A short ride through a laboratory thesis project

The interaction of microorganisms with pollutants has lead to the development of an exciting field of research for environmentalists. The unprecedented interest in bioremediation and its applications stems from the failure of aggressive and invasive treatment methods that disturb ecosystems by stuffing them with unnecessary quantities of chemicals. Therefore, the use of microorganisms represents a viable, cost-effective decontamination alternative that is based on natural processes and can be scaled up for industrial or other purposes.

In the following description I will try to familiarize you with my thesis work that is directly related to this field. This is done in two parts:

1) The first part will deal with the particularities of the laboratory work involved in the research, or what I like to call with the life of a “lab rat”.

2) In the second part I will present the outcomes of my work on the bioremediation of wastewater polluted with toxic azo dyes.

Part 1: The lab rat

Why lab work?

Science and policy, as we all came (and some will come) to know after the first year in Budapest is full with laws and theories that provide us with frameworks, which in turn guide our research and work. The hallmark for both science and policy is that these theories must be in agreement with observation. Evidence of this consistency can only be collected through investigating past case studies (literature reviews), researching current case studies on the field (field work) or by carrying out laboratory work. Laboratory work offers a unique opportunity for direct interaction with natural phenomena on a small scale while it allows the collection of data through the use of modern equipment. From a personal point of view, the laboratory work offered me opportunities such as:

    * I was trained in designing investigation or experimental work
    * I was engaged in scientific reasoning
    * I was able to work on and manipulate sophisticated equipment
    * I was able to record data, transform them, analyze them and discuss them with experienced researchers

From Newton and Galileo to the basement of the University of Manchester

Galileo and Newton were two of the first persons to establish experimental procedures in order to prove that their hypothesis is consistent with observation. It is said that the former used the tower of Pisa to measure the effect of weight on acceleration, while the latter was inspired by an apple hitting his head. Certainly both events are apocryphal and the truth is that both used highly advanced techniques (for that certain period) in order to prove their concepts.

Since that time science and technology has made tremendous advancements and the Williamson Research Centre for Molecular Environmental Science located at the basement of the Department of Earth Sciences (University of Manchester) is a vivid proof of this evolution. I will never forget what the MESPOM “Batch 1” experienced during our orientation week when we first visited this basement. There were so many machines, different microscopes, different laboratories and all these serious persons in white coats coming and going. The funniest part was the discussion between us after the meeting. Almost everyone had this big question mark marked in our eyes followed by questions such as: “Did you understand anything?”, while the most dispassionate ones were wondering: “Will we ever get to work with one of these?”

It was not more than 5 months after some of us were wondering around that basement with our shiny-white lab coats doing research for our thesis.

Time constraints

The time allocated for the research and write-up of the thesis work is usually viewed as a constraint for more of the students and especially for those who are planning to include lab work. Well, if you are planning to do equivalent work to that of Galileo’s and Newton’s then time might be a serious constraint. However, if you are more realistic and plan to do a small-scale research equivalent to that of a Master’s student then I can assure you that the amount of time allocated is adequate.

My research program can be divided in 3 periods:

Period 1 (the rat is introduced to the new place)

This was the first month when I received basic training on the techniques and the equipment that I had to use for the completion of my thesis. During this month I started to get familiar with basic microbiological techniques and I also started learning how to use the different equipment for different experiments. Along the way, I had lengthy discussions on the designing part of the project and I was given the opportunity to study existing literature in order to be able to realize why I use each technique. However, I was not yet able to take lots of initiatives and I was following what I was advised.

Period 2 (the rat explores the new place)

The period after the first month was the most adventurous one. Slowly, I was able to comprehend better what I was doing, understand the articles I was reading and finally I was able to manipulate every technique in order to facilitate my purposes. By this time I was spending lots of hours in the lab (the number can be scary for some students so I prefer not to mention it) and I was actually enjoying every moment. The funniest part is that by this time I was treating the bacterial cultures I was growing for the experiments as if they were my children:

    * I was emotionally attached to them
    * I was trying to keep them clean (uncontaminated)
    * When everything was going right, they were rewarding and fulfilling
    * When they were not growing for any reason my heart was breaking
    * It was difficult to trust any other person to look after them
    * I need to look after them at inconvenient times (weekends, holidays...)
    * I could not just throw them away when they were going sick

Period 3 (the rat makes the new place its home)

It was only 3 months after the day I started working on my project that I realized that I should leave the lab for a while and start writing. By that time I was a proper lab rat spending hours after hours, repeating the experiments and discovering new ones in order to prove my concept. I was now able to design my own experiments and lead the research to its ultimate objective. However, this is where the fun stops and the boring (at least in comparative terms) procedure of writing-up starts. No matter how boring this procedure was, it was absolutely essential since it helped me visualise my entire work and take a holistic view on the project. All of a sudden everything came together. All the articles I was reading at the time suddenly obtained the form of my literature review. All the graphs I had prepared were becoming the “results” section and everything was coming down to the deliverable. Of course, things were not that smooth and since I am not exactly the most punctual student on the planet I was running till the last moment.

