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On the site of the Tui mine at Te Aroha, there are several tailings containing high concentrations of heavy metals. From these tailings the bacterium Acidocella was isolated . This project focuses on the cadmium uptake by Acidocella, its resistance to this metal and operation of its defense mechanisms.
At the start of this project, the bacteria had to be identified by 16s RNA sequencing. 16s RNA is used kun je dat zo stellen? Ribosomen bestaan uit..by bacteria and other organisms as ribosomal RNA and it is very conserved, meaning there will not have been many mutations in the period of its evolution. The sequence of 16s RNA differs with different types of bacteria, and this is why it was used as an identifying method. Dit is wel erg nederlands engels, er zijn daar vast collega's die evn naar het engels willen kijken
In this project there is also an analysis of the optimal growth conditions for Acidocella focusing on pH and organic buffer and cadmium and zinc tolerance.
Later during the course of this project, there will be cadmium uptake experiments and acid-base titrations. In the last experiment, I intend to conduct a Surface Complexation Model (SCM) with the computer program FITMOD. With SCM one can observe the groups on the outer membrane of the cell, made of lipopolysacharides (LPS), responsible for the uptake of cadmium or zinc.
All the experiments will be done with two different cultures originating from the Acidocella culture. One culture is trained to grow on higher concentrations of Cd2+ while the other is not.
This report is a part of my graduation internship at the University of Waikato and is based on the first 10 weeks of my orientation period. In this report, I will be commenting upon the work undertaken as well as a brief review of some theoretical background.
Firstly, I would like to thank my professor Hugh Morgan and his wife Patricia Morgan, for guidance and support and their warmth and hospitality during my stay here. For helping me with some practical things at the lab, I would like to thank my colleagues Thom, Ariff and Steven. Last but not least, I would like to thank my girlfriend Crystal for proof reading this report.
Table of contents
Table of contents 4
1 Introduction 5
1.1 General 5
1.2.1 Optimum growth conditions and appearance 6
1.3 16S rRNA onderstaand stuk graag wat wetenschappelijker 7
1.4 Biosorption 7
1.4.1 Ionizable groups 8
1.5 Metal resistance mechanisms 9
1.5.1 Exclusion by permeability barrier 9
1.5.2 intracellular complexation 9
1.5.3 active transport efflux pumps out of the cytoplasm 9
1.5.4 enzymatic detoxification 9
1.5.5 reduction of the sensitivity 10
1.5.6 extra cellular complexation 10
1.7 FITMOD 10
2.1 Objective 11
3 Materials and methods 12
3.1 Non sensitive vs. Sensitive 12
3.2 Preparing 12
3.3 16S rRNA 12
3.4 Titrations 13
3.5 Cadmium ion adsorption 13
3.6 Atomic absorption spectroscopy 15
4.1 Optimum grow condition 16
4.1.1 pH 16
4.1.2 Organic buffers 16
4.1.3Cadmium and zinc tolerance 16
4.1 16s rRNA sequence analysis 16
4.2 Titrations 16
4.3 Cadmium ion adsorption 16
5 Discussion 17
6 Conclusions 17
7 Recommendations 17
8 References 18
9 Appendix 19
Protocollen heb ik niet bekeken, kan ik onvoldoende inschatten 19
9.1 Recipe GYE media 19
9.2 Cell harvest 19
9.3 Cell count 19
9.4 Preparation for 16S rRNA sequencing 20
9.5 Titrations 22
9.6 Cadmium ion adsorption 23
9.7 Measure cadmium concentration with AAS. 24
9.8 Raw data 24
16s rRNA sequences: 24
The Tui mine generated tailings of heavy metal minerals such as ZnSO4 and PbSO4 that were deposited during the last period of its operation of six years which ended in 1974. This has lead to contamination in Te Aroha. As a result, there is a lot of zinc and lead left in the soil and groundwater of this site (see Figure 1). These sulfates have caused acidy soils with low pH's as a result of high amounts of H2SO4 . Through adaptation, the bacteria in this soil have adapted to the extreme conditions with high amounts of heavy metals and low pH's.  ook dat is een veel voorkomend verschijnsel, zoek dat op en vermeld het hier. Vertel ook iets over de bacterieflora die je normal gesproken vindt in een dergelijke omgeving en gad an over naar jouw Acidocella
Figure 1 Tui mine Trace metals analysis.  hoe verhouden deze getallen zich tov normale omstandigheden?
