1.1 – Energy profile for the rate-limiting step in perchlorate reduction, abstraction of the first oxygen atom. (Urbansky and Schock, 1999).
- In addition to its resistance to reduction, ClO 4 has a relatively low charge density. Consequently, it does not generally form complexes with metals in the same way other anions do. Perchlorate is routinely employed as a counter-ion in the synthesis of metal compounds when a non-complexing anion is required (Urbansky, 2000). Some physical and chemical properties of perchlorate compounds are summarized in Table
1.1.
Perchlorate and chlorate degradation by two organisms isolated from wastewater 3
Chapter 1 – Literature study
Table 1.1 – Physical and chemical properties of selected perchlorate compounds.
Ammonium Sodium Potassium Perchloric Property Perchlorate Perchlorate perchlorate acid
Formula NH 4ClO4 NaClO 4 KClO 4 HClO 4 Formula 117.49 122.44 138.55 100.47 Weight Colourless White, crystals or white, White, orthorhombic crystalline Colourless, oily orthorhombic crystals; white powder; Colour /Form liquid crystals deliquescent colourless, crystals orthorhombic crystals Decomposes/ 480 oC 525 oC -112 oC Melting Point explodes Density 1.95 g/cm 3 2.52 g/cm 3 2.53 g/cm 3 1.768 g/cm 3 200 g/L of water 209.6 g/100mL of 15 g/L of water at Miscible in cold Solubility at 25 oC water at 25 oC 25 oC water
1.2.2. Perchlorate environmental occurrence
- As a strong oxidizing agent, ClO 4 is mostly used as ammonium perchlorate
(NH 4ClO 4) in the manufacturing of solid rocket fuel, missiles and explosives for various military munitions and also in industrial products (e.g., fireworks, air bag inflators and paint). Its production began in the United States in the mid-1940s, and since then large amounts of perchlorate waste effluents have been released to the environment. The
- presence of ClO 4 in water supplies and soils is also linked to the earlier use of Chilean nitrate as fertilizer (Urbansky et al., 2001), and recently its natural formation was
- reported (Dasgupta et al. 2005). It was showed that ClO 4 is readily formed by a variety of simulated atmospheric processes, for example, it is formed from chloride aerosol by electrical discharge and by exposing aqueous chloride to high concentrations of ozone.
Most of the affected regions have perchlorate concentrations below 0.5 g L -1, although concentrations as high as 3.7 g L -1 have been encountered in the United States.
Perchlorate and chlorate degradation by two organisms isolated from wastewater 4
Chapter 1 – Literature study
As of April 2003, there were more than 100 perchlorate users located in 40 states as shown in Figure 1.2 (Mayer, 2004). However, perchlorate are been used in a variety of operations all over the world. In Europe, perchlorate compounds are mostly produced in
Italy, France and Germany mainly as ammonium perchlorate to use as a solid propellant.
Figure 1.2 – Perchlorate manufacturers and users in US ( ●), April 2003 (Mayer, 2004).
Besides soil and groundwater, perchlorate is also ubiquitous in beverages and food products worldwide. Analysis of perchlorate in food and beverages samples
- showed higher concentrations than the values established by US EPA, 24.5 ppb ClO 4
(El Aribi et al., 2006). For example, America and Europe showed the highest perchlorate concentration in wine samples (Figure 1.3) and individually a rosé sample
- from Portugal showed the highest level with 50.25 ppb ClO 4 . Concerning beer samples
- perchlorate concentrations ranged from 0.03 to 10.663 ppb ClO 4 (average values of
Perchlorate and chlorate degradation by two organisms isolated from wastewater 5
Chapter 1 – Literature study triplicate samples). Also water samples were analyzed and linked with Portugal, perchlorate was found in tap and bottled water in Porto region with 0.041 and 5.098 ppb
- ClO 4 , respectively. Some of the other high concentrations that El Aribi and his colleagues reported include 145.6 ppb in Chilean apricots, 62.8 ppb in Mexican red tomatoes, 22 ppb in Chilean green grapes, and 39.9 ppb in raw Mexican asparagus. A surprising facet of the reported study is that perchlorate can remain in food even after it is cooked. Asparagus from Mexico had 39.9 ppb raw but retained 24.4 ppb after being boiled in water. This is a surprising result, because perchlorate is very soluble in water.
America
Europe
Australia Asia
Africa
0 1 2 3 4 5 6
- ClO 4 (ppb)
Figure 1.3 – Concentrations levels of perchlorate found in wine samples from various continents
(El Aribi et al., 2006).
The variation of perchlorate concentration from different continents, countries and even producers within the same region adds an additional dimension to the complexity of human exposure to perchlorate. Given that some of the levels of perchlorate found in drinking and food products are relatively high, it could be of health concern when considering all dietary sources.
Perchlorate and chlorate degradation by two organisms isolated from wastewater 6
Chapter 1 – Literature study
1.2.3. Health effects
Perchlorate competitively block thyroid iodine uptake and inhibit normal thyroid hormone production, which could lead to metabolic problems in adults and anomalous development in children (Greer et al., 2002). Preliminary toxicological studies have
- demonstrated that ClO 4 has a direct effect on iodine uptake by the thyroid gland at concentrations of 6 mg per kg of body weight per day resulting in fatal bone marrow
- disease (Greer et al., 2002). However, the long-term health effects of low levels of ClO 4 have not yet been established.
Chlorate is also a potential chlorine oxyanion pollutant which has been used as an herbicide in agriculture and it is used for the on-site generation of the bleaching agent chlorine dioxide (ClO 2) in the paper and pulp industry (Rosemarin et al., 1990). It can also be formed through the ozonation of drinking waters treated with chlorine
(Siddiqui, 1996). When fed to rats and mice in their drinking waters, the effect of
- chlorate and chlorite (ClO 2 ) causes oxidative damage to red blood cells, resulting in haemolytic anaemia and methaemoglobin formation (Stettler, 1977; Condie, 1986).
1.2.4. Legislation
The adverse health effects resultant from the ingestion of these anions are only observed at sufficient high doses. As mentioned before, the long-term health effects at levels currently encountered in the contaminant water sources have not yet been established. Perchlorate contamination was known to be a problem especially within the
- United States, but recent reports had shown ClO 4 contamination all over the world, especially in Europe and Middle East (El Aribi et al., 2006). Given the seriousness of
Perchlorate and chlorate degradation by two organisms isolated from wastewater 7
Chapter 1 – Literature study the potential adverse effects associated with these compounds and based on the report by the National Academy of Sciences (NAS) (National Research Council, 2005), the
- US Environmental Protection Agency (EPA) has established for ClO 4 an official reference dose (RfD) corresponding to a drinking water level of 24.5 ppb. Conversely, there are uncertainties in the toxicological database that is used to address the potential
- of ClO 4 to affect human health effects when present at low levels in drinking water.
- Consequently, as of April 2007, the EPA has not determined whether ClO 4 is present at sufficient levels in the environment to require a nation-wide regulation and how much should be allowed in drinking water. The World Health Organization (WHO) had not
- established drinking-water-quality guideline for ClO 4 as well. In the European communities there is no guide level for perchlorate. Although no other country has
- legislation regarding this matter, research on ClO 4 removal from drinking and wastewater is underway.
1.3. PERCHLORATE TREATMENT TECHNOLOGIES
Ideally, a technology should be able to handle concentrations ranging from ≤ 5
µg L -1 all the way to ~10 g L -1. The existing water treatment technologies for the
- removal of oxyanions like ClO 4 can be divided into physical, chemical and biological technologies. The physical are considered as a removal technology and chemical and biological as a destruction process. Within the first group, anion exchange (Gu et al.,
2003) as well as membrane processes such as electrodialysis (Roquebert et al., 2000), nanofiltration or reverse osmosis (Amy et al., 2003) are commonly used. However,
- ClO 4 is accumulated in a brine solution or a concentrated stream, which have to be
Perchlorate and chlorate degradation by two organisms isolated from wastewater 8
Chapter 1 – Literature study treated after disposal. Destruction is generally regarded as a preferable process because it eliminates the need for subsequent disposal of removed material, which is regarded as a hazard in this case.