Wow! This work sounds like C.S.I.!

For those of you that feel that the things I described earlier resemble the famous American series C.S.I. (Crime Scene Investigation) - perfect techniques going in, clean data coming out in seconds and no mistakes happening – I have to tell you, this is T.V.. Even though some of the techniques used in C.S.I. (chromatography, DNA amplification, DNA sequencing through Polymerase Chain Reaction, Inductive Coupled Plasma analysis and so on and so forth) are also used in the Williamson Research Centre, things in general do not work so smoothly and perfectly.

An ode to failure

When I was designing the experimental protocol during the first month of the research I was not experienced enough to predict all the procedural anomalies. Just as I said before, things did not work perfect all the time. The bacterial cultures were not growing for more than a week during the first month. I had to repeat a simple bacterial growth experiment 3 times in order to get acceptable results. I had to wait for more than 2 weeks in the middle of my experiments for the technician to fix the spectrophotometer. Once I even had to stop one of my experiments and repeat it all over just because I could not find any syringes and needles in the lab. But one thing is for sure. Success compensated me for each and every failure. Every time I was getting a proper graph the satisfaction was surpassing all past failures and was giving me strength to continue.

Part 2: The thesis project

For those who are more interested in my thesis project, here you can find some technical information on this. The title of the project is:

Optimization strategies for the industrial applications of Shewanella oneidensis MR-1: The case of azo dyes

For those who already understood the meaning of the title, the following information will sound fairly simple. For those who think that the title is rather complicated, the following information will be more exciting and interesting.

A brief introduction

Instead of an introduction I will try to provide a small analysis of the title and the project undertaken by The University of Manchester and The Centre for Process Innovation (CPI) located in Teeside (close to Newcastle). The CPI is a U.K. based research and development centre that is closely cooperating with Universities and the global industry. They supported my project by providing ideas and information on the applied-industrial potentials of my project. In turn, I shared the outcomes of my experiments with them.

The microorganism used in the study

Ultimately, what I did is to harness natural processes in order to treat wastewater polluted with azo dyes. The microorganism selected for this research is Shewanella oneidensis strain MR-1. The Shewanella genus currently comprises 45 species all of which are Gram-negative bacteria that belong in the -γ- (gama) subgroup of the phylum proteobacteria of the order Alteromonadales. Shewanella species are Gram-negative, with a bacilli shape. In the field of bioremediation Shewanella species are frequently used for several reasons:

    * They are abundant in nature and in all natural environments (soil, fresh water, sea water etc).
    * Their metabolic versatility is fascinating. They are able to utilize a number of substances including metals and radionuclides for production of metabolic energy (e.g. Manganese, Iron, Chromium, Uranium, Technetium, Mercury and of course Azo dyes). Their metabolic versatility forms a very good explanation for their abundance (iron and manganese amongst others are abundant).
    * Finally, the fact that the complete genome of some of the Shewanella species is decoded offers us unique opportunities for the future. In particular, the genome of Shewanella oneidensis MR-1 was decoded in 2002. In this way researchers can identify the genes responsible for dye reduction and thus reveal the genetic mechanism that is behind this phenomenon.

The part of the title that refers to “Optimization strategies” reveals the form of work that I did. In particular, when someone refers to optimization strategies for the use of this or that bacteria he/she mainly refers to work from the scratch. In this sense, even though the final work intended to prove the “decoloration” concept, the experimental work started from the basic metabolic activities of Shewanella oneidensis MR-1.

Azo dyes

Colour is a trademark of human life. Industries related to textile, plastics, tannery, paper and printing, electroplating and a number of others use colour as one of the most illustrious characteristic of their products.

Colorants are highly coloured substances used to impart colour to potential substrates. Colorants are divided into soluble dyes and insoluble pigments. Moreover, dyes can be categorized into two main categories. Firstly, according to the chemical group (chromophores) responsible for absorbing light at a certain wavelength and eventually giving the colour. Dyes are categorized as azo, tri-arylmethane and anthraquinone. According to their mode of application to the fabric they can be categorized as direct, reactive, acid, disperse, mordant and vat.

It was demonstrated in 1858 that reacting an aromatic amine (Ar-NH2) with nitrous acid (HNO2) yields an unstable diazonium ion (ArN=N+). From this, coloured compounds containing an azo bond (-N=N-) can be formed (below are to examples). The first commercially available azo dye was Aniline Yellow (4-aminoazobenzene).