Acidocella is a Gram negative bacterium which forms straight or slightly curved rods with rounded and tapered ends, a SEM Photo is shown in figure 2. The cell wall contains lipopolysaccharides (LPS). With the exception of some strains, most of the strains are motile due to the presence of polar or lateral flagella. They are strictly aerobic and cannot make spores or capsules. The colonies are white, cream or light brown. Acidocella is strictly acidophilic and the maximum pH for growth is 6.
The strains for this project are isolated from the Tui mine where high amounts of heavy metals are traced. Figure 1 in the introduction shows the trace metal analyse, noticeable is that on one point in the mine very high amounts of zinc are polluted, up to 500 mg/L. The bacterium used for this project is resistant to very high amounts of zinc as it was isolated from one of these tailings.
1.2.1 Optimum growth conditions and appearance
Acidocella is an acidophil and therefore the pH level is an important condition for its growth and has to be lower than 6. Other conditions such as the growth of the bacteria at the presence of organic acids like citric acid and acetic acid are also be measured, because they can be used as a buffer in growth media. It has been established that Acidocella grows within a pH range from pH 3 to 6; some strains can grow on pH 2.5. 
Figure 2 Scanning electron micrograph showing general cell morphology of Acidocella 
1.3 16S rRNA onderstaand stuk graag wat wetenschappelijker
A new standard for identifying bacteria started developing in the 1980's. It was published that phylogenetic relationships of bacteria, and all other life-forms, could be determined by comparing a stable fraction of the genetic code. 16S rRNA is ribosomal RNA which means that it is a part of the structure of a ribosome. The 16S rRNA gene can be compared not only between all bacteria but also with the 16S rRNA gene of Archaea and the 18S rRNA gene of Eukarya as well. 16S rRNA gene is not identical for all organisms (different taxonomic groups can have different rates of alteration): the rates could fluctuate at times through the evolution, and the rates might be changed at different sites throughout the 16S rRNA gene. 16S rRNA gene sequence data on an individual strain with a close neighbour exhibiting a comparison score of â‰¥97% are regarded as being of the same species. There is a large number of strains where the 16S rRNA gene sequence has been determined. Most of these sequences are stored in the database of the National Center for Biotechnology Information (NCBI). The computer algorithm Basic Local Alignment Search Tool (BLAST) can be used to search this sequence database for 16s rRNA gene sequence similarities. With 16S rRNA sequencing a distinction can be made between different Acidocella species, which would be difficult with the common methods for identifying bacteria. [2,4,5,6]
Heavy metals interact with cellular components through covalent and ionic bonding. They can damage cell membranes, interrupt cellular functions, modify enzyme specificity and damage the structure of DNA. They can react with sulphydryl groups to form mercaptide compounds or they can act as oxidizing agents, which can lead to enzyme inhibition or deactivation in two different ways. If the sulphydryl groups are located within the active site, when heavy metals bind on these groups, the active site will be concealed. Alternately, when the sulphydryl group is used for structure enhancement by the enzyme, in case of heavy metal binding, the sulphydryl groups will lose the electrical negativity used for holding different parts of the enzyme together. Some microorganisms have adapted to the presence of heavy metals by developing a range of resistance mechanisms. There are several metal resistance mechanisms such as: exclusion by permeability barrier, intra- and extra-cellular complexation, active transport efflux pumps, enzymatic detoxification and reduction of the sensitivity of cellular targets to metal ions.
The normal manner in which metal concentrations are enhanced by the cell occurs through the membrane transport systems. Cat ions are transported into the cell by constant expressed divalent cat ion uptake systems of a broad specificity. Essential heavy metals, as well nonessential heavy metals will be up taken with nonspecific uptake systems.welke?
