Perchlorate and chlorate degradation by two organisms ... · Perchlorate and chlorate degradation...

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.

Perchlorate and chlorate degradation by two organisms isolated from wastewater ii

ABSTRACT

The biological removal of perchlorate (ClO4-) and chlorate (ClO3

-) 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 iii

TABLE OF CONTENT

CHAPTER 1. LITERATURE STUDY………………………………………… 1

1.1. Introduction………………………………………………………………. 1

1.2. Perchlorate as a pollutant.....……………………………………………… 2

1.2.1. Properties...........…………………………………………………...

1.2.2. Perchlorate environmental occurence....……………………………

1.2.3. Health effects..……………………………………………………...

1.2.4. Legislation...........................................................................................

.

2

4

7

7

1.3. Perchlorate treatment technologies……………...…………………………. 8

1.3.1. Physical processes..……………………..…...……………………..

1.3.2. Chemical processes………..……………………………………....

1.3.3. Biological processes................…..…………………………….......

9

13

14

1.4. The microbiology and biochemistry of perchlorate reduction………….. 16

1.4.1. Perchlorate reducing bacteria..………………………………………

1.4.2. Electron donors used by PRB for growth…………...………........

1.4.3. Nutritional requirements for PRB……..…………………………...

1.4.4. Biological perchlorate reduction……………………………………

1.4.5. Enzymes responsible for (per)chlorate reduction…………………..

1.4.6. Factors that interfere with perchlorate enzyme induction…………

16

19

19

20

21

25

1.5. Outline of the thesis………………………………………………………... 26

CHAPTER 2. MATERIALS AND METHODS….………………………………

27

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 iv

2.4. Morphology………………………………………………………………… 29

2.5. 16S ribosomal DNA extraction and sequencing…………………………… 29

2.5.1. Extraction and confirmation 16S ribosomal DNA……………………

2.5.2. PCR amplification and purification…………………………………

29

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………………………………….…..................

2.9.2. Specific uptake rate…………..……………………………………….

2.9.3. Substrate uptake yield………..……………………………………..

2.9.4. Chloride formation yield……………………………………………

2.9.5. Biomass yield……………………………………………………….

33

33

33

33

33

CHAPTER 3. RESULTS AND DISCUSSION …………………………..……… 34

3.1. Results………………………………………..……………………….……. 34

3.1.1. Morphological and genetic characterization of the isolates…..............

3.1.2. Growth kinetics………………..……………………………………

34

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 v

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 vi

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 ClO4- by

Dechlorospirillum sp. DB. Note the different concentration scale for

ClO3-. 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 ClO3- 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 ClO4- + 5mM of ClO3

- 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


- by Dechlorospirillum sp.

DB.………...……………………………………………………………

39

Figure 3.6: Acetate and perchlorate uptake as function of time during the reduction

of 10mM of ClO4- 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 ClO3- 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


- by Dechlorosoma sp.

PCC. Dry weight (DW) as a function of time is also represented.……..

42

Chapter 1 – Literature study

Perchlorate and chlorate degradation by two organisms isolated from wastewater

1

Chapter 1. LITERATURE STUDY

1.1. INTRODUCTION

Perchlorate (ClO4-) and chlorate (ClO3

-) 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, ClO4- and chlorate are also a

health concern, as they can cause serious diseases such cancer. In the medium-term the

removal of ClO4- 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 ClO4- 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 ClO4- so as to optimize conditions



2

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 ClO4-, 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, ClO4- 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).

Nevertheless, ClO4- is slightly weaker than dichromate (Cr2O7

2-) or

permanganate (MnO4-) and its redox reaction is extremely non-labile, i.e. reacts slowly

with most reducing agents. The reduction of ClO4- can only be observed in concentrated

strong acid. In 0.1 to 4.0 M acid solution, ClO4- is not reduced by common reagents

such as thiosulfate, sulfite, or iron(II). In fact, the redox behaviour of ClO4- 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

ClO4– + 8H

+ + 8e

- ↔ Cl

– + 4H2O, E° = 1.287 V (1)



3

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 Ea 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, ClO4- 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.