1.3.1. Physical processes
Physical removal processes work exactly as the name suggests. They physically
- - separate ClO 4 ion from water. As these techniques do not destroy the ClO 4 , they create
- a subsequent need for disposal and treatment of both the ClO 4 and any waste products of the process. In addition, all of these techniques currently suffer from lack of
- selectivity. Along with the ClO 4 , they tend to remove or replace unacceptably large quantities of beneficial dissolved salts or their components parts. Although these technologies are all well-established, they will be difficult to use in large systems,
- mainly because of the low concentration of ClO 4 in the source water and the lack of selectivity. Moreover, their use is limited even in small water systems by pre-treatment and post-treatment factors.
Anion Exchange
- Anion exchange is a technology frequently used to remove ClO 4 from drinking water, groundwater, surface water, and environmental media at full scale. Anion exchange resins are usually packed into a column, and as contaminated water is passed through the column, contaminant ions are exchanged for other ions such as chlorides or hydroxides in the resin (Figure 1.4).
Perchlorate and chlorate degradation by two organisms isolated from wastewater 9
Chapter 1 – Literature study
Figure 1.4 - Mechanism of anion exchange – chloride for perchlorate.
- The most commonly used anion exchange media for ClO4 removal are synthetic
- and strongly basic resins. This technology has been used at sites to reduce ClO 4 concentrations to less than 3 µg/L (Gu et al., 2003). Its effectiveness is sensitive to a variety of untreated water contaminants and characteristics. It has also been used as a
- polishing step for other water treatment processes such as biological treatment of ClO 4 .
In general, ion exchange is often preceded by treatments such as filtration and oil-water separation to remove organics, suspended solids, and other contaminants that can foul the resins and reduce their effectiveness. The main drawback of this technology is the periodic regeneration of resins to remove the adsorbed contaminants and replenish the exchanged ions. The regeneration process results in a backwash solution, a waste regenerating solution and a waste rinse water that need to be treated afterwards. The lack of selectivity should also be considered as a disadvantage of this process.
Reverse osmosis
- Reverse osmosis (RO) is a membrane technique also used for ClO 4 removal
(Urbansky, 1998). Reverse osmosis is a physical separation method based on the principle of osmosis. In this technology, high pressure is applied to reverse the osmosis process and force water molecules to pass through the semi-permeable membrane out of
Perchlorate and chlorate degradation by two organisms isolated from wastewater 10
Chapter 1 – Literature study the perchlorate contaminated water (Figure 1.5). As a result, two channels of water are formed in the reverse osmosis system. One is treated water from the freshwater side of
- the system and the other is concentrate or salty water containing ClO 4 , which is subject to further treatment prior to disposal.
Figure 1.5 - Reverse osmosis (RO). The influent water is forced through a membrane that is impermeable to dissolved salts.
- RO membranes are capable of removing 80% or more of the ClO 4 . Membrane filtration point-of-use devices can be practical options for homeowners, small businesses, or isolated users. Over again, the lack of selectivity and the concentrate disposal are the most disadvantages of this process. Membrane corruption should also be considered as a trouble in this technology.
Electrodialysis
- Electrodialysis is another physical method for removing ClO 4 . This technology
- applies an electric current to remove ClO 4 . Perchlorate-contaminated water is exposed to an electric current as it passes through a semi-permeable membrane (Figure 1.6). This
- separates ClO 4 ions from contaminated groundwater and surface water. The technology
Perchlorate and chlorate degradation by two organisms isolated from wastewater 11
Chapter 1 – Literature study produces alternate channels of nearly deionised water (the diluate or dialyzate) and salty water (the concentrate). The diluate is used, and the concentrate undergoes further treatment prior to disposal (Roquebert et al., 2000; Urbansky and Schock, 1999).
Figure 1.6 - Electrodialysis. Water flows through alternate semipermeable membranes while under the influence of an electric field.
1.3.2. Chemical processes
Chemical and electrochemical reduction
- From the description of the oxidation-reduction reactions of ClO 4 above, it is clear that chemical reduction will play no role in drinking water treatment in the near future. Chemical reduction is simply too slow. Unless safe new catalysts become available, this appears unlike to change. Common reductants like iron metal, thiosulfate, sulfite, iodide, and ferrous ions, do not react at any observable rate, and the more reactive species are too toxic (and still to sluggish). In addition, any reductant will necessarily have oxidized by-products. The toxicity of the by-products must be
Perchlorate and chlorate degradation by two organisms isolated from wastewater 12
Chapter 1 – Literature study considered and consequently there is more hope for electrochemical reduction. A decided advantage of electrochemical reduction is the large amount of control over kinetics that results from control of the operating potential. Although electrochemical technologies are well established for other industries such electroplating of metals and electrodialysis of brine, they have not yet found a place in drinking water treatment.
Nevertheless, it should also be considered the electricity consumption and the high operation costs of this technology.
Figure 1.7 - Simple electrolytic cell of the reduction of perchlorate.
1.3.3. Biological processes
- - o The high reduction potential of chlorate and perchlorate (ClO 3 /Cl E = 1.03 V;
- - o ClO 4 /Cl E = 1.287 V) makes them ideal electron acceptors for microbial metabolism.
In this way, biological reduction appears to hold the most promise for large-scale
- treatment of perchlorate-laden waters, since ClO 4 can be biologically degraded under suitable conditions. Some technology current been used in this purpose are the bioreactor and membrane bioreactors. The latter combines biological with physical
- processes to remove ClO 4 from contaminated water.
Perchlorate and chlorate degradation by two organisms isolated from wastewater 13
Chapter 1 – Literature study
Bioreactor
- A bioreactor frequently serves as a technology for removing ClO 4 from contaminated groundwater and surface water at full scale. This technology uses microorganisms capable of reducing perchlorate into innocuous chloride and oxygen in the presence of an electron donor and an appropriate medium to support microbial growth. Contaminated water is placed in direct contact with microbes that selectively
- degrade the contaminant of concern. Bioreactors have been used at sites to reduce ClO 4 concentrations less than 4 µg/L (Urbansky and Schock, 1999). An example of this system is the fluidized bed bioreactor. They are made up of suspended sand or granular- activated carbon media to support microbial activity and growth of biomass. The activated carbon media are selected to produce a low-concentration effluent (i.e., at part-per-billion levels) and provide larger surface area for growth of microorganisms.
The fluidized bed expands with the increased growth of biofilms on the media particles.
The result of this biological growth is a system capable of additional degradative performance for target contaminants. Normally, the treated effluent is suitable for discharge, but when applied for drinking water treatment, the effluent from bioreactors might require further treatment to remove biosolids present in the effluent.
Membrane Bioreactor
Combining physical removal with biological degradation, the ion exchange membrane bioreactor (IEMB) is an integrated process that combines the transport of charged pollutants from water streams through an appropriated ion exchange membrane
Perchlorate and chlorate degradation by two organisms isolated from wastewater 14
Chapter 1 – Literature study with their simultaneous biodegradation by a suitable microbial culture in a separate
- compartment (Figure 1.8). This process was successfully tested for drinking water ClO 4
- removal from 100ppb to 4ppb of ClO 4 (Matos et al., 2006).
Figure 1.8 - Schematic diagram of ion transport and bioreduction in the ion exchange membrane bioreactor (Matos et al., 2006).