Bedenk dat je bij de verdediging alles moet kunnen verklaren wat je hebt opgeschreven
Cadmium is a divalent metal which can disturb or destroy important cellular functions by binding to sulfhydryl groups on essential proteins. Several bacteria have developed a resistance to cadmium, for example S. aureus, Bacillus subtillis, and E. Coli. Cadmium resistance can be given by numerous mechanisms which are described in the following paragraphs, except for the enzymatic detoxifications.  
1.4.1 Ionizable groups
All the metal ions have to pass the cell wall, before they access the plasma membrane and cell cytoplasm. The cell wall consists of a diversity of peptidoglycan and proteins. This offers a number of negatively charged groups, which are able to attract and bind metallic cat ions based on electrostatic forces. Because bacteria are very small they have an high surface to volume ratio and thus an high amount of negatively charged groups compared with the volume. The cell wall of microorganisms can have a high ion exchange ability by the presence of strongly complexing metal functional groups such as: carboxyl, aldehyde, sulfhydryl, and phosphoryl groups. These groups are shown in the figure below.
Figure 3 Functional groups that strongly complex metals.
It is known that some biological structures have different metal ion binding affinities. For example nitrogens of the aminoacid histidine can attract CuÂ2+ even as Zn2+ shows an affinity for the sulfhydryl groups present on the amino acid cysteine. Because of these metal affinities, the harmful metals can be caught by this groups. [9,10,11]
1.5 Metal resistance mechanisms
To protect the microorganisms against heavy metals, there are several metal resistance mechanisms developed by bacteria. Some of them are explained in the following subparagraphs.
1.5.1 Exclusion by permeability barrier
Alterations in the cell wall, membrane, or envelope of a microorganism are examples of metal elimination by permeability blockade. This mechanism is a way for the organism to protect metal-sensitive, essential cellular components. One way to do this is altering a protein in the cell wall which is involved by taking up heavy metals, another way is making extra binding sites to the outer membrane or envelope. This mechanism is used by several bacteria like: E. coli, P. putida, A. viscous, Klebsiela aerogenes and Pseudomonas putida 
1.5.2 intracellular complexation
Intracellular complexation is the accumulation of metals inside the cytoplasm. In this way the heavy metal ions will bind to a protein, which transcription is induced by the heavy metal ions. The heavy metal is caught by this protein and cannot bind on vital proteins of the cell anymore. There are a few cyanobacterial strains known who use this, furthermore there is P. putida and Micobacterium scrofulaceum who are able to use this mechanism. 
1.5.3 active transport efflux pumps out of the cytoplasm
For cadmium active transport out of the cytoplasm is the most prominent metal resistance system. Active transport or efflux systems represent the largest category of metal resistance systems. Microorganisms use active transport mechanisms to export toxic metals from their cytoplasm. Cadmium can come in to the cell via divalent ion transport systems, these systems are normally used by the cell to uptake essential metals. Active transport via efflux pumps can be regulated by one or more operons, the presence of cadmium will induce the transcription of the proteins made by these operons. By this, an active efflux pump will be made which transports cadmium from the inside to the outside of the cell. Several bacteria who use this mechanism: E. Coli, S. Aureus, Bacillus sp. Listeria sp. and Alcaligenus eutrophus 
1.5.4 enzymatic detoxification
An enzyme detoxifies a heavy metal to a less toxic form. For instance the metal mercury (toxic because it binds to and inactivates essential thiols that are part of enzymes and proteins) can be detoxified by Gram-positive and Gram-negative bacteria. Those bacteria contain a set of genes that form a HgÂ2+ resistance operon. This operon not only detoxifies Hg2+ but also transports and self-regulates resistance. The mercury ions are transported into the cytoplasm by special transport proteins and in the cytoplasm they are detoxified by special enzymes by changing the chemical structure of de mercury salt. Examples of bacteria who use this mechanism are: S. aureus, Bacillus sp, E. coli, P. aeruginosa, Serratiamarcescens and Thiobacillus ferrooxidans 
1.5.5 reduction of the sensitivity
Microorganisms can show mutations that decrease the sensitivity and damage of DNA or they produce metal resistant components or change pathways. For example, bacteria can induce the transcription for a certain protein which is affected by an heavy metal in order to have enough of good working protein. Gram-negative organisms can endure more cadmium than Gram-positive organisms, this is shown in continuing protein synthesis when exposed to cadmium. Bacteria who use this mechanism: E. coli, Pseudomonas sp. S. aureus, S. faecium and B. subtilis 
1.5.6 extra cellular complexation
Extracellular complexation is until now only found in several species of yeast and fungi, in bacteria it has only been hypothesized. This mechanisms works by the excretion of proteins, the metal ions will bind on these proteins outside the cell and thus will not enter the cell. For example glutathione is excreted by Saccharomyces cerevisea to bind copper ions outside the cell, so that the metal cannot enter the cell. Other organisms use other components than glutathione. 