4

Table 1.1 – Physical and chemical properties of selected perchlorate compounds.

Property Ammonium

Perchlorate

Sodium

Perchlorate

Potassium

perchlorate

Perchloric

acid

Formula NH4ClO4 NaClO4 KClO4 HClO4

Formula

Weight 117.49 122.44 138.55 100.47

Colour /Form White,

orthorhombic

crystals

White,

orthorhombic

crystals; white

deliquescent

crystals

Colourless

crystals or white,

crystalline

powder;

colourless,

orthorhombic

crystals

Colourless, oily

liquid

Melting Point Decomposes/

explodes 480

oC 525

oC -112

oC

Density 1.95 g/cm3 2.52 g/cm

3 2.53 g/cm

3 1.768 g/cm

3

Solubility 200 g/L of water

at 25oC

209.6 g/100mL of

water at 25oC

15 g/L of water at

25oC

Miscible in cold

water

1.2.2. Perchlorate environmental occurrence

As a strong oxidizing agent, ClO4- is mostly used as ammonium perchlorate

(NH4ClO4) 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 ClO4- 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 ClO4- 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.



5

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 ClO4-

(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 ClO4-. Concerning beer samples

perchlorate concentrations ranged from 0.03 to 10.663 ppb ClO4- (average values of



6

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

ClO4-, 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.

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.

0 1 2 3 4 5 6

Africa

Asia

Australia

Europe

America

ClO4- (ppb)



7

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 ClO4- 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 ClO4-

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 (ClO2) 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 (ClO2-) 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 ClO4- contamination all over the world,

especially in Europe and Middle East (El Aribi et al., 2006). Given the seriousness of



8

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 ClO4- 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 ClO4- to affect human health effects when present at low levels in drinking water.

Consequently, as of April 2007, the EPA has not determined whether ClO4- 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 ClO4- as well. In the European

communities there is no guide level for perchlorate. Although no other country has

legislation regarding this matter, research on ClO4- 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 ClO4- 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,

ClO4- is accumulated in a brine solution or a concentrated stream, which have to be



9

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 ClO4- ion from water. As these techniques do not destroy the ClO4

-, they create

a subsequent need for disposal and treatment of both the ClO4- and any waste products

of the process. In addition, all of these techniques currently suffer from lack of

selectivity. Along with the ClO4-, 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 ClO4- 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 ClO4- 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).



10

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 ClO4-

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 ClO4-.

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 ClO4- 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



11

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 ClO4-, 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 ClO4-. 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 ClO4-. This technology

applies an electric current to remove ClO4-. Perchlorate-contaminated water is exposed

to an electric current as it passes through a semi-permeable membrane (Figure 1.6). This

separates ClO4- ions from contaminated groundwater and surface water. The technology



12

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 ClO4- 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



13

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

The high reduction potential of chlorate and perchlorate (ClO3-/Cl

- E

o = 1.03 V;

ClO4-/Cl

- E

o = 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 ClO4- 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 ClO4- from contaminated water.



14

Bioreactor

A bioreactor frequently serves as a technology for removing ClO4- 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 ClO4-

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



15

with their simultaneous biodegradation by a suitable microbial culture in a separate

compartment (Figure 1.8). This process was successfully tested for drinking water ClO4-

removal from 100ppb to 4ppb of ClO4- (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 ClO4- itself or combined with physical removal gather

operational conditions which makes this process the most promising technology for

ClO4- 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 ClO4-, but there is little ongoing research directed toward the physiology of

perchlorate reducing bacteria (PRBs).



16

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 ClO4- 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 ClO4-, 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 ClO4- 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).



17

Table 1.2 – Perchlorate respiring bacterial isolates.

