Perchlorate and chlorate degradation by two organisms isolated ...

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 (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

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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………………………………………………………...

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

26

27

2.1. Source of organisms……………………...……………………………….

27

2.2. Media.............................................................................................................

27

2.3. Bacterial isolation procedures and culturing conditions ………………….

28


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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


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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…………………………………………………….. Figure 1.4: Mechanism of anion exchange – chloride for perchlorate.……………..

6 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

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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 reduction of 5mM of ClO4- + 5mM of ClO3- 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 reduction of 5mM of ClO4- + 5mM of ClO3- by Dechlorosoma sp. PCC. Dry weight (DW) as a function of time is also represented.……..


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Chapter 1 – Literature study

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


1

Chapter 1 – Literature study for maximal destruction while minimizing by-product formation, wasteful sidereactions 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).

ClO4– + 8H+ + 8e- ↔ Cl– + 4H2O, E° = 1.287 V

(1)

Nevertheless, ClO4- is slightly weaker than dichromate (Cr2O72-) 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


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