The experimental part

The experimental work can be summarized in the following points:

   1. The first experiment was the “optimization of the anaerobic planktonic, growth.” This was a simple growth experiment in which, I grew bacteria in liquid medium under different conditions. The different conditions were related to the amount of energy source and the amount of a specific Terminal Electron Acceptor (TEA). The TEA is the substance that corresponds to the role that iron, manganese, uranium etc. play in nature. In other words, it is necessary for the bacterial growth. For the “decoloration experiments” the TEA would be a dye. The purpose of this experiment was to identify the optimum ratio of energy source to TEA and employ this for all future experiments.
   2. For bioremediation applications microorganisms can be used in two modes. Either in a “free” phase (planktonic) or attached to a support material. For industrial applications (e.g. bioreactors) it is very common to select materials such as activated carbon or pumice where bacteria settle on. For my experiments I used three materials for cell immobilization: Pallets of activated carbon, pumice and grains of coke. Once bacterial cells find a niche on them they settle and start forming biofilms according to the procedure presented below:

3. The third set of experiments included decoloration experiments using planktonic cells and the results are presented below:

Quartz cuvettes containing Direct Blue 53 and cells of Shewanella oneidensis MR-1. Figure (a) shows the cuvettes before incoculation (transportation of cells in the cuvette). Figure (b) shows the cuvettes after 30 minutes of incubation with Shewanella oneidensis MR-1. Figure (c) shows the result after 1 hour when the colour is totally removed.

Quartz cuvettes containing Direct Blue 53 and cells of Shewanella oneidensis MR-1. Figure (a) shows the cuvettes before incoculation (transportation of cells in the cuvette). Figure (b) shows the cuvettes after 30 minutes of incubation with Shewanella oneidensis MR-1. Figure (c) shows the result after 1 hour when the colour is totally removed.

4. The forth experiment included decoloration using immobilized cells/biofilms. The purpose of the decoloration experiments (both using planktonic and immobilized cells) was to optimize the decoloration process. The oprimization was mainly related to selecting the optimum age of the cells, the addition of a chemical mediator that would enhance decoloration rates and several other techniques.
The lab techniques:

Here is a brief overview of the lab techniques I used for all the experiments:

Analysis Using a UV-visible spectrophotometer

Absorption was monitored using an Analytik Jena AG SPECORD 600 UV-vis spectrophotometer controlled by WinASPECT software (version 2.0.4.112). The spectrophotometer was fitted with the 8-fold cell changer attachment the temperature of which was controlled at 30˚C using circulated heated water.

Analysis Using Environmental Scanning Electron Microscopy

Environmental scanning electron microscopy (ESEM) is used in materials’ science for characterization of the surface of materials. It was used in this study for the analysis of the surface of the support material (activated carbon and coke). Moreover, it was used for observing biofilms formed by Shewanella oneidensis MR-1 on the materials’ surface. ESEM was carried out using a Philips XL30 ESEM-FEG (FEI Company)

Analysis Using Confocal Scanning Electron Microscopy

Among the most versatile and effective approaches especially for the observation of biofilms, is Confocal Laser Scanning Microscopy (CLSM). The microscope used for the study is the Leica SP5, which is a completely filter free system.

Analysis Using High Performance Liquid Chromatography

Chromatography is a commonly-used method in forensics to analyze fibers and blood samples. Liquid chromatography is used to analyze liquid samples for identification of metal ions and organic compounds. HPLC analysis of samples (500 ul) was carried out using a Gemini 5u C18 110A Phenomenex® column (250 x 10.00 mm,Phenomenex, UK) fitted to a Dionex GP50 gradient pump and a UV-vis detector Dionex UVD170U (Dionex, UK).

Some interesting findings

Apart from the complicated (and certainly boring) graphs I managed to get some exciting results mainly through the use of the imaging techniques (ESEM and CLSM).

The ESEM was used in order to understand the surface of the materials I used. This would provide me with a more clear picture of the biofilm formation process.

Further on, the CLSM was used to track down biofilms and to give me a clear picture of the first stages of formation (monolayers). For this procedure a fluorescent dye was employed in order to stain the bacteria and separate it from the background material. Then, by playing with different wavelengths I managed (after 3 days of observation and continuous failures) to visualize them:

To summarize, planktonic and immobilized cells of Shewanella oneidensis MR-1 were able to reduce solutions of the azo dye Direct Blue 53 and give colorless solutions under anaerobic conditions. Anaerobic biological degradation of azo dyes possesses several advantages over physical and chemical treatment and is in demand.

 Sound biotechnological applications that can be scaled-up will be extremely useful in remediating the environmental issues created by the release of effluents containing azo dyes.
To respond to this challenge a series of experimental strategies were adopted for the optimization of the activity of Shewanella oneidensis MR-1 against azo dyes.

I hope you enjoyed the tour that describes my lab work and some of my interesting findings of the research. Certainly the lab work will attract some and will frighten some others. Anyway, it has been an amazing experience and since I was given the opportunity both from the University of Manchester and the CPI to continue working on this project as a research assistant there is only one thing I can say for the coming time. I am looking forward!