Isolate Electron

acceptor Reference

Vibrio dechloraticans Cuznesove B-1168 ClO4-, ClO3

-, NO3

- Korenkov et al. (1976)

Wolinella succinogenes HAP-1 ClO4-, ClO3

-, NO3

- Wallace et al. (1996)

Dechloromonas agitata ClO4-, ClO3

-, O2 Achenbach et al. (2001)

GR-1 ClO4-, ClO3

-, NO3

-, O2, Mn (IV) Rikken et al. (1996)

Dechloromonas sp. HZ ClO4-, ClO3

-, NO3

-, O2 Zhang et al. (2002)

Dechlorosoma suillum ClO4-, ClO3

-, NO3

-, O2 Achenbach et al. (2001)

AB-1 ClO3-, NO3

- Bliven et al. (1996)

Dechlorospirillum JB116 ClO4-, ClO3

-, NO3

- Bardiya and Bae (2006)

Dechlorosoma sp. KJ ClO4-, ClO3

-, O2 Logan et al. (2001b)

Dechlorosoma sp. PDX ClO4-, ClO3

-, O2 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).



18

Figure 1.9 – Phylogenetic distribution of (per)chlorate and chlorate reducing microorganisms

based on total 16S rDNA (Coates and Achenbach, 2004)



19

1.4.2. Electron donors used by PRB for growth

Acetate (CH3COO-) has been most frequently used as a single substrate for

heterotrophic ClO4- 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 ClO4- degradation (Xu et al., 2003). Recent

molecular studies of the genetic systems associated with ClO4- 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 ClO4-. In support

of this, growth and ClO4- reduction were completely inhibited when an active



20

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.

The ClO4- 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).

ClO4- � ClO3

- � ClO2

- � O2 + Cl

-



21

Chlorate produced by the perchlorate reductase should compete with ClO4- for

the catalytic site of the (per)chorate-reductase enzyme, presumably slowing the ClO4-

reduction rate. While some amount of chlorate accumulation is possible, it only has

been reported for a mixed culture growing on nitrate and ClO4- (Nerenberg et al., 2002)

and for a pure culture growing on ClO4- (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)



22

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 40oC) 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, pcrABCD, encoding two structural

subunits (pcrA and pcrB), a cytochrome (pcrC), and a molybdenum chaperone

subunit (pcrD). 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



23

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 H2O2 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



24

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 30oC (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



25

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 ClO4-, although this question will require further research

to resolve. Improved understanding of the biological ClO4- reduction kinetics and its

biochemical mechanisms will lead to better biological remediation processes. Since

there are hardly any ClO4- degradation kinetic data available, the present work was

focused on isolation of possible new ClO4- 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



26

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

ClO4- and ClO3

- degradation kinetic data available, it was studied the kinetic

characterization of the isolates obtained. Furthermore, the improved understanding of

the biological ClO4- reduction will lead to better biological remediation processes.

Chapter 2 – Materials and methods


27

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).

Reagent Basal

Medium SL-10

Medium

KL

Medium

SLA K2HPO4 1.55

NaH2PO4.H2O 0.85

NH4Cl 0.25

MgSO4.7H2O 0.1

HCl (37%) 10 ml

Na2SeO3 0.0017

Na2SeO3.5H2O 0.15 0.1

FeCl2.4H2O 1.5 18

FeSO4.7H2O 4

Na2MoO4.2H2O 0.036 0.4 0.3

NiCl2.6H2O 0.024 0.1 0.1

EDTA 3

H3BO3 0.6 5

ZnCl2 0.07 1

MnCl2.4H2O 0.1 0.7

CoCl2.6H2O 0.19 2.5

CuCl2.2H2O 0.002 0.1



28

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 O2-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 37oC under constant shaking

(100mot/min). A concentration of 5mM ClO4- 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 ClO4- concentration was increased to 10 mM (Figure 2.1).

Figure 2.1 – Schematic representation of the reactor used for batch tests.

10% (volume)

5 mM 10 mM

10% (volume)

5 mM 10 mM



29

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



30

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 94oC for 5 minutes followed by

three steps repeated 30 times. Step 1: 94oC for 30 seconds; step 2: 48

oC for 30 seconds;

and step 3: 72oC 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.



31

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.