1.6 Surface Complexation Model (SCM)
A Surface Complexation Model (SCM) describes the adsorption and desorption of metal ions to potentional ligands into and onto the organism's cell wall. An SCM is based on a number of molecular scale thermodynamic reactions, each relating adsorption of a particular dissolved chemical species to a particular type of cell wall functional group (ionizable group) using a single stability constant (K). hoe verhoudt zich dit tot wat je hierboven hebt beschreven? Tot welk def mechanisme zijn deze ionizable groepen gerelateerd?With a SCM the concentrations and the amount of proton removals ''deprotonation'' constants can be described as all different ionizable groups on the cell wall have their own specifications for proton binding and removal. Some earlier studies have showed that different bacterial species display similar types of the reactive surface functional groups and generally show similar actions to certain metals. In theory cadmium can bind to all negatively charged ionizable groups but one study suggests that there are bacteria which can have a different type of functional group what can be involved in cadmium biosorption. en wat is nu de boodschap?
SCM's can be made from the acid-base titration and cadmium adsorption parameters by the computer program FITMOD. A SCM describes the electrostatic and chemical interactions, such as pH and ionic strength, conditions between the liquid (10 mM NaNO3) and the cell. FITMOD can determine the concentrations and deprotonation constants of the binding sites on bacteria. FITMOD assumes the ionisable functional groups to be distributed equally in a volume. It calculates the overall variation of the error in the chemical mass balance equations and by doing so, it provides a quantitative approximation of the accuracy of the fit. When the overall variation is between 1 and 20, the fits to data are indicated as reasonable. 
2 Project goals
This project contains five different parts:
- Identifying the organism by sequencing the 16S-rRNA to make sure that it is Acidocella and determining the optimum growth conditions such as pH and maximum concentration of zinc and cadmium which it can tolerate.
- Determining the major ionizable groups in the proteins of the cell surface.
- Determining cadmium uptake at different pH's and cell concentrations.
- Analyzing the major ionizable groups on base of the cadmium uptake to determine the active groups in heavy metal adsorption.
- Determining if isolations at the Tui mine are already adapted to locally high concentrations of heavy metals and what numbers of Acidocella are present at different locations in the Tui mine spoils.???
One of the bacteria isolated from this site is a species of Acidocella. This bacterium is acidophilic and the optimum growth condition is in a low pH environment such as the Tui site. One of the objectives of this project is to discover how this bacterium is adapted to these conditions. The focus question guiding this project is ''how can Acidocella survive high concentrations of heavy metals?''gaat het vooral om de zware metalen of de combinatie zware metalen zuur milieu?The heavy metals being referred to are zinc and lead, but we will also use cadmium as a reference for heavy metals. The assumption is that there are different ionizable groups on the cell wall of the bacterium where the metal ions can bind to. Another objective is to compare the determinations of ionizable groups on the cell wall of Acidocella cells which have been adapted to very high concentrations of zinc and cadmium with strains which have not been exposed to the heavy metals and which on initial contact probably would be killed.
3 Materials and methods
3.1 Non sensitive vs. Sensitive
The meaning was that two different cultures of Acidocella would be used for all the experiments, both originating from one culture. The first one is trained to grow on high concentrations of Cd2+ by first incubating them on a low concentration of Cd2+ and transfer them each time to a higher concentration of Cd2+ after growth. The other culture is the original culture itself and is not trained to grow in high concentrations of Cd2+.