- Biological degradation of ClO 4 itself or combined with physical removal gather operational conditions which makes this process the most promising technology for
- ClO 4 treatment, as it has low operation costs and high selectivity. Several drinking water, wastewater, and in-situ treatment systems are being developed to biologically
- remove ClO 4 , but there is little ongoing research directed toward the physiology of perchlorate reducing bacteria (PRBs).
Perchlorate and chlorate degradation by two organisms isolated from wastewater 15
Chapter 1 – Literature study
1.4. THE MICROBIOLOGY AND BIOCHEMISTRY OF
PERCHLORATE REDUCTION
1.4.1. Perchlorate reducing bacteria
The first studies published on the biological reduction of chlorine oxyanions indicated that microorganisms rapidly reduced chlorate that was applied as an herbicide for thistle control (Aslander, 1928). Initial investigation of the microbiology of chlorate reduction suggested that it was mediated by nitrate-respiring organisms in the environment and chlorate uptake and reduction was simply a competitive reaction for the nitrate reductase system of these bacteria (Coates and Achenbach, 2004; de Groot et
- al., 1969). The ability of bacteria to use ClO 4 as a terminal electron acceptor was not reported until 1976 (Romanenko et al., 1976). Initially, it was supposed that all chlorate
- reducing bacteria (CRB) were able to reduce ClO 4 , leading to the early speculation of the abbreviation (per)chlorate. However, current studies have provided evidence that not all CRB are perchlorate reducing bacteria (PRB) and consequently there is a subset of
- CRB that cannot use ClO 4 as an electron acceptor for respiration (Logan et al., 2001b).
PRB are nearly ubiquitous and have now been isolate from a broad diversity of environments, including rivers, sediments, soils, farm animal waste lagoons and wastewater treatment plants (Bruce et al., 1999; Wolterink et al., 2002; Achenbach et al., 2001; Waller et al., 2004; Bardiya et al., 2006). More than fifty dissimilatory
(per)chlorate-reducing bacteria are now in pure culture and this number continues to increase (Table 1.2 and Figure 1.9).
Perchlorate and chlorate degradation by two organisms isolated from wastewater 16
Chapter 1 – Literature study
Table 1.2 – Perchlorate respiring bacterial isolates.
Electron Isolate Reference acceptor
- - - Vibrio dechloraticans Cuznesove B-1168 ClO 4 , ClO 3 , NO 3 Korenkov et al. (1976) - - - Wolinella succinogenes HAP-1 ClO 4 , ClO 3 , NO 3 Wallace et al. (1996) - - Dechloromonas agitata ClO 4 , ClO 3 , O 2 Achenbach et al. (2001) - - - GR-1 ClO 4 , ClO 3 , NO 3 , O 2, Mn (IV) Rikken et al. (1996) - - - Dechloromonas sp. HZ ClO 4 , ClO 3 , NO 3 , O 2 Zhang et al. (2002) - - - Dechlorosoma suillum ClO 4 , ClO 3 , NO 3 , O 2 Achenbach et al. (2001) - - AB-1 ClO 3 , NO 3 Bliven et al. (1996) - - - Dechlorospirillum JB116 ClO 4 , ClO 3 , NO 3 Bardiya and Bae (2006) - - Dechlorosoma sp. KJ ClO 4 , ClO 3 , O 2 Logan et al. (2001b) - - Dechlorosoma sp. PDX ClO 4 , ClO 3 , O 2 Logan et al. (2001b)
Some PRB strains reported in the literature include: Vibrio dechloraticans
Cuznesove B-1168 (Korenkov et al., 1976), Wolinella succinogenes HAP-1 (Wallace et al., 1996), Dechlorosoma suillum (Achenbach et al., 2001) and isolates GR-1 (Rikken et al., 1996) and perclace (Herman et al., 1998). These PRB are mainly Gram-negative, facultative anaerobes or microaerophilic and non-fermenting. Analysis of the 16S rDNA demonstrated that these organisms are phylogenetically diverse with members in the α-,
β-, γ-, and ε-subclasses of the Proteobacteria (Coates et al., 1999; Wallace et al., 1996).
Most of them belong to the β-subclass of the Proteobacteria and are members of the genus Dechloromonas or Dechlorosoma (Coates et al., 1999).
Perchlorate and chlorate degradation by two organisms isolated from wastewater 17
Chapter 1 – Literature study
Figure 1.9 – Phylogenetic distribution of (per)chlorate and chlorate reducing microorganisms based on total 16S rDNA (Coates and Achenbach, 2004)
Perchlorate and chlorate degradation by two organisms isolated from wastewater 18
Chapter 1 – Literature study
1.4.2. Electron donors used by PRB for growth
- Acetate (CH 3COO ) has been most frequently used as a single substrate for
- heterotrophic ClO 4 reduction (Wu et al., 2007). Other alternative electron donors include a wide variety of organic substrates, including alcohols, carboxylic acids and simple volatile fatty acids, such as lactate, propionate, butyrate, or valerate. For autotrophic PBR growth, hydrogen (Nerenberg et al., 2002; Nerenberg et al., 2006), both soluble and insoluble ferrous iron and hydrogen sulphide could also be used as a growth substrate.
1.4.3. Nutritional requirements for PRB
There is no detailed information on the best medium to use for PRB or what trace nutrients or metals are needed for growth, but iron, molybdenum, and selenium
- appear to be important for PRB growth and ClO 4 degradation (Xu et al., 2003). Recent
- molecular studies of the genetic systems associated with ClO 4 reduction indicated the presence of a molybdenum-dependent chaperone gene in association with the genes encoding chlorite dismutase (CD) and perchlorate reductase in Dechloromonas aromatica strain RCB and Pseudomonas sp. strain PK (Bender et al., 2002).
Furthermore, the perchlorate reductase enzyme purified from strain GR-1 contained 1 mol of molybdenum per mol of the heterodimeric molecule (Kengen et al., 1999)
- suggesting that molybdenum play a functional role in the reduction of ClO 4 . In support
- of this, growth and ClO 4 reduction were completely inhibited when an active
Perchlorate and chlorate degradation by two organisms isolated from wastewater 19
Chapter 1 – Literature study perchlorate-respiring culture of D. aromatica was transferred into medium from where molybdenum was omitted (Chaudhuri et al., 2002).
1.4.4. Biological perchlorate reduction
Understanding the respiratory pathways used by bacteria will be important to the long term operation of biological reactors. In 1996, Rikken and his colleagues proposed a three step mechanism of perchlorate reduction.
- - - - ClO 4 ClO 3 ClO 2 O 2 + Cl
- The ClO 4 reduction pathway consist in three steps: the first two steps via two electrons transfers with (per)chlorate reductase, which sequentially reduces perchlorate to chlorate, then chlorate to chlorite (Kengen et al., 1999; Bender et al., 2005). The third step with chlorite dismutase, which transforms chlorite into chloride and oxygen by disproportionation, does not consume electrons and therefore does not directly produce energy for the cells (van Ginkel et al., 1996; Bender et al., 2002).
Figure 1.10 – Schematic of perchlorate-reducing pathway based on accepted roles of (per)chlorate reductase and chlorite dismutase enzymes (Nerenberg et al., 2006).
Perchlorate and chlorate degradation by two organisms isolated from wastewater 20
Chapter 1 – Literature study
- Chlorate produced by the perchlorate reductase should compete with ClO 4 for
- the catalytic site of the (per)chorate-reductase enzyme, presumably slowing the ClO 4 reduction rate. While some amount of chlorate accumulation is possible, it only has
- been reported for a mixed culture growing on nitrate and ClO 4 (Nerenberg et al., 2002)
- and for a pure culture growing on ClO 4 (Nerenberg et al., 2006).