32

The medium was made O2-free by flushing continuous Argon (Ar) during more

than 12h. The growth kinetics was conduct at controlled temperature (37oC) 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 (OD600nm) 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 30µl. 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 Na2CO3 at

a flow rate of 1ml min-1

. The injection loop volume was 30µl and the suppressor

controller was set at 50 mA for the analysis.



33

2.9. CALCULATIONS

2.9.1. Specific growth rate

The specific growth rate was determined based on the cell dry weight (DW) as

function of time: t

DW

∆

∆=

)ln(µ

2.9.2. Specific uptake rate

The following formula was used to determine the specific uptake rate for acetate,

perchlorate and chlorate: -t

SqS

∆

∆=

2.9.3. Electron acceptor yield over acetate

The following formula was used to determine the chloride formation yield:

−−∆

−∆=

−COOCH

acceptor

COOCH

acceptore

eY

33

2.9.4. Chloride yield over electron acceptor

The following formula was used to determine the chloride formation yield:

−∆

−∆=

−−

Cl

acceptor

Clacceptore

eY

2.9.5. Biomass yield

The following formula was used to determine the biomass yield for acetate,

perchlorate and chlorate: S

XY

SX

∆

∆=

Chapter 3 – Results and discussion


34

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%



35

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 ClO4- )

Dechlorospirillum sp. DB was grown on basal medium + KL solution amended

with 20mM of CH3COO- and 10 mM of ClO4

- (Figure 3.2).



36

0

5

10

15

20

25

0 1 2 3 4 5 6 7 8 9 10 11 12

Time (h)

CH

3C

OO

- , C

lO4- (m

M)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

ClO

3- (

mM

), D

W (

g/L

)

ClO4-

CH3COO-

ClO3-

DW

Figure 3.2 – Acetate and perchlorate uptake and transient accumulation of chlorate as function

of time during the reduction of 10mM of ClO4- by Dechlorospirillum sp. DB. Note the different

concentration scale for ClO3-. 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

OD600nm 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.



37

Dechlorospirillum sp. DB (10mM ClO3- )

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 ClO3-

and 20mM of CH3COO-.

0

5

10

15

20

25

0 1 2 3 4 5 6 7 8 9 10Time (h)

CH

3C

OO

- , C

lO3

- , C

l- (m

M)

0

0.1

0.2

0.3

0.4

0.5

0.6

DW

(g

/l)

ClO3-

CH3COO-

Cl-

DW (g/l)

Figure 3.3 – Acetate and chlorate uptake as function of time during the reduction of 10mM of

ClO3- 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.



38

Dechlorospirillum sp. DB (5mM ClO4- + 5mM ClO3

- )

In order to study the growth kinetic of Dechlorospirillum sp. DB with

perchlorate and chlorate, it was performed a kinetic with 5mM ClO4- and 5mM ClO3

-

simultaneously in the media. Again, basal medium + KL was used as growth media and

CH3COO- was used in a concentration of 20mM (Figure 3.4).

0

5

10

15

20

25

0 1 2 3 4 5 6 7 8 9 10

Time (h)

CH

3C

OO

- , C

lO4

- , C

lO3- ,

Cl- (

mM

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

DW

(g

/L)

ClO3-CH3COO-Cl-ClO4-DW

Figure 3.4 – Acetate, perchlorate and chlorate uptake as function of time during the reduction of

5mM of ClO4- + 5mM of ClO3

- 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.



39

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.

0

1

2

3

4

5

6

0 1 2 3 4 5 6 7 8 9 10Time (h)

mM

ClO3-

ClO4-

Figure 3.5 – Perchlorate and chlorate uptake as function of time during the reduction of 5mM of

ClO4- + 5mM of ClO3

- by Dechlorospirillum sp. DB.

The same batch tests were performed with Dechlorosoma sp. PCC. It was

performed a kinetic study with 10mM of ClO4-, one with 10mM of ClO3

- and a last one

with 5mM ClO4- + 5mM ClO3

-. In this case, the medium used in all tests was basal

medium + SLA as described before.



40

Dechlorosoma sp. PCC (10mM ClO4- )

In this kinetic the basal medium + SLA was amended with 10mM of ClO4- and

20mM of CH3COO-.