Firstly, the project commenced with growing the cells in GYE media with a low concentration of zinc sulfate (20 mM) as the heavy metal. Each time, the growth was measured with an optical density at 660 nm. With a result of 0.6 or more, the cells were transferred to GYE media with a higher concentration of zinc sulfate gaat het dan om de isolatie van tolerante stamen?. In the first steps of this process, the concentration inducements occurred with steps of 20 mM. When the concentration had reached the value of 100 mM, the concentration inducements were done with 50 mM of each transfer. All the titration and adsorption experiments have been performed three times, taking account of each culture, the original Acidocella A3 as well the zinc sulfate incubated Acidocella A3z!!!!
Before starting with the titration or adsorption experimentsprobeer het zakelijk en wetenschappelijk te houden, het is geen krantenartikel, a cell harvest had to be done for each experiment. By incubating a certain volume of GYE media, a required amount of cells could be harvested at a relative centrifugal force (RCF) of 10415. After harvesting, four or five times a cell wash was executed in a solution of 10 mM NaNO3. The total amount of cells was measured in the end. The cell concentration required for the experiment could be set with the outcome of this measurement. A NaNO3 solution of 10 mM at a pH of 3,0 was used for this.
3.3 16S rRNA
DNA samples were obtained from the whole genome of Acidocella. This was done by incubating 3 ml of cells with a CTAB DNA extraction buffer (100 mM Tris HCL, 1,4 mM NaCl, 20 mM EDTA, 2% w/v cetyltrimethylammoniumbromide (CTAB), 1% polyvenylpiriladine at pH 8,0) and 0,4% w/v 2-mercaptoethanol for 20 min at 100ËšC. After this, the nucleic acids were extracted with Phenol, followed by chloroform and iso-amyl alcohol (24:1). At the end of the extraction, a precipitation was done with iso-propyl alcohol at -80ËšC.
After extracting the genome of Acidocella, microbial 16S rRNA genes could be amplified. For this, a mix of genomic DNA, PCR buffer (10 mM Tris HCL, 50 mM KCL), 1,5 mM MgCl2, 0,2 mM dNTP, 1,0 ÂµM of each primer (27f and 1522r), Bovine Serum Albumin (BSA) and Taq polymerase were made. The mixture was treated with a PCR machine on the following temperature program: 30 sec 94ËšC denaturation, 30 sec 55ËšC annealing, 72ËšC amplification for 36 cycles.
In the final stageszakelijk en wetenschappelijk, an electrophoresis was performed to separate the amplification product from the reagents and byproducts. Firstly, the electrophoresis of the PCR product was conducted in 1% agarose gel and TAE buffer. The gel was stained in Ethidium bromide for 10 min and then observed under UV light (302 nm). The band containing the amplified 16S rRNA gene was dissected out of the agarose gel and transferred to a new tube. The tube is placed at -20ËšC for 10 min and then squeezed in order to separate the liquid from the gel. This liquid is again transferred to a new tube and purified with phenol and chloroform. After precipitation with iso-propyl alcohol at -80ËšC the pallet is dissolved in a small amount of TE buffer and sent to the sequence department.
For making a SCM several parameters are needed. The first parameter is the pH against the concentration of an added acid. In this project a culture with a concentration of 50 g/l wet weight will be set with HNO3 to a pH of 2 till 3 in a 10 mM solution of sodium nitrate. Then an acid-base titration will be performed with an automatic titration system which prints out the results. This allows an indication of the buffering capacity of the culture.
After setting the wet weight concentration with 10 mM NaNO3 to 50 g/l, the sample could be loaded to a sample beaker provided by the automatic titter machine (Mettler Toledo, DL22). All the titrations were conducted under the presence of nitrogen bubbles and with a sample volume of 25 ml. The samples were titrated twice. The first titration started from a pH of two up to a pH of five and the other titration started from a pH of three going up to nine. While titrating, the samples were continuously stirred in the presence of nitrogen bubbles. The titrants used were HNO3 (0.2 mM) and NaOH with a concentration of 0,1 mM as weaker base or with a concentration of 0,5 mM as a stronger base. The weaker base was used for the titrations with the pH range from three to nine and the stronger base was used for the pH range from two to five.
