Biofilm Formation asignment
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1. Four bacterial strains were isolated from wastewater and tested for their ability to form biofilms under metal stress conditions using microtiter plate and metal reduction assays. 2. All strains showed increased biofilm formation under metal stress compared to no metal stress, demonstrating their ability to resist metal toxicity through biofilm production. 3. The microtiter plate assay found that biofilm formation increased over 3, 5, and 7 days of growth and was highest under metal stressed conditions for all strains.
Biofilm Formation asignment
- 1. Submitted to : Maam Saba Submitted by : M.Phil. I Group: 03 Report : Wastewater Analysis Department Of Microbiology And Molecular Genetics University Of The Punjab
- 2. 1 Group Members Sr. No. Members Roll No: 1 Ayesha Siddiqa 01 2 Khaleeqa Ahsan 02 3 Shafiqa Shehzadi 03 4 Nigarish Umer 07 5 Rabiya Ikram 08 6 Asma Kalsoom 09 7 Naima Khan 10 8 Mansoor 13
- 3. 2 Introduction Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics. Figure 1 The formation of a biofilm occurs in several stages, comprising the development, maturation and disassembly of the bacterial community. Studies of biofilm development of single-strain microorganisms have focused mainly on clinically relevant bacteria or strains involved in food spoilage, but areas such as high-grade chemical production, microorganisms in water distribution systems and bioaugmentation through natural genetic transformation have also been covered. The development and persistence of biofilms are affected not only by the surrounding environment but also by the variety of species present. Microtiter Plate Assay The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms
- 4. 3 grown in microtiter dishes do develop some properties of mature biofilms, such an antibiotic tolerance and resistance to immune system effectors. This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. This assay was used to study biofilm formation by the four different strains isolated from wastewater. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms. All of the organisms showed biofilm formation on surfaces of polystyrene within 24-48h. The biofilm formation was studied under metal stress conditions and showed increased slime production in metal stress as compared to strains without metal stress. So, under stress conditions these bacteria showed large biofilm production and their ability to resist metal stress. Reduction Assay Industrialization and technological advancements, the hallmarks of civilization have been increasing heavy metal releases to the environment, that pose a significant threat to environment and public health because of their toxicity, accumulation in the food chain and persistence in nature. Heavy metals are toxic because they interfere with the normal biochemical reactions of the human body. The release of heavy metals into the environment causes an environmental pollution problem because they are nonbiodegradable and hence accumulate in living organisms. Some metals (e.g., Cd, Co, Cr, Cu, Fe, Ni, Pb and Zn) are essential metals, which serve as microelements; they are used for redox reactions to stabilize molecules through electrostatic interactions, as components of various enzymes, and as regulators of osmotic pressure. However, the presence of high concentrations of heavy metals in the environment can be detrimental to a variety of living species. The most important features that distinguish heavy metals from other toxic pollutants are their nonbiodegradability and propensity in living materials. Metal processing, finishing and plating are the main sources of metal wastes; however, there are also many other processes from which heavy metals originate. Metal uptake by microorganisms is a complicated process that depends on the chemistry of the metal ions, surface features of the microorganisms, cell physiology and physicochemical influences from the environment, e.g. pH, temperature and metal concentration. Heavy metals are released into the environment from a wide range of natural and anthropogenic sources. The rate of influx of these heavy metals into the environment far exceeds their removal by natural processes, thus leading to the accumulation of heavy metals in the environment, with aquatic ecosystems normally at the receiving end. The main problems caused by the release of
- 5. 4 toxic pollutants into receiving waters are toxicity to aquatic organisms and restrictions on the human use of these waters. As a result of natural and industrial processes heavy metals are increasingly found in microbial habitats. Thus, microbes have evolved several mechanisms to tolerate the presence of heavy metals (by efflux, complexation, or reduction of metal ions) and they also use them as terminal electron acceptors in anaerobic respiration. The intake and subsequent efflux of heavy metal ions by microbes usually include a redox reaction involving the metal, and some bacteria can use such reactions for energy and growth. Therefore, bacteria that are resistant to and grow on metals also play an important role in the biogeochemical cycling of those metal ions. As a result of these characteristics, microbes play an important role in cleaning up or remediating metal-contaminated environments. Chromium and nickel are released into the environment by a large number of processes such as electroplating, leather tanning, wood preservation, pulp processing, steel manufacturing, etc., and the concentration levels of chromium and nickel in the environment widely varies. These two metals are of major concern because of their larger usages in developing countries and their non- degradability nature. Hexavalent chromium is highly soluble in water and carcinogenic to human. Ni (II) is more toxic and carcinogenic metal when compared with Ni (IV). Due to their toxic effects on living systems stringent limits have been stipulated for the discharge of chromium and nickel into the environment. The aim of this study was to determine whether the bacteria isolated from the wastewaters have the ability to produce biofilms in the metal stress and if so, then to what level they are producing the biofilms. So for that purpose two experiments were performed i.e. microtiter plate and metal reduction assay.
- 6. 5 Figure 2 Biofilm formation by bacterial isolates on a microtiter plate 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 3 day 5 day 7 day S1+S S1 S2+S S2 S3+S S3 S4+S S4