0

5

10

15

20

25

0 2 4 6 8 10 12 14 16

Time (h)

CH

3C

OO

- , C

lO4

- (m

M)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

DW

(g

/L)

ClO4

CH3COO

DW

Figure 3.6 – Acetate and perchlorate uptake as function of time during the reduction of 10mM of

ClO4- 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 ClO3-.



41

Dechlorosoma sp. PCC (10mM ClO3- )

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 ClO3- and

20mM of CH3COO-.

0

5

10

15

20

25

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Time (h)

CH

3C

OO

- , C

lO3- ,

Cl- (

mM

)

0,0

0,2

0,4

0,6

0,8

1,0

DW

(g

/L)

ClO3-

CH3COO-

Cl-

DW (g/l)

Figure 3.7 – Acetate and chlorate uptake as function of time during the reduction of 10mM of

ClO3- 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.



42

Dechlorosoma sp. PCC (5mM ClO4- + 5mM ClO3

- )

It was also performed a kinetic study with 5mM ClO4- and 5mM ClO3

-

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 CH3COO- was used in a

concentration of 20mM.

0

5

10

15

20

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Time (h)

CH

3C

OO

- , C

lO4

- , C

lO3- (m

M)

0,0

0,2

0,4

0,6

0,8

1,0

1,2

DW

(g

/L)

ClO4-

CH3COO-

ClO3-

DW (g/l)

Figure 3.8 – Acetate, perchlorate and chlorate uptake as function of time during the reduction of

5mM of ClO4- + 5mM of ClO3

- 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



43

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 (qCH3COO-), specific perchlorate reduction rates (qClO4-), specific chlorate

reduction rates (qClO3-) and biomass yield (g [DW] / gCH3COO-) 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



44

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.

Isolate Electron

Acceptor µµµµ (h

-1) Reference

Chlorate 0.21 Dechlorospirillum sp. DB

Perchlorate 0.17 This study

Chlorate 0.13 Dechlorosoma sp. PCC

Perchlorate 0.17 This study

Chlorate 0.26 Dechlorosoma sp. KJ

Perchlorate 0.14 Logan et al., (2001)

Chlorate 0.21 Dechlorosoma sp. PDX

Perchlorate 0.21 Logan et al., (2001)

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



45

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] / gCH3COO-) 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



46

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.

Isolate Electron

Acceptor

Cell yield (g [DW] /

g CH3COO-) Reference

Chlorate 0.47± 0.01 Dechlorospirillum sp. DB

Perchlorate 0.94 ± 0.03 This study

Chlorate 0.82± 0.02 Dechlorosoma sp. PCC

Perchlorate 0.70 ± 0.01 This study

Oxygen 0.46 ± 0.07

Chlorate 0.44 ± 0.05 Dechlorosoma sp. KJ

Perchlorate 0.50 ± 0.08

Logan et al., (2001b)

Oxygen 0.27 ± 0.01

Chlorate 0.28 ± 0.01 Azospira oryzae strain GR1

Perchlorate 0.24 ± 0.01

Rikken et al., (1996)

Oxygen 0.13 ± 0.04 AB1

Chlorate 0.10 ± 0.04 Olsen S., (1997)

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.



48

Chapter 4 – Conclusions and further research


49

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

Chapter 4 – Conclusions and further research


50

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.

Bibliography

Perchlorate and chlorate degradation by two organisms isolated from wastewater 52

BIBLIOGRAPHY

Achenbach, L. A., U. Michaelidou, R. A. Bruce, J. Fryman and J. D. Coates. 2001.

Dechloromonas agitata gen. nov., sp. nov. and Dechlorosoma suillum gen. nov.,

sp. nov., two novel environmental dominant (per)chlorate-reducing bacteria and

their phylogenetic position. Int. J. Syst. Evol. Microbiol. 51:527-533.