1.4.5. Enzymes responsible for (per)chlorate reduction
Perchlorate reductase
Perchlorate reductase and chlorite dismutase are the only enzymes in the perchlorate reduction pathway that have been isolated and characterized (van Ginkel et al., 1996; Coates et al., 1999; Kengen et al., 1999; Okeke et al., 2003) (Figure 1.11).
However, concerning chlorate reducers, a chlorate reductase from a Pseudomonas chloritidismutans that is only able to use chlorate as a terminal electron acceptor, was also isolated and characterized (Wolterink et al., 2003).
Figure 1.11 – Model of the pathway involved in the respiratory reduction of (per)chlorate by
(per)chlorate reducing bacteria (Achenbach et al., 2006)
Perchlorate and chlorate degradation by two organisms isolated from wastewater 21
Chapter 1 – Literature study
To date, few data are available for perchlorate reductase. The first isolated perchlorate reductase belongs to strain GR-1 (Kengen et al., 1999). It was found a single enzyme able to catalyze both chlorate and perchlorate. The oxygen-sensitive enzyme is located in the periplasm and have and apparent molecular mass of 420 kDa, with subunits of 95 kDa and 40 kDa in an α3β3 composition. Metal analysis of this enzyme showed the presence of 11 mol of iron, 1 mol of molybdenum, and 1 mol of selenium per mol of heterodimer. Few years later, another perchlorate reductase was purified and characterized. The molecular analysis from the bacterium Perclace revealed two subunits of 35 kDa and 75 kDa, less than reported for the two subunits of strain GR-1, which can be due to posttranslational modification of the protein in each bacterium.
Very little information has been published on the temperature and pH activity/stability profiles of perchlorate reductases. Perclace perchlorate reductase displayed a wide range of temperature activity (20 to 40 oC) but is most active at 25 to 35 oC. The enzyme was also relatively active in a wide range of pH (Okeke et al., 2003). Genetically, the perchlorate reductase operon (pcr) has recently been identified in the genome of two perchlorate-reducing bacteria, Dechloromonas agitata and Dechloromonas aromatica .
There are four genes in the transcriptional unit, pcr ABCD, encoding two structural subunits ( pcr A and pcr B), a cytochrome ( pcr C), and a molybdenum chaperone subunit ( pcr D). Amino acid sequence analysis of the products encoded by the pcr operon indicated similarities to subunits of microbial nitrate reductase, selenate reductase, dimethyl sulphide dehydrogenase, ethylbenzene dehydrogenase and chlorate reductase, all members of the type II DMSO (dimethyl sulfoxide) reductase family.
Transcriptional analysis indicated that pcr gene cluster is expressed in anaerobic perchlorate and chlorate grown cultures of D. agitata . However, aerobic cultures with perchlorate, chlorate, or nitrate as the electron acceptor displayed no induction of pcr
Perchlorate and chlorate degradation by two organisms isolated from wastewater 22
Chapter 1 – Literature study transcription, indicating the ability of oxygen to completely inhibit expression of the perchlorate reductase operon. Conversely, the D. agitata cld gene exhibits basal expression under aerobic conditions, thus further implicating separate regulation of chlorite dismutase and perchlorate reductase.
Chlorite dismutase
A central step in the reductive pathway of perchlorate and chlorate that is common to all (per)chlorate reducing bacteria is the dismutation of chlorite into chloride and molecular oxygen catalysed by chlorite dismutase (van Ginkel et al., 1996). The oxygen production during (per)chlorate reduction and subsequently chlorite dismutation is, besides photosynthesis and the detoxification of H 2O2 by catalases, the only known biological oxygen generating pathway.
Figure 1.12 – Model of the pathway involved in the reduction of chlorite by perchlorate reducing bacteria (Coates and Achenbach, 2004)
A logical consequence of this oxygen production is, that (per)chlorate-reducing bacteria are not strictly anaerobic bacteria. The chlorite dismutase from GR-1 has a molecular mass of 140 kDa and consists of four 32 kDa subunits, each one containing
Perchlorate and chlorate degradation by two organisms isolated from wastewater 23
Chapter 1 – Literature study
0.9 molecule of protoheme IX and 0.7 molecule of iron. In this strain the enzyme displays maxima for activity at pH 6.0 and 30 oC (van Ginkel et al., 1996). Chlorite dismutase from I. dechloratnas was also purified and the results revealed a tetrameric enzyme of 115 kDa (Stenklo et al., 2001). The chlorite dismutase gene is present in all
(per)chlorate reducers and, as such, detection of this gene is unable to distinguish between perchlorate reducing bacteria and those that can only reduce chlorate. The chlorite dismutase gene ( cld ) was isolated and characterized from D. agitata and from
Ideonella dechloratans . In the case of D. agitata , the chlorite dismutase gene is basally expressed under aerobic conditions. In contrast, chlorite dismutase expression is constitutive in the chlorate reducing microorganisms Pseudomonas strain PDA and
Pseudomonas strain PK.(Xu et al., 2004)
Chlorate reductase
Up to now, at least three enzymes that can reduce chlorate have been purified and characterized. A chlorate reductase C had been purified from the denitrifying strain
Proteus mirabilis (Oltmann et al., 1976), as well from Pseudomonas chloritidismutans
(Wolterink et al., 2003) and Pseudomonas sp. PDA (Steinberg et al., 2005). Comparison with the periplasmic perchlorate reductase of strain GR-1 showed that the cytoplasmic chlorate reductase of P. chloritidismutans reduced only chlorate and bromate.
Differences were also found in N-terminal sequences, molecular weight, and subunit composition. However, the metal analysis and electron paramagnetic resonance measurements showed the presence of iron and molybdenum, which are also found in other dissimilatory oxyanions reductase. The chlorate reductase from PDA had three
Perchlorate and chlorate degradation by two organisms isolated from wastewater 24
Chapter 1 – Literature study subunits (60, 48 and 27 kDa). Concerning genetic studies, a gene cluster of a chlorate reductase from I. dechloratans was also analysed (Thorell et al., 2003)
Evidence for separate enzymes is provided indirectly by the fact that not all CRB
- are capable of respiration with ClO 4 , although this question will require further research
- to resolve. Improved understanding of the biological ClO 4 reduction kinetics and its biochemical mechanisms will lead to better biological remediation processes. Since
- there are hardly any ClO 4 degradation kinetic data available, the present work was
- focused on isolation of possible new ClO 4 reducing bacteria as well as its kinetic characterization.
1.4.6. Factors that interfere with perchlorate enzyme induction
Many environmental factors have been shown to affect microbial (per)chlorate reduction, including trace elements, pH, salt concentration, and presence of other electron acceptors. Several lines of evidence suggest that the optimal pH for perchlorate reduction occurs around neutral pH. The Dechloromonas and Azospira species generally grow optimally at pH values near neutrality in freshwater environments.
Concerning salinity, to date no microorganism isolated has been demonstrated to reduce perchlorate in salinities greater than 2% (Logan et al, 2001a). This presents a problem for the biological treatment of waste brine concentrated with perchlorate collected by ion-exchange processes, suggesting that the metabolism is limited by the chloride content. An enrichment culture from the Great Salt Lake was able to carry out perchlorate reduction in salt brines as concentrated as 11% NaCl (Logan et al., 2001) but perchlorate removal efficiency was not reported while another enrichment culture
Perchlorate and chlorate degradation by two organisms isolated from wastewater 25
Chapter 1 – Literature study developed from marine sediments was reported to reduce 70 to 90 mg.L -1 perchlorate at
6% NaCl within 24 hours (Cang et al., 2004).