3.5 Cadmium ion adsorption
Another parameter for making an SCM is the uptake of cadmium by Acidocella. The culture will be incubated with a certain concentration of Cd2+ followed by centrifugation to separate the cells from the media. The amount of Cd2+ which is adsorbed by the cells can be calculated by measuring the cadmium concentration in the supernatant comparing to the added amount by an atomic flame adsorption spectrometer. An earlier study welke? referentie?suggested that at pH's lower than 3,0 cadmium ions removal can be inhibited as a result of a competition between hydrogen and cadmium ions on the ionisable groups on the cell wall with an significant predominance of hydrogen ions. Hypothetically, when the pH increases, the negative charge density of the groups on the cell wall will increase as well due to deprotonation of metal binding sites and thus an increase in the metal adsorption will occur. It can be expected that cadmium concentrations from samples incubated at higher pH's than 7,0 will be lowhet ging toch over zure milieu's? This could be be due to cadmium hydroxide which may start precipitating from the solutions at higher pH values. 
Before starting with the cadmium ion adsorption experiment, a certain amount of cells are obtained by
Figure 4 Flowchart cadmium ion adsorptioninoculating nine litters of GYE media separated over 12 flasks of two liter. These are incubated, harvested and washed as described as in paragraph 6,1. The total obtained amount of cells is assumed as a 200% in order that cell suspensions could be made with NaNO3 (10 mM) of 100%, which is the standard, following by cell suspensions of 50%, 25%, 10% and 0%. 9 ml of cell suspension was transferred to seven vessels for each cell suspension and 1 ml of a solution of 50 ppm Cd(NO3)2 in 0,05% HNO3 is added to each tube in order to acquire a concentration of 5 ppm Cd(NO3)2 in each vessel. For every cell suspension, the pH in the vessels is set to 2, 3, 4 ,5 ,6 ,7 and 8, with NaOH and HNO3. After this, the vessels could be incubated at 30ËšC in a shake incubator for two hours. After this incubation time, all of the vessels are centrifuged at ^$%^^%$ RCF?, the supernatant was transported to 50 ml Falcon tubes and 100 Âµl of HNO3 (7%) was added to lower the pH of each tube to less than two. In the end, all the Falcon tubes were set to the weight of the heaviest Falcon tube since Falcon tubes are not significantly different in weight and a total common end volume is shared. Some tubes, especially the ones from the higher cell concentrations, needed an extra centrifuge round to make the supernatant clear enough for use on de flame Atomic Absorption Spectroscope (Avanta, GBC). All of the Falcon tubes were measured for Cd concentration with the AAS after setting up a calibration curve with standard Cd(NO3)2 in 0,5% HNO3 solutions, 10 ppm CdÂ2+, 7,5 ppm CdÂ2+ÂÂ, 5 ppm CdÂ2+, 4 ppm CdÂ2+, 3 ppm CdÂ2+, 2 ppm CdÂ2+ and 1 ppm CdÂ2+. The concentration of adsorption was then calculated. In figure 4 you geen persoonlijk taalgebruikcan see a flowchart of this experiment.
3.6 Atomic absorption spectroscopy
With Atomic Absorption Spectroscopy (AAS) it is possible to measure concentrations of ions in liquids or solids. AAS is based on the absorption of light by gas phase atoms and because of this, the samples must be vaporized in a flame or graphite heater. The light that will be absorbed can be ultraviolet or visible light. Due to the absorption of light by the atoms, transitions to higher electronic energy levels will occur.. The results are shown in numbers on a computer and are determined from the amount of absorption. After calibrating the instrument, a working curve can be made which is used to transfer the absorption numbers to real concentrations.
4.1 Optimum grow condition
4.1.2 Organic buffers
4.1.3Cadmium and zinc tolerance
4.1 16s rRNA sequence analysis
4.3 Cadmium ion adsorption
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