Achenbach, L. A., K. S. Bender, Y. Sun and J. D. Coates. 2006. The biochemistry

and genetics of microbial perchlorate reduction. In Perchlorate: Environmental

occurrence, interaction and treatment. Chapter. 13, pag. 297-310, eds. Baohua Gu

and John D. Coates, Springer Science+Business Media, NY, USA.

Amy, G., Y. Yoon, J. Yoon, and M. Song. 2003. Treatability of perchlorate-containing

water by RO, NF and UF membranes. AWWA Research Foundation, USA.

Aslander, A. 1928. Experiments on the eradication of Canada thistle Cirsium arvense

with chlorates and other herbicides. J. Agric. Res. 36:915-935.

Bardiya, N. and J-H. Bae. 2006. Isolation and characterization of Dechlorospirillum

anomalous strain JB116 from a sewage treatment plant. Microbiol. Res. In Press.

Bender, K. S., S. M. O’Connor, R. Chakraborty, J. D. Coates, and L. A.

Achenbach. 2002. Sequencing and transcriptional analysis of the chlorite

dismutase gene of Dechloromonas agitata and its use as a metabolic probe. Appl.

Environ. Microbiol., 68:4820-4826.

Bibliography


Bender, K. S., M. R. Rice, W. H. Fugate, J. D. Coates and L. A. Achenbach. 2004.

Metabolic primers for detection of (per)chlorate-reducing bacteria in the

environment and phylogenetic analysis of cld gene sequences. App. Environ.

Microbiol. 70:5651-5658.

Bender, K. S., C. Shang, R. Chakraborty, S. M. Belchik, J. D. Coates, and L. A.

Achenbach. 2005. Identification, characterization, and classification of genes

encoding perchlorate reductase. J. Bacteriol. 187:5090-5096.

Bliven, A.R. 1996. Chlorate respiring bacteria: isolation, identification, and effects on

environmentally significant substrates. M.S. Thesis, Department of Chemical and

Environmental Engineering, University of Arizona, Tucson, AZ.

Bruce, R. A., L. A. Achenbach, and J. D. Coates. 1999. Reduction of (per)chlorate by

a novel organism isolated from paper mill waste. Environ. Microbiol. 1:319-329.

Cang, Y., D. J. Roberts, and D. A. Clifford 2004. Development of cultures capable of

reducing perchlorate and nitrate in high salt solutions. Wat Res. 38:3322-3330.

Chaudhuri S. K., S. M. O’Connor, R. L. Gustavson, L. A. Achenbach, and J. D.

Coates. 2002. Environmental factors that control microbial perchlorate reduction.

Appl. Environ. Microbiol. 68:4425-4430.

Bibliography


Coates, J. D., U. Michaelidou, R. A. Bruce, S. M. O´Connor, J. N. Crespi and L. A.

Achenbach. 1999. Ubiquity and diversity of dissimilatory (per)-chlorate-reducing

bacteria. Appl. Environ. Microbiol. 65:5234-5241.

Coates, J. D. And Laurie Achenbach. 2004. Microbial perchlorate reduction: rocket-

fuelled metabolism. Nature reviews Microbiology 2: 569-580.

Condie, L. 1986. Toxicological problems associated with chlorine dioxide. J. Am.

Water Works Assoc. 78:73-78.

Dasgupta, P. K., P. K. Martinnelango, W. A. Jackson, T. A. Anderson, K. Tian, R.

W. Tock and S. Rajagopalan. 2005. The origin of naturally occuring perchlorate:

the role of atmospheric processes. Environ. Sci. Technol. 39:1569-1575.

de Groot, G. N. and A. H. Stouthamer. 1969. Regulation of reductase formation in

Proteus mirabillis. I. formation of reductases and enzymes of the formic

hydrogenlyase complex in the wild type and in chlorate-resistant mutants. Arch.

Microbiol. 66:220-233.

El Aribi, H., Y. J. C. Le Blanc, S. Antonsen and T. Sakuma. 2006. Analysis of

perchlorate in foods and beverages by ion chromatography coupled with tandem

mass spectrometry (IC-ESI-MS/MS). Anal. Chim. Acta, 567:39-47.