Regarding the presence of other final electron acceptors, oxygen and nitrate can be inhibitors of perchlorate reduction (Chaudhuri et al., 2002). Furthermore, molecular studies focused on the identification of the gene encoding the perchlorate reductase demonstrated that its expression was down regulated by the presence of atmospheric oxygen (Bender et al., 2005).
1.5. OUTLINE OF THE THESIS
The present research work was focus on the isolation, purification and characterization of possible new perchlorate reducing bacteria. Since there is hardly any
- - ClO 4 and ClO 3 degradation kinetic data available, it was studied the kinetic characterization of the isolates obtained. Furthermore, the improved understanding of
- the biological ClO 4 reduction will lead to better biological remediation processes.
Perchlorate and chlorate degradation by two organisms isolated from wastewater 26
Chapter 2 – Materials and methods
Chapter 2. MATERIALS AND METHODS
2.1 SOURCE OF ORGANISMS
A sludge sample was collected at the anaerobic digestor from Beirolas wastewater treatment plant (Portugal). The primary inoculum used to start the enrichment was obtained by the dilution of the sludge sample (1:10 in 0.6% NaCl).
2.2 MEDIA
The media used in all tests performed are summarized in Table 2.1
Table 2.1 – Media and reagents used for enrichment and isolation (g/L).
Basal Medium Medium Reagent SL-10 Medium KL SLA
K2HPO 4 1.55 NaH 2PO 4.H 2O 0.85 NH 4Cl 0.25 MgSO 4.7H 2O 0.1 HCl (37%) 10 ml Na 2SeO 3 0.0017 Na 2SeO 3.5H 2O 0.15 0.1
FeCl 2.4H 2O 1.5 18 FeSO 4.7H 2O 4 Na 2MoO 4.2H 2O 0.036 0.4 0.3 NiCl 2.6H 2O 0.024 0.1 0.1 EDTA 3
H3BO 3 0.6 5 ZnCl 2 0.07 1 MnCl 2.4H 2O 0.1 0.7 CoCl 2.6H 2O 0.19 2.5 CuCl 2.2H 2O 0.002 0.1
Perchlorate and chlorate degradation by two organisms isolated from wastewater 27
Chapter 2 – Materials and methods
All media were prepared using ultrapure water (Milli Q system) and research grade chemicals in the amounts indicated in grams per liter. For the enrichment, the basal medium was amended with 1 mL/L of mineral solution SL-10. For growth kinetics of the isolate (per1), 1 mL/L of medium KL was added to the basal medium and for the isolate (per2), 1 mL/L of medium SLA was added to the basal medium. Sodium acetate was used as the sole electron donor in 1:2 molar ratio to sodium perchlorate and/or sodium chlorate, final electrons acceptor. Solid agar plates were prepared by adding 15 g/L agar on the medium previous described.
2.3. BACTERIAL ISOLATION PROCEDURES AND CULTURING
CONDITIONS
All the enrichment was performed under anaerobic conditions with basal media
+ SL-10. The media was made O 2-free by flushing continuous Argon and were prepared in 50mL bottles capped with butyl rubbers stoppers, crimped with aluminium capsules and sterilized by autoclaving. Incubation was carried out 37 oC under constant shaking
- (100mot/min). A concentration of 5mM ClO 4 was selected to start the enrichment.
During a period of two months, continuous transfers (10% by volume) were made in sterilize conditions in a laminar flow. Cultures became turbid in 7 to 14 days. For
- further enrichment, the ClO 4 concentration was increased to 10 mM (Figure 2.1).
10% (volume)
5 mM 10 mM
Figure 2.1 – Schematic representation of the reactor used for batch tests.
Perchlorate and chlorate degradation by two organisms isolated from wastewater 28
Chapter 2 – Materials and methods
Subsequently, two different selection methods were applied to reach pure cultures. In the first one, a sample was serially diluted to 10 -3 and spread onto agar plates. The anaerobic growth in agar plates was performed with kit Microbiologie
Anaerocult® mini. Select colonies were picked and then re-grown in fresh liquid Basal medium + SL-10. The second method applied consisted in the continuous transfers of the enriched liquid culture at exponential phase to fresh Basal medium + SL-10 during a period of 20 days.
2.4. MORPHOLOGY
The enrichments were followed by optical microscopy (phase contrast) examination and the purity of the isolates obtained was confirmed latter by molecular methods.
2.5. 16S RIBOSOMAL DNA EXTRACTION AND SEQUENCING
2.5.1. Extraction and confirmation
DNA extraction of the isolates was performed using FastDNA ® SPIN Kit (for soil), according to the manufacturer instructions (Bio101 systems, Q-biogen, USA).
Some changes were performed in order to adjust the kit to our sample. To the Lysing matrix E Tube was added 500 µl of centrifuged cell pellet, 650 µl sodium phosphate buffer and 80 µl MT buffer. The tube was processed in FastPrep® Instrument at speed
Perchlorate and chlorate degradation by two organisms isolated from wastewater 29
Chapter 2 – Materials and methods
4.5. The supernatant was transfer to 2 new tubes (600 µl to each one) and 500 µl of
Binding Matrix was also added to each tube. The supernatant (600 µl) was discarded and the remaining amount was resuspended in the Binding Matrix. The extraction was confirmed by gel electrophoresis. The agarose solution was prepared in TAE (tris- acetate EDTA) with a final concentration of 1%. The solution was heated during 50 seconds in the microwave and was poured in a gel tray to allow cool at room temperature. To stain the gel, ethidium bromide was incorporated before gel polymerization. After polymerization, DNA samples and the mass ladder (1 kb) were loaded into the sample well. A loading dye (bromophenol blue) was used together with samples and the mass ladder. The gel was run at 100V for a period of 40min. The gel was then visualized directly upon UV light.
2.5.2. PCR amplification and purification
The 16S ribosomal DNAs were amplified by conventional PCR. The amplification program included initial denaturation at 94 oC for 5 minutes followed by three steps repeated 30 times. Step 1: 94 oC for 30 seconds; step 2: 48 oC for 30 seconds; and step 3: 72 oC for 2 minutes. The final elongation was at 72 oC for 5 minutes. The primers 27f and 1492r, and Taq polymerase (Invritogen) were used in this amplification. The PCR products were purified by gel electrophoresis and then cleaned with QIAquick® PCR purification kit (250). The gel electrophoresis was prepared in the same conditions as mentioned before for DNA extraction confirmation, with the exception of the run time and voltage, 1h and 80V, respectively. The purified products were then sequenced by BaseClear, DNA sequencing services, The Netherlands.
Perchlorate and chlorate degradation by two organisms isolated from wastewater 30
Chapter 2 – Materials and methods
2.6. PHYLOGENETIC ANALYSIS
For establishing the identity of the isolates by 16S rDNA nucleotide-nucleotide sequence homology, the BLAST (Basic Local Alignment Search Tool) network service, via the nucleotide collection (nr/nt) database at the National Center for Biotechnological
Information (NCBI) was used. (http://www.ncbi.nih.gov , March 2007).
2.7. BATCH GROWTH KINETICS
After confirmation of purity the isolate Dechlorospirillum sp. DB (per1) show to grow better on basal medium + medium KL and Dechlorosoma sp. PCC (per2) on basal medium + medium SLA. These were the media used to grow the isolates in batch tests.
The batch tests were performed in a reactor filled with 0.5 L (Figure 2.2) of the appropriate medium with electron acceptor and donor.
Figure 2.2 – Schematic representation of the reactor used for batch tests.
Perchlorate and chlorate degradation by two organisms isolated from wastewater 31
Chapter 2 – Materials and methods
The medium was made O 2-free by flushing continuous Argon (Ar) during more than 12h. The growth kinetics was conduct at controlled temperature (37 oC) and the redox potential was measured with a redox electrode in-situ . The pH measurements were made ex-situ and samples (5 mL) were taken in sterilized conditions, at regular time intervals for further analysis. To keep a positive pressure inside the reactor, Ar was supplied every time that it was taken a sample.
2.8. ANALYTICAL TECHNIQUES
Culture growth was monitored by optical density at 600nm (OD 600nm ) with a spectrophotometer and converted to dry weight (DW) using a calibration curve. The
DW determination was made using the method described elsewhere (Olsson and
Nielsen, 1997). The anions concentration was analyzed by HPLC. The concentration of perchlorate was determined by ion chromatography equipped with an Ion Pac AS16 column and a AG16 guard column (4mm, Dionex), a self-regenerating suppressor (SRS
Ultra II), and an autosampler. The eluting perchlorate was detected by a conductivity detector (Dionex) and the suppressor controller was set at 100 mA for the analysis. The samples were analyzed with a 50mM NaOH mobile phase at a flow rate of 1 ml min -1.
The injection loop volume was 30 µl. The chlorate, chlorite, chloride and acetate were determined with the same ion chromatography system described before. An Ion Pac
AS9 column and a AG9 guard column were used. The eluent used was 9 mM Na 2CO 3 at a flow rate of 1ml min -1. The injection loop volume was 30 µl and the suppressor controller was set at 50 mA for the analysis.
Perchlorate and chlorate degradation by two organisms isolated from wastewater 32
Chapter 2 – Materials and methods
2.9. CALCULATIONS
2.9.1. Specific growth rate
The specific growth rate was determined based on the cell dry weight (DW) as
∆ ln( DW ) function of time: µ = ∆t
2.9.2. Specific uptake rate
The following formula was used to determine the specific uptake rate for acetate,
∆S perchlorate and chlorate: - q = S ∆t
2.9.3. Electron acceptor yield over acetate
The following formula was used to determine the chloride formation yield:
∆e − acceptor Ye−acceptor = − − CH 3COO ∆ CH 3COO
2.9.4. Chloride yield over electron acceptor
The following formula was used to determine the chloride formation yield:
∆e − acceptor Ye−acceptor = Cl − ∆ Cl −
2.9.5. Biomass yield
The following formula was used to determine the biomass yield for acetate,
∆X perchlorate and chlorate: YX = S ∆S
Perchlorate and chlorate degradation by two organisms isolated from wastewater 33
Chapter 3 – Results and discussion
Chapter 3. RESULTS AND DISCUSSION
3.1. RESULTS
3.1.1. Morphological and genetic characterization of the isolates
By optical microscopy examination (Phase contrast) it was observed a spirillum- shaped enriched culture (per1) obtained from the first selection method. The individual cells were highly motile and occasionally growing as clusters. The size of the cells was
8.49×2.21 (±1.36×0.57) µm. Through the second isolation method applied it was achieved a rod-shaped enriched culture (per2). The cells were in its majority non-motile and clusters were not observed. The size of the cells was 4.97×1.43 (± 0.85×0.28) µm.
A B
Figure 3.1 – Optical microscopy observation of the enriched cultures; A: (per1) and B: (per2)
(100x).
The purity of the two isolates was confirmed by genetic characterization of 16S rDNA. The BLAST search showed that the microorganism (per1) shared a 99%
Perchlorate and chlorate degradation by two organisms isolated from wastewater 34
Chapter 3 – Results and discussion sequence similarities to the 16S rDNA of Dechlorospirillum sp. DB. Concerning the genetic characterization by 16S rDNA of the microorganism (per2), the sequencing showed that the microorganism shared a 99% sequence similarities to the 16S rDNA of
Dechlorosoma sp. PCC.
Both isolates have already its sequence deposited. Dechlorospirillum sp. DB has its complete sequence of 16S rDNA with the accession number AY530551 (Bender et al., 2004). Phylogenetically belongs to the α-subclasse of Proteobacteria and was first isolated during a cld (chlorite dismutase) primer development (J. Coates, unpublished data). The chlorite dismutase gene was also sequenced (Bender et al., 2004), but no kinetic parameters were determined so far.
Concerning Dechlorosoma sp. PCC, it have the 16S rDNA partially sequenced with the accession number AY126453 (Nerenberg et al., 2002), but no other publication related with this bacteria is available at the moment. Phylogenetically belongs to the β- subclasse of Proteobacteria .
.
3.1.2. Growth kinetics
- Dechlorospirillum sp. DB (10mM ClO 4 )
Dechlorospirillum sp. DB was grown on basal medium + KL solution amended
- - with 20mM of CH 3COO and 10 mM of ClO 4 (Figure 3.2).
Perchlorate and chlorate degradation by two organisms isolated from wastewater 35
Chapter 3 – Results and discussion
25 ClO4- 1.8 CH3COO- ClO3- 1.6 DW 20 1.4
1.2 (mM) -
4 15 1.0 , ClO - 0.8 10 (mM), (g/L) DW -
COO 0.6 3 3
CH 0.4 ClO 5 0.2
0 0.0 0 1 2 3 4 5 6 7 8 9101112
Time (h)
Figure 3.2 – Acetate and perchlorate uptake and transient accumulation of chlorate as function - of time during the reduction of 10mM of ClO 4 by Dechlorospirillum sp. DB. Note the different - concentration scale for ClO 3 . Dry Weight (DW) as a function of time is also represented.
Over perchlorate reduction by Dechlorospirillum sp. DB it was observed accumulation and subsequent degradation of the intermediate chlorate. Around 3.4% on a molar basis of perchlorate concentration was accumulated as chlorate.
The chloride (Cl -) formation was also detected during perchlorate reduction, indicating a completely conversion of perchlorate into innocuous chloride (data not shown). The specific acetate uptake rate was two times higher compared with perchlorate uptake rate (Table 3.3). This information showed that the molar ratio acetate to perchlorate was 2:1 as it was expected. Concerning pH, it was observed over the growth a slightly increased from 6.99 to 7.33, which was not significant (data not shown). The maximum cell dry weight obtained was 1.53 g/L corresponding to an
OD 600nm of 1.08. During the kinetic, some samples were examined by optical microscopy and no changes were observed in the shape of the microorganisms, neither other bacteria were present, which confirmed the purity of the isolate.
Perchlorate and chlorate degradation by two organisms isolated from wastewater 36
Chapter 3 – Results and discussion
- Dechlorospirillum sp. DB (10mM ClO 3 )
The study of chlorate reduction by Dechlorospirillum sp. DB is represented in
- Figure 3.3. In this kinetic the basal medium + KL was amended with 10mM of ClO 3
- and 20mM of CH 3COO .
25 0.6 ClO3- CH3COO- Cl- 0.5 20 DW (g/l) (mM)
- 0.4 15 , Cl - 3 0.3 , ClO - 10 (g/l) DW 0.2 COO 3
CH 5 0.1
0 0 0 1 2 3 4 56 7 8 910 Time (h)
Figure 3.3 – Acetate and chlorate uptake as function of time during the reduction of 10mM of - ClO 3 by Dechlorospirillum sp. DB. Dry weight (DW) and chloride formation as a function of time are also represented.
Dechlorospirillum sp. DB showed the ability to reduce chlorate in the same concentration and conditions as used for perchlorate reduction. A completely reduction of chlorate into chloride was observed. The maximum value achieved for cell dry weight was 0.49 g/L, demonstrating a lower biomass yield compared with perchlorate reduction. The pH ranged from 6.96 to 7.29.