Bibliography


Greer, M. A., G. Goodman, R. C. Pleus and S. E. Greer. 2002. Health effects

assessment for environmental perchlorate contamination: the dose response for

inhibition of thyroidal radioiodine uptake in humans. Environ Health Presp.

110:927-937.

Gu, B., G. M. Brown and Y-K. Ku 2003. “Treatment of Perchlorate-Contaminated

Groundwater Using Highly Selective, Regenerable Ion-Exchange Technology: A

Pilot-Scale Demonstration.” Remediation. Spring 2002.

Herman, D. C., and W. T. Frankenberger. 1998. Microbial-mediated reduction of

perchlorate in groundwater. J. Environ. Qual. 27: 750-754

Kengen, S. W. M., G. B. Rikken, W. R. Hagen, C. G. Van Ginkel, and A. J. M.

Stams. 1999. Purification and characterization of (per)chlorate reductase from the

chlorate-respiring strain GR-1. J. Bacteriol. 181:6706-6711.

Korenkov, V. N., V. I. Romanenko, S. I. Kuznetsov and J. V. Voronnov. 1976.

Process for purification of industrial waste waters from perchlorate and chlorates.

U. S. patent 3,943,055.

Logan, B. E., J. Wu and R.F. Unz 2001a. Biological perchlorate reduction in high-

salinity solutions. Wat. Res. 35:3034-3038.

Logan, B. E., Zhang, H., Mulvaney, P., Milner, M. G., Head, I. M. and Unz, R. F.

2001b. Kinetics of perchlorate- and chlorate-respiring bacteria. App. Environ.

Microbiol. 67:2499-2506.

Bibliography


Matos, C.T., S. Velizarov, J.G. Crespo, M. A. Reis. 2006. Simultaneous removal of

perchlorate and nitrate from drinking water using the ion exchange membrane

bioreactor concept. Water Res. 40:231-40.

Mayer, Kevin. 2004. Perchlorate in the Environment. Presentation at American

Chemical Society Meeting, Agricultural Chemical Division.

Michaelidou, U., Achenbach, L.A., and Coates, J.D. (2000). Isolation and

characterization of two novel (per)chlorate-reducing bacteria from swine waste

lagoons. Kluwer Academic/Plenum Publishers, NY. 271-283 In Perchlorate in the

Environment (E. Urbansky, ed).

Nerenberg, R., B. E. Rittmann, and I. Najm. 2002. Perchlorate reduction in a

hydrogen-based membrane-biofilm reactor. Journal AWWA. 94:103-111.

Nerenberg, R., Y. Kawagoshi, and B. E. Rittmann. 2006. Kinetics of a hydrogen-

oxidizing, perchlorate-reducing bacterium. Wat. Res. 40:3290-3296.

Okeke, B. C. and W. T. Frankenberger Jr. 2003. Molecular analysis of perchlorate

reductase from a perchlorate-respiring bacterium Perc1ace. Microbiol. Res.

158:337-344.

Olsen, S., 1997. Consortium and pure culture kinetics of chlorate reducing organisms.

M. S. thesis. University Arizona. Tucson.

Bibliography


Olsson, L., J. Nielsen. 1997. On-line and in situ monitoring of biomass in submerged

cultivations. TibTech. Vol 15.

Oltmann, L. F., W. N. Reijnders and A. H. Stoughamer. 1976 Characterization of

purified nitrate reductase A and chlorate reductase C from Proteus mirabilis. Arch.

Microbiol. 111:25-35.

Rikken, G. B., A. G. M. Kroon and C. G. van Ginkel. 1996. Transformation of

(per)chlorate into chloride by a newly isolated bacterium: reduction and

dismutation. Appl. Microbiol. Biotechnol. 45:420-426.

Romanenko, V. I., V. N. Korenkov, and S. I. Kuznetsov. 1976. Bacterial

decomposition of ammonium perchlorate. Mikrobiologiya 45:204-209.

Roquebert, V., S. Booth, R. S. Cushing, G. Crozes and E. Hansen 2000.

Electrodialysis reversal (EDR) and ion exchange as polishing treatment for

perchlorate treatment. Desalination 131:285-291.