Perchlorate and chlorate degradation by two organisms isolated from wastewater 37
Chapter 3 – Results and discussion
- - Dechlorospirillum sp. DB (5mM ClO 4 + 5mM ClO 3 )
In order to study the growth kinetic of Dechlorospirillum sp. DB with
- - perchlorate and chlorate, it was performed a kinetic with 5mM ClO 4 and 5mM ClO 3 simultaneously in the media. Again, basal medium + KL was used as growth media and
- CH 3COO was used in a concentration of 20mM (Figure 3.4).
25 1.2 ClO3- CH3COO- Cl- 1.0 20 ClO4- (mM)
- DW , Cl
- 0.8 3 15 , ClO
- 0.6 4
10 (g/L) DW
, ClO 0.4 -
COO 5 3 0.2 CH
0 0.0 0 1 2 3 4 56 7 8 910
Time (h)
Figure 3.4 – Acetate, perchlorate and chlorate uptake as function of time during the reduction of - - 5mM of ClO 4 + 5mM of ClO 3 by Dechlorospirillum sp. DB. Dry weight (DW) and chloride formation as a function of time are also represented.
Concerning acetate, it can be observed two different uptake rates, near related with chlorate and perchlorate reduction respectively and similar to each substrate individually (Table 3.3). The chloride produced was identical to the sum of perchlorate and chlorate amounts, showing once more a completely conversion of both electron donors in chloride. The pH showed again a small variation starting with 7.02 and ended with 7.34. The maximum cell dry weight produced was 1.11 g/L.
Perchlorate and chlorate degradation by two organisms isolated from wastewater 38
Chapter 3 – Results and discussion
In this kinetic it should be stressed the observed preference for chlorate when both chlorate and perchlorate were present in the media. From Figure 3.5 it can be observed in more detail the reduction of the electrons acceptor, perchlorate and chlorate.
Perchlorate was not reduced unless chlorate was almost reduced. This observation probably indicates chlorate inhibition over perchlorate reduction when both were present at the same concentration of 5mM.
6 ClO3- ClO4- 5
4
3 mM
2
1
0 0 1 2 3 4 5 6 7 8 910 Time (h)
Figure 3.5 – Perchlorate and chlorate uptake as function of time during the reduction of 5mM of - - ClO 4 + 5mM of ClO 3 by Dechlorospirillum sp. DB.
The same batch tests were performed with Dechlorosoma sp. PCC. It was
- - performed a kinetic study with 10mM of ClO 4 , one with 10mM of ClO 3 and a last one
- - with 5mM ClO 4 + 5mM ClO 3 . In this case, the medium used in all tests was basal medium + SLA as described before.
Perchlorate and chlorate degradation by two organisms isolated from wastewater 39
Chapter 3 – Results and discussion
- Dechlorosoma sp. PCC (10mM ClO 4 )
- In this kinetic the basal medium + SLA was amended with 10mM of ClO 4 and
- 20mM of CH 3COO .
25 1.0 ClO4 0.9 CH3COO 20 DW 0.8 0.7 (mM) -
4 15 0.6
0.5 , ClO -
10 0.4 (g/L) DW COO 3 0.3 CH 5 0.2 0.1
0 0.0 0 2 4 6 8 10 12 14 16 Time (h)
Figure 3.6 – Acetate and perchlorate uptake as function of time during the reduction of 10mM of - ClO 4 by Dechlorosoma sp. PCC. Dry weight (DW) as function of time is also represented.
The results showed that concerning perchlorate reduction, Dechlorosoma sp.
PCC had similar behaviour compared with Dechlorospirillum sp. DB. The chloride
(Cl -) formation was detected during perchlorate reduction, indicating a completely conversion of perchlorate into innocuous chloride (data not shown). The biomass production was 0.89 g/L in cell dry weight and conversely to Dechlorospirillum sp. DB,
- no chlorate accumulation was observed for the same detection limit of 0.06mM ClO 3 .
Perchlorate and chlorate degradation by two organisms isolated from wastewater 40
Chapter 3 – Results and discussion
- Dechlorosoma sp. PCC (10mM ClO 3 )
The study of chlorate reduction by Dechlorosoma sp. PCC is shown in Figure
- 3.7. For this kinetic the basal medium + SLA was amended with 10mM of ClO 3 and
- 20mM of CH 3COO .
25 1,0 ClO3- CH3COO- Cl- 20 DW (g/l) 0,8 (mM) - 15 0,6 , Cl - 3 , ClO - 10 0,4 (g/L) DW COO 3
CH 5 0,2
0 0,0 0 1 2 3 4 5 6 7 8 9 101112131415 Time (h)
Figure 3.7 – Acetate and chlorate uptake as function of time during the reduction of 10mM of - ClO 3 by Dechlorosoma sp. PCC. Dry weight (DW) and chloride formation as function of time is also represented.
Dechlorosoma sp. PCC can also reduce chlorate as a single electron donor.
Chlorate was completely reduced to chloride, which proves the total reduction of chlorate into chloride. Biomass produced was less than the observed in perchlorate reduction and the maximum value of cell dry weight was 0.75g/L. The pH ranged from
6.96 to 7.29.
Perchlorate and chlorate degradation by two organisms isolated from wastewater 41
Chapter 3 – Results and discussion
- - Dechlorosoma sp. PCC (5mM ClO 4 + 5mM ClO 3 )
- - It was also performed a kinetic study with 5mM ClO 4 and 5mM ClO 3 simultaneously in the media, in order to study the growth kinetic of Dechlorosoma sp.
- PCC. The basal medium + SLA was used as growth media and CH 3COO was used in a concentration of 20mM.
ClO4- 20 CH3COO- 1,2 ClO3- DW (g/l) 1,0 15 (mM)
3- 0,8 , ClO - 4 10 0,6 DW (g/L) DW , ClO - 0,4 COO
3 5
CH 0,2
0 0,0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Time (h)
Figure 3.8 – Acetate, perchlorate and chlorate uptake as function of time during the reduction of - - 5mM of ClO 4 + 5mM of ClO 3 by Dechlorosoma sp. PCC. Dry weight (DW) as a function of time is also represented.
Again in this kinetic study, it should be stressed the observed preference for chlorate when chlorate and perchlorate are present in the same media. It can also be observed two different acetate uptake rates, near related with chlorate and perchlorate reduction respectively, as it was observed for Dechlorospirillum sp. DB. Once more, a
Perchlorate and chlorate degradation by two organisms isolated from wastewater 42
Chapter 3 – Results and discussion completely conversion of both electron donors into chloride was observed (data not shown). The maximum cell dry weight produced was 1.17g/L.
3.2. DISCUSSION
Batch cultures of Dechlorospirillum sp. DB and Dechlorosoma sp. PCC were performed and kinetic parameters such as specific growth rate ( µmax ), specific acetate uptake rates (q CH3COO-), specific perchlorate reduction rates (q ClO4-), specific chlorate
- reduction rates (q ClO3-) and biomass yield (g [DW] / gCH 3COO ) were determined. The values determined were summarized in Table 3.3.
Comparing Dechlorospirillum sp. DB with Dechlorosoma sp. PCC the specific growth rate determined for perchlorate reduction showed no significant difference.
However, comparing both isolates during chlorate reduction, Dechlorospirillum sp. DB showed a specific growth rate higher than Dechlorosoma sp. PCC. Comparing perchlorate and also chlorate reduction for each isolate individually, different values were found. Dechlorospirillum sp. DB showed higher specific growth rate for chlorate, while Dechlorosoma sp. PCC showed for perchlorate reduction. The difference found in specific growth rate for chlorate and perchlorate reduction within the same bacteria, possibly will indicate different mechanisms involved in each reduction. However, this result was not yet conclusive to predict if the same enzyme is responsible for both reduction or if different enzymes are present. Regarding the kinetics when both electron acceptors were present, it was observed in Dechlorospirillum sp. DB an increased of the specific growth rate compared with each electron acceptor separately and a mean value
Perchlorate and chlorate degradation by two organisms isolated from wastewater 43
Chapter 3 – Results and discussion for Dechlorosoma sp. PCC. The specific growth rates values find in this work were also compared with those reported by others (Table 3.1).
Table 3.1 – Specific growth rates of described perchlorate and chlorate reducing bacteria. Electron Isolate µµµ (h -1) Reference Acceptor Chlorate 0.21 Dechlorospirillum sp. DB This study Perchlorate 0.17 Chlorate 0.13 Dechlorosoma sp. PCC This study Perchlorate 0.17 Chlorate 0.26 Dechlorosoma sp. KJ Logan et al., (2001) Perchlorate 0.14 Chlorate 0.21 Dechlorosoma sp. PDX Logan et al., (2001) Perchlorate 0.21 Pseudomonas sp. PDA Chlorate 0.18 Logan et al., (2001) Pseudomonas sp. PDB Chlorate 0.26 Logan et al., (2001) Azospira oryzae strain GR1 Chlorate 0.10 Rikkenl et al., (1996) AB1 Chlorate 0.012 Olsen S., (1997) Perclace Perchlorate 0.07 Herman and Frankenberger, (1998) Dechloromonas agitata strain CKB Chlorate 0.28 Bruce at al., (1999)
The specific growth rates determined in this study were within the values found in the literature, ranging from 0.07 to 0.26 h -1.
Different uptake rates were found for both electron donor and acceptors. The acetate uptake rate for all kinetic studies was always twice than the value for uptake rate of perchlorate or chlorate. This fact means that the ratio acetate for electron acceptor was approximately 2:1 what is in accordance with the literature.
Regarding Dechlorospirillum sp. DB the highest uptake rate was observed for chlorate as the sole electron acceptor. Conversely, the uptake rates determined for
Dechlorosoma sp. PCC were higher for perchlorate as a sole electron acceptor. This fact
Perchlorate and chlorate degradation by two organisms isolated from wastewater 44
Chapter 3 – Results and discussion was in agreement with the values found for the specific growth rate, as mentioned before.
It was also observed for Dechlorospirillum sp. DB that comparing perchlorate and chlorate uptake rates separately with the ones when both were present, the value for perchlorate reduction was very similar, while for chlorate reduction was half value. In this case it can be suggested that chlorate uptake rate could be influenced by chlorate concentration. On the other hand, perchlorate could have an inhibitor effect over chlorate reduction for the concentration used, although chlorate was preferentially reduced. For Dechlorosoma sp. PCC, the uptake rates found when perchlorate and chlorate were present together at 5mM each one, were both different compared with the kinetic study with the electrons acceptor present individually. It seems that uptake rate decreased with perchlorate concentration decrease and that the uptake rate increased with chlorate concentration decrease. In this case it can be suggested that both perchlorate and chlorate uptake rate could be influenced by concentration.
In all batch test performed it was observed a completely conversion of the electron acceptors used in each kinetic into innocuous chloride, further confirming that chlorite dismutation occurred in the isolates of this study.
- The biomass yields for acetate (g [DW] / gCH 3COO ) calculated in this work were generally higher than those reported by others in the literature (Table 3.2). For
Dechlorosoma sp. PCC the biomass yield for acetate was similar for perchlorate and chlorate reduction individually. The same was verified in two other perchlorate reducing bacteria found in the literature, in which no significant changes were found related with
Perchlorate and chlorate degradation by two organisms isolated from wastewater 45
Chapter 3 – Results and discussion perchlorate and chlorate reduction. Concerning Dechlorospirillum sp. DB the biomass yield determined for chlorate was half value of perchlorate.
Table 3.2 – Biomass yields in the presence of different electron acceptors determined in this study and reported by others.
Electron Cell yield (g [DW] / Isolate Reference - Acceptor g CH 3COO ) Chlorate 0.47± 0.01 Dechlorospirillum sp. DB This study Perchlorate 0.94 ± 0.03 Chlorate 0.82± 0.02 Dechlorosoma sp. PCC This study Perchlorate 0.70 ± 0.01 Oxygen 0.46 ± 0.07 Dechlorosoma sp. KJ Chlorate 0.44 ± 0.05 Logan et al., (2001b) Perchlorate 0.50 ± 0.08 Oxygen 0.27 ± 0.01 Azospira oryzae strain GR1 Chlorate 0.28 ± 0.01 Rikken et al., (1996) Perchlorate 0.24 ± 0.01 Oxygen 0.13 ± 0.04 AB1 Olsen S., (1997) Chlorate 0.10 ± 0.04
Among the biomass yield found in the literature it was observed that
Dechlorospirillum sp. DB showed highest values for perchlorate reduction and that
Dechlorosoma sp. PCC showed highest values for chlorate reduction compared with the other isolates.
Chlorate accumulation can be explained based on the existence of two enzymes responsible for the conversion of perchlorate into chlorite, in which the conversion of chlorate into chlorite was the rate-limiting step. If a unique enzyme was present, then chlorate accumulation could be explained based on the idea that chlorate affinity decreased when perchlorate is present at 10mM.
Perchlorate and chlorate degradation by two organisms isolated from wastewater 46
Chapter 3 – Results and discussion
Perchlorate and chlorate degradation by two organisms isolated from wastewater 48
Chapter 4 – Conclusions and further research
Chapter 4. CONCLUSIONS AND FURTHER RESEARCH
Two different bacteria were isolated using two different selection methods. The genetic characterization of 16S rDNA showed that both isolates have already its sequence deposited, but no other description was made. The purity of the isolates was easily confirmed by genetic characterization with 16S rDNA sequence homology.
Regarding more characterization of these two isolates, for future work it should be done a 16S rDNA sequence homology for phylogenetic tree construction. This will further allow relating these isolates with other perchlorate reducing bacteria. Concerning description of these bacteria it should be done the G + C content, cytochrome oxidase presence, catalase activity, Gram staining, pH, salinity and temperature range for optimal growth, fatty acid profile, electron donor/acceptor use and also microbial size should be performed.
It should be stressed that both bacteria were able to couple complete reduction of the electron acceptors with growth. This was confirmed by the increase of biomass in all kinetics performed.
The bacteria isolated showed different perchlorate reduction system. The main evidence was the transient accumulation of chlorate by Dechlorospirillum sp. DB during perchlorate reduction, which was not observed in Dechlorosoma sp. PCC.
However, the results were not conclusive to predict if one enzyme was responsible for both reduction (perchlorate and chlorate) or if different enzymes were present. Chlorate accumulation during perchlorate reduction was hardly studied. For further investigation, the enzymes involved in the perchlorate reduction pathway of these two bacteria, should be purified and studied concerning its biochemical characterization. Furthermore, for future enzymatic studies it will be necessary a large scale biomass production of these
Perchlorate and chlorate degradation by two organisms isolated from wastewater 49
Chapter 4 – Conclusions and further research bacteria, based in kinetic parameters determined in this research work such as biomass yield. Dechlorospirillum sp. DB showed the highest biomass yield even compared with other found in the literature.
Kinetic studies starting with different perchlorate and chlorate concentration should be done to observe the effect of the initial concentration over the reduction of each electron acceptor.
Regarding the batch test done with chlorate and perchlorate present in the same medium, it was observed the preference for chlorate over perchlorate in both bacteria.
This observation could also indicate that chlorate inhibit perchlorate at 5mM, although this finding was not in agreement with chlorate accumulation observed during perchlorate reduction in the first test. For a better understanding of the effect of chlorate during perchlorate reduction it should be test chlorate spike during perchlorate reduction.
Perchlorate and chlorate degradation by two organisms isolated from wastewater 50
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