Rosemarin, A., K. Lehtinen and M. Notini. 1990. Effects of treated and untreated

softwood pupl mill effluents on Baltic sea algae and invertebrates in model

ecosystems. Nord Pulp Paper Res J. 2:83-87.

Sidiqqui, M. 1996. Chlorine-ozone interactions: formation of chlorate. Water Res. 30:

2160-2170

Bibliography


Steinberg, L. M., J. J. Trimble and B. E. Logan. 2005. Enzymes responsible for

chlorate reduction by Pseudomonas sp. are different from those used for

perchlorate reduction by Azospira sp. FEMS Microbiol. Lett. 247:153-159.

Stenklo, K., H. D. Thorell, H. Bergins, R. Aasa and T. Nilsson 2001. Chlorite

dismutase from Idonella dechlorotans. J. Biol. Inorg. Chem. 6:601-607.

Thorell HD, Stenklo K, Karlsson J, Nilsson T. 2003. A gene cluster for chlorate

metabolism in Ideonella dechloratans. Appl Environ Microbiol. 69:5585-92.

Urbansky, E. T. 1998. Perchlorate chemistry: implications for analysis and

remediation. Biorem. J. 2: 81-95.

Urbansky, E. T., M. R. Shock. 1999. Issues in managing the risks associated with

perchlorate in drinking water. J. Environ Managem 56: 79–95.

Urbansky, E. T. 2000. Perchlorate as an environmental contaminant. Environ Sci &

Pollut Res. 9:187-192.

Urbansky, E.T., S.K. Brown, M.L. Magnuson, C.A. Kelty. 2001. Perchlorate levels

in samples of sodium nitrate fertilizer derived from Chilean caliche. Environ.

Pollution 112: 299-302.

Bibliography


van Ginkel, C. G., Rikken, G. B., A. G. M. Kroon and S. W. M. Kengen 1996.

Purification and characterization of chlorite dismutase: a novel oxygen-generating

enzyme. Arch. Microbiol. 166:321-326.

Wallace, W., T. Ward, A. Breen and H. Attaway. 1996. Identification of an

anaerobic bacterium which reduces perchlorate and chlorate as Wolinella

succinogenes. J. Ind. Microbiol. 16:68-72.

Waller, A. S., Cox, E. E. and Edwards, E. A. 2004. Perchlorate-reducing

microorganism isolated from contaminated sites. Environ. Microbiol. 6:517-527.

Wolterink, A. F. W. M., A. B. Jonker, S. W. M. Kengen and A. J. M. Stams. 2002.

Pseudomonas chloritidismutans sp. nov., a non-denitrifying, chlorate-reducing

bacterium. Int. J. Syst. Evol. Microbiol. 52:2183-2190.

Wolterink, A. F. W. M., E. Schiltz, P-L. Hagedoorn, W. R. Hagen, S. W. M.

Kengen and A. J. M. Stams. 2003. Characterization of the Chlorate Reductase

from Pseudomonas chloritidismutans. J. Bacteriol. 185:3210-3213.

Wu, D., P. He, X. Xu., M. Zhou, Z. Zhang, and Z. Houda. 2007. The effect of

various reaction parameters on bioremediation of perchlorate-contaminated water.

J. Hazardous Materials. In press.

Xu, J., Y. Song, B. Min, L. Steinberg and B. E. Logan. 2003. Microbial degradation

of perchlorate: principles and applications. Environ. Eng. Sci. 20:405-422.

Bibliography


Xu , J., J. Trimble, L. steinberg and B. E. Logan. 2004. Chlorate and nitrate

reduction pathways are separately induced in the perchlorate-respiring bacterium

Dechlorosoma sp KJ and chlorate-respiring bacterium Pseudomonas sp. PDA. Wat.

Res. 38: 673-680

Zhang H., M.A. Bruns, and B.E. Logan. 2002. Perchlorate reduction by a novel

chemolithoautotrophic hydrogen-oxidizing bacterium. Environ. Microbiol., 4:570-576.

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